a comparative study of fluorescence resonance … · 2018-07-10 · date: 15.09.2014 certificate...

A COMPARATIVE STUDY OF FLUORESCENCE RESONANCE

ENERGY TRANSFER (FRET) IN SOLUTION PHASE AND IN

HYBRID FILMS PREPARED BY LB AND LBL TECHNIQUES.

THESIS SUBMITTED FOR THE AWARD OF DEGREE OF DOCTOR OF

PHILOSOPHY IN PHYSICS IN PARTIAL FULFILMENT OF THE

RESEARCH REQUIREMENTS

By

DIBYENDU DEY

DEPARTMENT: PHYSICS

FACULTY: SCIENCE

UNIVERSITY: TRIPURA UNIVERSITY

ADDRESS: SURYAMANINAGAR - 799022

TRIPURA (WEST), INDIA

REGISTRATION NUMBER: F.TU/REG/Ph.D/10/(12-14)11, dated: 26/07/2011

YEAR OF SUBMISSION: 2014

A COMPARATIVE STUDY OF FLUORESCENCE RESONANCE

ENERGY TRANSFER (FRET) IN SOLUTION PHASE AND IN

HYBRID FILMS PREPARED BY LB AND LBL TECHNIQUES.

THESIS SUBMITTED FOR THE AWARD OF DEGREE OF DOCTOR OF

PHILOSOPHY IN PHYSICS IN PARTIAL FULFILMENT OF THE

RESEARCH REQUIREMENTS

By

DIBYENDU DEY

Under the supervision of:

Dr. Syed Arshad Hussain

Assistant Professor, Department of Physics

DEPARTMENT: PHYSICS

FACULTY: SCIENCE

UNIVERSITY: TRIPURA UNIVERSITY

ADDRESS: SURYAMANINAGAR - 799022

TRIPURA (WEST), INDIA

REGISTRATION NUMBER: F.TU/REG/Ph.D/10/(12-14)11, dated: 26/07/2011

YEAR OF SUBMISSION: 2014

DEDICATED TO MY PARENTS AND SUPERVISOR

Date: 15.09.2014

Certificate This is to certify that Mr. Dibyendu Dey has completed his research work presented in

this thesis entitled “A comparative study of Fluorescence Resonance Energy

Transfer (FRET) in solution phase and in hybrid films prepared by LB and LBL

techniques.” under the supervision of Dr. S. A. Hussain. This is absolutely based on

his own work in accordance with the approved plan of work. He has got his name

registered on 26/07/2011 (F.TU/REG/Ph.D/10/(12-14)11) for the award of Ph.D.

degree in Physics of Tripura University. He has presented his public seminar on

13.05.2014. All the members of the Ph.D. committee recommended Mr. Dey to

prepare and submit his Ph.D. thesis without any further alteration. This has been

approved by P.G Board of studies, Physics, in the meeting held on 13.05.2014. He has

not submitted any part of this work elsewhere to achieve any diploma/ degree or any

other academic award before. As he has fulfilled all the formalities for submission of

Ph.D. thesis as per the regulations of the Tripura University, this Ph.D. thesis may be

considered for necessary action by the University.

(Dr. Syed Arshad Hussain) (Prof. Barin Kumar De) Ph.D. Supervisor Chairman, P.G Board of studies Assistant Professor Head Department of Physics Department of Physics Tripura University Tripura University Email: [email protected] Email: [email protected]

Ph.D. Thesis: Dibyendu Dey

Acknowledgement i

Acknowledgement It is a great pleasure to convey my gratitude to all the people who contributed

a lot throughout my research work in my humble acknowledgment. I would like to

express my deep gratitude to my supervisor Dr. Syed Arshad Hussain for providing

excellent experimental conditions, for his care and many of productive discussions

about this research work on FRET.

I would also like to express my sincere gratitude to Prof. D. Bhattacharjee for

his suggestions and motivation throughout my research work.

I wish to express my thanks to Prof. Barin Kumar De, Head, Department of

Physics, Tripura University, for his kind help and support in various ways. I am also

thankful to Dr. Anirban Guha, Dr. Surya Chattopadhyaya and Dr. Ratan Das for their

moral support and valuable suggestions.

My special gratitude goes to all of my lab mates, Mr. Santanu Chakraborty,

Mrs. Mitu Saha, Miss Joyashree Bhattacharjee, Dr. Sekhar Charraborty, Miss Jaba

Saha, Mr. Arpan Datta Roy, Mr. Pintu Debnath, Mr. Ashish Shil, Md. Nurul Islam,

Dr. Dhananjay Dey, Mrs. Soma Banik, Mr. Chandan Debnath, Mrs. Namita Das, for

their discussions, lab assistance and their constant support.

I am also thankful to all research scholar of Atmospheric and Sferics research

group Department of Physics and surface science research group of Department of

Chemistry T.U. for their constant help and support. I am thankful to all staff of

Physics Department for their support.

I am grateful to Tripura University for providing me all the journals & Lab

facilities needed to carry out my research work.

At this point, I would also like to give my warmest thanks to my parents who

always supported and encouraged me without a second thought in everything I have

ever done. Without the love and support of my family, this would have been a very

hard journey. My love for them is eternal.

I also apologize and thank all those whom I missed in the acknowledgment.

The 15th September, 2014

Agartala, India . (Dibyendu Dey)


List of Publications ii

List of Publications

List of publications related to my Ph.D. thesis

1. Effect of nanoclay laponite and pH on the energy transfer between fluorescent

dyes.

Dibyendu Dey, D. Bhattacharjee, S. Chakraborty, S.A. Hussain

Published in Journal of Photochemistry Photobiology A-Chemistry 252 (2013)

174– 182.

2. Development of hard water sensor using fluorescence resonance energy transfer.


Published in Sensors and Actuators B: Chemical 184 (2013) 268-273.

3. Development of a DNA sensor using a molecular logic gate,

D. Bhattacharjee, Dibyendu Dey, S. Chakraborty, S.A. Hussain, S. Sinha,

Published in Journal of Biological Physics 39 (2013) 387-394.

4. Development of an Ion-Sensor using Fluorescence Resonance Energy Transfer,

Dibyendu Dey, Jaba Saha, Arpan Datta Roy, D. Bhattacharjee, Syed Arshad Hussain


5. A comparative study of FRET between Acriflavine and Rhodamine B on to thin

film.

Dibyendu Dey, D. Bhattacharjee, Syed Arshad Hussain

(Paper based on this work is under preparation)

6. Molecular Logic Gates using FRET Phenomenon.

Dibyendu Dey, D. Bhattacharjee, S. Chakraborty and Syed Arshad Hussain

Published in the Proceedings of the Conference on Recent Trends of Research in

Physics (CRTRP-2012) (ISBN: 978-81-904362-9-8 (2013))


List of Publications iii

List of publications where I have substantial contributions

1. J-aggregates of thiacyanine dye organized in LB films: Effect of irradiation of light.

Syed Arshad Hussain, Dibyendu Dey, S. Chakraborty, D. Bhattacharjee

Published in Journal of Luminescence 131 (2011) 1655–1660.

2. Effect of DNA denaturation on FRET between laser dyes



3. Study of Hysteresis during pH and Temperature Changes of Acriflavine: A

Gateway to Optrode.

Soma Banik, Dibyendu Dey, D. Bhattacharjee, Syed Arshad Hussain.

Published in Invertis Journal of Science and Technology 7 (2014) 96-103.

4. Development of a sensor to study the DNA conformation using molecular logic

gates

Arpan Datta Roy, Dibyendu Dey, Jaba Saha, Santanu Chakraborty, D. Bhattacharjee,

Syed Arshad Hussain

(Communicated for publication in Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy).

5. Fluorescence Resonance Energy Transfer (FRET) sensor

Syed Arshad Hussain, Dibyendu Dey, Sekhar Chakraborty, Jaba Saha, Arpan Datta

Roy, Santanu Chakraborty, Pintu Debnath, D. Bhattacharjee.

Published in Journal of Spectroscopy and Dynamics 5 (2015) 1-16.


List of seminars iv

List of seminars attend

1. State level seminar on Frontier Areas of Chemistry held at Department of

Chemistry, Tripura University on September 3rd, 2010. Presented paper entitled

“Study of DNA conformation based on FRET between Acriflavine and Rhodamine B”.

2. 7th National conference in Physics held at Department of Physics Manipur

University during October 5-6, 2010. Presented paper entitled “Enhancement of

FRET between laser dyes in presence of clay”.

3. International conference on emerging areas of chemistry (IEAC-2011) held at

Department of Chemistry Tripura University during January 12-14, 2011.

Presented paper entitled “Design of DNA sensor using FRET between laser dyes”.

4. UGC Sponsored National seminar on contemporary trends of research in

physical sciences held at Department of Physics, Gurucharan College Silchar,

Assam during February 11-12 2011. Presented paper entitled “A comparative study

between the spectroscopic characteristics of two laser dyes in self assembled ultrathin

films”.

5. National seminar on emerging trends in science and technology held at ICFAI

University Tripura during March 25-26, 2011. Presented paper entitled “Effect of

clay on FRET between two laser dyes Pyrene and Acriflavine”.

6. A National seminar on condensed matter physics held at Department of

Physics, Gauhati University during August 24-26, 2011. Presented paper entitled

“Design of Hard water sensor using FRET between laser dyes”.

7. Fifteenth national conference on surfactants, emulsions and biocolloids-2011

(NATCOSEB XV) held at department of Chemistry Tripura University during

December 27-29, 2011. Presented paper entitled “Design of pH sensor using FRET

between Acriflavine and Rhodamine B”.


List of seminars v

8. National Conference on Recent Trends of Research in Physics &

Departmental Reunion – 2012 (CRTRP2012). 3 – 4th February, 2012. Organized

by Department of Physics, Tripura University, Suryamaninagar. Presented paper

entitled “Construction of basic and universal molecular logic gates on the basis of pH

sensetiveness of dsDNA”

9. National seminar on green chemistry and nanoscience: theory and

applications held at department of Chemistry MBB College during July 20-21,

2012. Presented paper entitled “Design of Optrode for the sensing of pH and

Temperature”.

10. International conference on material science (ICMS-2013) held at

Department of Physics Tripura University during February 21-23, 2013.

Presented paper entitled “Design of molecular logic gate for the sensing of different

ions using FRET”.


Synopsis vi

Synopsis

Fluorescence Resonance Energy Transfer (FRET) is an electrodynamic

phenomenon occurs through the transfer of exited state energy from donor to

acceptor. It has vast applications in the field of medical diagnostic, DNA analyses,

and study of molecular level interaction and for various sensing applications. The

theoretical analysis was well developed by Förster. FRET process is distance

dependent between donor and acceptor. So for the FRET applications it is very

important to control the intermolecular separation between the FRET pair.

Introduction of negatively charged clay particles are very useful for concentrating the

cationic dye molecules on to their surfaces and hence influence the FRET efficiency.

FRET sensors are popular tools for studying intracellular processes and to

identify the presence of different analytes. FRET sensors are based on the influence of

different analyte as well as their conformational changes on the FRET efficiency for a

particular donor-acceptor pair. Therefore, it is very important to identify new FRET

pair and to quantify the FRET parameters in different condition in order to

demonstrate their possible application. The main purpose of the present thesis work is

to investigate the FRET between organic laser dyes and to study the influence of clay

on FRET parameters and demonstrate their possible sensing applications. Based on

the results of my pre-Ph.D. thesis work we have demonstrated pH-sensor, hard water

sensor, ion sensor and DNA sensor. It has been observed that incorporation of clay

particles enhances the sensing efficiency.

The main objectives of this thesis are:-

1. To identify FRET (D-A) pair in order to explore their possible applications.

2. To study the FRET phenomenon among different molecules in solution and

organized in the restricted geometry of ultra thin films.

3. To demonstrate the parameters changing which the efficiency and extent of

energy transfer can be controlled.

4. To compare the FRET efficiency among D-A pair in ultra thin films,

solution and in bulk.

5. To demonstrate the applications of FRET in designing ratiometric sensors.


Synopsis vii

The findings of my pre-Ph.D. research work will be presented in the

Ph.D. thesis in ten chapters as follows:-

Chapter 1 states about the motivations and objectives of the present thesis work.

Chapter 2 contains the general background and the necessary preliminaries required

to understand the results and discussions throughout the thesis.

Chapter 3 contains the International and National status of the recent research

activities related to the topic of the present thesis work.

Chapter 4 deals with the details about the materials and different experimental

techniques used for sample preparation as well as characterizations throughout the

present thesis work.

Chapter 5 contains the study of FRET between two dyes Acriflavine (Acf) and

Rhodamine B (RhB) in solution and ultrathin films in presence and absence of clay

mineral laponite. UV–Vis absorption and fluorescence spectroscopy studies suggest

both the dyes present mainly as monomer in solution and films. Energy transfer

occurred from Acf to RhB in both solution and films. The energy transfer efficiency

increases in presence of clay laponite and the maximum efficiency were 92.50% and

55.71% in clay dispersion and in films respectively. Presence of laponite particles

onto film was confirmed by atomic force microscopy investigations with a surface

coverage of more than 75%.

Chapter 6 reports the change in FRET efficiency between two laser dyes with the

variation of solvent pH. Energy transfer efficiency was pH sensitive and it varies from

4.5% to 44.45% in mixed dye solution for a change in pH from 3.0 to 12.0. With

proper calibration it is possible to use the present system under investigation to sense

pH over a wide range from 3.0 to 12.0.

Chapter 7 mainly concern with a method for the sensing of water hardness by

determining the concentration of calcium and magnesium ions in water, based on

FRET process. The principle of the proposed sensor is based on the change of FRET

efficiency between two laser dyes Acf and RhB in presence of permanent hard water

components (CaCl2 and MgCl2). Nanodimensional clay platelet laponite was used to

enhance the efficiency of the sensor.

Chapter 8 mainly concern with a method for the sensing of different ions by

determining the concentration of corresponding salts (KCl, NaCl, MgCl2, CaCl2,

FeCl3, FeSO4, and AlCl3) in water, based on FRET process. The principle of the

proposed sensor is based on the change of FRET efficiency between two laser dyes


Synopsis viii

Acf and RhB in presence of different ions (K+, Na+, Mg2+, Ca2+, Fe2+, Fe3+, Al3+).

Nanodimensional clay platelet laponite was used to enhance the efficiency of the

sensor.

Chapter 9 reports the increase in FRET efficiency between two laser dyes in the

presence of deoxyribonucleic acid (DNA). Two types of molecular logic gates have

been designed where DNA acts as input signal and fluorescence intensity of different

bands are taken as output signal. Use of these logic gates as a DNA sensor has been

demonstrated.

Chapter 10 contains overall conclusion and future prospect of my research.


Contents ix

Contents Cover Page

Dedication

Certificate

Acknowledgement i

List of publications ii

List of seminars/workshops attend iv

Synopsis vi

Contents ix

Introduction and Objective

1.1 Motivation 2

1.2 Thesis Objectives 3

REFERENCES 3

Background and Overview

2.1 Fluorescence Resonance Energy Transfer (FRET) 5

2.1.1 Principle and theoretical consideration of FRET 5

2.1.2 Methods for FRET detection 8

2.2 Influence of Clay on FRET 10

2.2.1 Properties of clay minerals 10

2.2.2 Influence of clay on FRET 12

2.3 FRET as molecular spy 13

2.4 Sensing technology using FRET ..14

REFERENCES 15

International and National status

3.1 International status 18

3.2 National status 21

REFERENCES 22

Experimental Techniques

4.1 Introduction to the experimental techniques 26

Chapter 1 1-3

Chapter 2 4-16

Chapter 3 17-24

Chapter 4 24-45


Contents x

4.2 Materials used 26

4.2.1 Dyes used as FRET pair 26

4.2.2 Clay mineral 27

4.2.3 Double stranded DNA 27

4.2.4 Matrix materials for film preparation 27

4.2.5 Other materials 28

4.3 Solution preparation 28

4.4 Film preparation 28 4.4.1 Langmuir-Blodgett technique 28

4.4.2 Layer-by-Layer (LbL) self assembled technique 31

4.5 Molecular Spectroscopy 33

4.5.1 Ultraviolet-Visible (UV-Vis) absorption spectroscopy 33

4.5.2 Fluorescence spectroscopy 37

4.5.3 Atomic Force Microscopy (AFM) 41

REFERENCES 44

Comparative study of FRET between two laser dyes Acriflavine and Rhodamine

B in solution phase and in hybrid ultrathin films prepared by LbL and LB

techniques.

5.1 Introduction 47

5.2 Experimental 48

5.2.1. Solution preparation 48

5.2.2. Film preparation 48

5.3 Results and discussion 50

5.3.1 The UV–Vis absorption and steady state fluorescence spectroscopy 50

5.3.2 FRET between Acf and RhB in solution in presence and absence of nanoclay laponite 51

5.3.3 FRET between Acf and RhB in ultrathin films in presence and absence of nanoclay laponite 53

5.3.4 Atomic Force Microscopy study 56

5.3.5 Effect of spacer molecules on FRET between Acf and RhB in ultrathin

films 57

5.4 Conclusion 59

REFERENCES 60

Chapter 5 46-61


Contents xi

Development of pH sensor using fluorescence resonance energy transfer

6.1 Introduction 63

6.2 Experimental 63

6.2.1 Solution preparation 63


6.3.1. Effect of pH on FRET 64

6.3.2 Sensing of pH of aqueous solution by FRET 66

6.4 Conclusion 67 REFERENCES 67

Development of hard water sensor using fluorescence resonance energy transfer

7.1 Introduction 70

7.2 Experimental 71



7.3.1 Sensing of Hard Water by FRET 71

7.3.2 Variation of salt concentration 75

7.3.3 Design of sensor 76

7.4 Conclusion 77

REFERENCES 77

Development of an ion sensor using fluorescence resonance energy transfer

8.1 Introduction 79

8.2 Experimental 80



8.3.1 Sensing of ions by FRET 80

8.3.2 Ions with variable size 82

8.3.3 Ions with variable valency 82

8.3.4 Effect of variation of salt concentration on FRET efficiency 83

8.3.5 Design of ion sensor 85

8.4 Conclusion 86

Chapter 6 62-68

Chapter 7 69-77

Chapter 8 78-88


Contents xii

REFERENCES 87

Development of a DNA sensor using a molecular logic gate

9.1 Introduction 90

9.2 Experimental 90



9.3.1 FRET between Acf and RhB in the presence of DNA 91

9.3.2 Design of molecular logic gates 93

9.3.3 Design of NOT gate as DNA sensor 93

9.3.4 Design of YES-NOT gate as DNA sensor 93

9.4. Conclusion 94

REFERENCES 95

Overall conclusion and future prospect 99

Reprints of publications (Cover page only)

Chapter 9 89-96

Chapter 10 97-99


Chapter 1: Motivation and objectives 1

Chapter-1

Motivation and objectives

This chapter states about the motivations and objectives of

the present thesis work.



Chapter-1

Motivation and objectives 1.1. Motivation

Fluorescence Resonance Energy Transfer (FRET) is an ideal tool to measure

the separation between an excited donor (D) and an acceptor (A). As the extent of

such dipole interaction is sensitive to the D-A distance this technique is widely used

for studying the structural dynamics of bio-molecules [1-7]. One of the important

methods for sensing of different chemical and biological materials is fluorescent

sensors. But for this type of sensors change in fluorescent intensity could very well be

perturbed by environmental factors. The introduction of ratiometric sensors can

minimize this environmental perturbation, because it measures the ratio of two

emissions in different environments. The design of ratiometric sensors can be done by

using FRET. The advantage of FRET is that the ratio between two fluorescence

intensities is independent of external factors such as fluctuation of excitation source

and concentration. FRET observed the changes in the emission intensity ratio of two

fluophores which is favourable in increasing the signal selectivity. FRET sensors

became popular tools for studying intracellular processes and to identify the presence

of different analytes. FRET based sensors are based on the influence of different

analyte as well as their conformational changes on the FRET efficiency for a

particular D-A pair [8-13]. Therefore it is very important to identify new FRET pair

and to quantify the FRET parameters in different condition in order to demonstrate

their possible applications of such system.

On the other hand, molecules can be confined onto the clay surfaces and

within the clay interlayer causing an increase in molecular proximity, which is vital

for FRET efficiency. Therefore it is very important to identify and study FRET

between molecules in organo-clay hybrid system in order to explore their possible

applications.

The main purpose of the present thesis work is to investigate the FRET

between organic laser dyes and to study the influence of clay on FRET parameters

and to demonstrate their possible applications. Based on the results of my pre-Ph.D.

thesis work we have demonstrated pH-sensor, hard water sensor, ion sensor and DNA



sensor etc. It has been observed that incorporation of clay particles enhances the

sensing efficiency.

1.2. Thesis Objectives The main objectives of this thesis are:

1. To identify FRET (D-A) pair in order to explore their possible applications.

2. To study the FRET phenomenon among different molecules in solution and

organized in the restricted geometry of ultra thin films.

3. To demonstrate the parameters changing which the efficiency and extent of

energy transfer can be controlled.

4. To compare the FRET efficiency among D-A pair in ultra thin films,

solution and in bulk.

5. To demonstrate the applications of FRET in designing ratiometric sensors.

References 1. T. H. Förster, Z. Naturforsch 4A (1949) 321. 2. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd edn, Springer, New York, (2006). 3. D. M. Chudakov, S. Lukyanov, K. A. Lukyanov, Trends Biotechnol. 23 (2005) 605. 4. B. N. Giepmans, S. R. Adams, M. H. Ellisman, R. Y. Tsien, Science 312 (2006) 217. 5. A. Miyawaki, Dev.Cell 4 (2003) 295. 6. J. Zhang, R. E. Campbell, A. Y. Ting, R. Y. Tsien, Nat. Rev. Mol. Cell. Biol. 3 (2002) 906. 7. B. A. Pollok R. Heim, Trends Cell. Biol. 9 (1999) 57. 8. C. Ma, F. Zeng, L. Huang, S. Wu, J. Phys. Chem. B 115 (2011) 874. 9. A. R. Clap, I. L. Medintz, H. Mattoussi, Phys. Chem. Chem. Phys. 7 (2006) 47. 10. T. Pons, I. L. Medintz, K. E. Sapsford, S. Higashiya, A. F. Grimes, D. S. English, H. Mattoussi, Nano Lett. 7 (2007) 3157. 11. T. Pons, I. L. Medintz, X. Wang, H. Mattoussi, J. Am. Chem. Soc. 128 (2006) 15324. 12. X. L. Zhang, Y. Xiao, X. H. Qian, Angew. Chem. Int. Ed. 47 (2008) 8025. 13. H. Ueyama, M. Takagi, S. Takenaka, J. Am. Chem. Soc. 124 (2002) 14286.


Chapter 2: Background and Overview 4

Chapter-2

Background and Overview

This chapter contains the general background and the

necessary preliminaries required to understand the results and

discussions throughout the thesis.



Chapter-2

Background and Overview 2.1. Fluorescence Resonance Energy Transfer (FRET)

FRET is a non-radiative transfer of energy from the excited state donor

fluorophore (D) to an acceptor fluorophore (A). The theoretical analysis was well

developed by Förster [1, 2]. The rate of energy transfer depends upon the extent of

spectral overlapping area of the emission spectrum of donor with the absorption

spectrum of the acceptor, the relative orientation of the donor and acceptor transition

dipole moments and the distance between these molecules [1-3]. Due to its sensitivity

to distance, FRET has been used to investigate molecular level interactions [3].

Fluorescence emission rate of energy transfer has wide applications in biomedical,

protein folding, RNA/DNA identification and their energy transfer processes [4-10].

FRET mechanism is also important in other phenomenon, such as

photosynthesis kinetics, chemical reactions and Brownian dynamics [11-13]. Another

important application of FRET phenomenon is in dye lasers. If a dye laser has to be

used as an ideal source, its spectral region needs to be extended. FRET can be used to

extend the spectral range of the laser dyes. The use of energy transfer in dye lasers is

also helpful in minimizing the photo quenching effects and thereby increasing the

laser efficiency.

2.1.1. Principle and theoretical consideration of FRET

In the process of FRET, initially a donor fluorophore (D) absorbs the energy

due to the excitation of incident light and transfer the excitation energy to a nearby

chromophore, the acceptor (A) (Fig. 2.1).

hAAADAD

DhD

*

**

*

Fig. 2.1. Basic principle of Fluorescence Resonance Energy Transfer (FRET). Here h is the Planck’s constant and is the frequency of the radiation.



Energy transfer manifests itself through decrease or quenching of the donor

fluorescence and a reduction of excited state lifetime accompanied also by an increase

in acceptor fluorescence intensity. Fig. 2.2 is a Jablonski diagram that illustrates the

coupled transitions involved between the donor emission and acceptor absorbance in

FRET. In presence of suitable acceptor, the donor fluorophore can transfer its excited

state energy directly to the acceptor without emitting a photon. The first step

represents the absorption of energy by the donor molecule. The donor molecule gets

excited to the singlet state, S1D from the ground state, S0

D. Several excited states are

available to the donor; however, vibrational relaxation to S1D by internal conversion is

rapid, ensuring that a majority of emission occurs from this state. Various energy

states are possible for the donor in excited states, including spontaneous emission and

nonradiative processes. Nonradiative energy transfer between the donor and acceptor

can occur when a suitable acceptor fluorophore is placed nearby the donor molecule.

This transfer involves a resonance between the singlet-singlet electronic transitions of

the two fluorophores, generated by coupling of the emission transition dipole moment

of the donor and the absorption transition dipole moment of the acceptor. Therefore,

the FRET efficiency and the range of distances over which it can be observed are

determined by the spectral properties of a given pair of donor and acceptor.

Fig. 2.2. Jablonski diagram of FRET process.

There are few criteria that must be satisfied in order for FRET to occur. These

are: (i) the fluorescence emission spectrum of the donor molecule must overlap the

absorption or excitation spectrum of the acceptor chromophore. The degree of overlap



is referred to as spectral overlap integral (J). (ii) The two fluorophores (donor and

acceptor) must be in the close proximity to one another (typically 1 to 10 nanometer).

(iii) The transition dipole orientations of the donor and acceptor must be

approximately parallel to each other. (iv) The fluorescence lifetime of the donor

molecule must be of sufficient duration to allow the FRET to occur.

Solving the enigma surrounding fluorescence quenching experiments revealed

the phenomenon of FRET and led J. Perrin [14] to propose dipole–dipole interactions

as the mechanism via which molecules can interact without collisions at distances

greater than their molecular diameters. Some 20 years later, Förster [15, 16] built

upon Perrin’s idea to put forward an elegant theory which provided a quantitative

explanation for the non-radiative energy transfer in terms of his famous expression

given by

6

01 1TD

Rk rr

Where, Tk r is the rate of energy transfer from donor to acceptor, r is the

distance between donor and acceptor and 0R is the well-known Förster radius. The

distance at which resonance energy transfer is 50% efficient, is called the Förster

distance. At r = R0, the transfer efficiency is 50% and at this distance the donor

emission would be decreased to half of its intensity in the absence of acceptor.

The value of R0 can be defined by the following expression [17-20]

2

6 40 5 4

0

9000 ln102

128D

D A

kR F d

Nn

Where,

FD= the normalized fluorescence intensity of the donor.

εA = the extinction coefficient of the acceptor (in M-1cm-1).

λ = the wavelength (in nm).

D = the fluorescence quantum yield of the donor in the absence of acceptor.

n = is the refractive index of the medium.

k2 = orientation factor of transition dipole moment between donor and acceptor.

N = Avogadro number.

The integral part of equation (2) is known as the spectral overlap integral J(λ) and is

given by



34

0

dFJ AD

Therefore the above definition of R0 in equation (2) can be rewritten in terms of J(λ)

with units M-1cm-1nm4 as 12 4 6

0 0.2108 ( ) (4)DR k n J Where R0 is in units of Å

The energy transfer efficiency can be termed as [72-74]

1 5

1T D T

T D D T

k r k rE

k r k r

This is the fraction of the transfer rate to the total decay rate of the donor. Using

equation (1) and (5) E can be expressed as

666

0

60

rR

RE

The efficiency of the energy transfer (E) can also be expressed as

71 D

DA

FFE

Where FDA is the relative fluorescence intensity of donor in the presence of acceptor

and FD is the fluorescence intensity of donor in the absence of acceptor. This equation

is equivalent to equation 5 [20].

2.1.2. Methods for FRET detection

FRET can be detected and quantified in a number of ways. As the process

FRET can result in both, a decrease in donor-fluorescence and an increase in

fluorescence of the acceptor, a ratio metric determination of the two signals can be

done. Also in presence of acceptor, a change in fluorescence lifetime of donor occurs.

Therefore, it is possible to detect the FRET by observing the change in fluorescence

intensity of donor and acceptor or the change in donor lifetime in presence of

acceptor.

(a) Measurement of fluorescence of Donor and Acceptor

During FRET process the donor fluorescence intensity decreases in presence

of acceptor compared to the fluorescence intensity of pure donor. On the other hand

simultaneous increase in acceptor fluorescence intensity occurs [12, 13].



To investigate the FRET process, the fluorescence spectra of pure donor, pure

acceptor and their mixture were measured (figure 2.3). In all the cases the exciting

wavelength is selected to be very close to the absorption monomer of the donor, so

that only the donor molecules are excited and the acceptor molecules cannot absorb

light directly. From figure it can be observed that the fluorescence intensity of donor

is much higher, whereas the acceptor fluorescence intensity is very less. But in case of

mixture, the fluorescence intensity of donor decreases and that of acceptor increases

compared to their pure counter parts. Inset of figure 2.3 shows the excitation spectra

measured with excitation wavelength fixed at donor and acceptor fluorescence

maximum in case of D-A mixture. Interestingly both the excitation spectra are almost

similar and possess characteristic absorption bands of donor monomers. This confirms

that the acceptor fluorescence is mainly due to the light absorption by donor and

corresponding transfer to acceptor monomer. Thus FRET between donor to acceptor

has been confirmed.

Fig. 2.3. Change in fluorescence intensity due to FRET. Inset shows the Excitation

spectra for D-A mixture with emission wavelength at donor (I) and acceptor (II) nm.

(b) Change in fluorescence lifetime

During the FRET process the lifetime of donor in presence of acceptor

decreases with respect to the lifetime of pure donor [12]. Therefore it is possible to



detect the FRET process by measuring the lifetime of pure donor and that of donor in

presence of acceptor. Figure 2.4 shows the fluorescence decay curve of a typical

donor in absence and presence of acceptor. It is observed that the fluorescence

lifetime of pure donor is much higher than that of donor in presence of acceptor. The

results confirm FRET from donor to acceptor.

Fig. 2.4. Change in fluorescence lifetime due to occurring of FRET.

2.2. Influence of Clay on FRET 2.2.1. Properties of clay minerals

Nano dymentional clay particles play an important role in modern technology.

Clay minerals are characterized by certain properties, including (i) a layer structure

with one dimension in the nanometer range; the thickness of the 1:1 (TO) layer is

about 0.7 nm, and that of the 2:1 (TOT) layer is about 1 nm (shown in figure 2.5), (ii)

the anisotropy of the layers or particles, (iii) the existence of several types of surfaces:

external basal (planar) and edge surfaces (iv) as well as internal (interlayer) surfaces

(v) the ease with which the external, and often also the internal, surface can be

modified (by adsorption, ion exchange, or grafting), (vi) plasticity, and (vii) hardening

on drying or firing; this applies to most (but not all) clay minerals. Due to unique

features of clay, such as ion exchange properties, swelling and intercalation

behaviour, and their lower cost compared to synthesized materials, clay minerals have



drawn much attention in material science. For preparation of thin films, these

properties of the clay particles are most relevant.

One of the important properties of clay minerals is cation exchange capacity

(CEC) and it depends upon available surface area, crystal size, pH and the type of

exchangeable cation. Generally, it is expressed in microequivalents per gram (µeq g-1).

However, due to isomorphous substitution reaction of metal atoms in the lattice

structure of clay, an overall negative charge on individual clay layers appears. But the

cations of interlayer region compensate these charges. These interlayer cations are

exchangeable in nature and may take places with other cations under suitable

circumstances. There is variability in the charge-balancing interlayer cations, such as

Na+ and Ca2+ in naturally occurring clay minerals. Interestingly, these simple small

cations can be exchanged for more complex species such as alkyl-ammonium

molecules. Depending on the valency as well as size of the cations, adsorption of

cations in the interlayer region (intercalation) takes place. Divalent cations are bound

more firmly than monovalent cations but less than that of trivalent ones. However,

larger cations are preferentially adsorbed under the same condition for monovalent

cations. In addition, the silicate surfaces of the tetrahedral sheets of clay minerals are

relatively hydrophobic allowing the intercalation of neutral organic molecules such as

polyether. Various existing clay minerals exhibit different layer arrangement,

substitutions and composition. Generally, clay minerals are classified by various

structures, like allophone, kaolinite, halloysite, smectite, illite, chlorite, vermiculite,

attapulgite–palygorskite–sepiolite and mixed layer minerals.

Fig. 2.5. Structure of basic 2:1 clay minerals.



An important property of smectites clays is swelling. As smectite clays adsorb

water or dry, like a stack of papers that can become taller, they are able to expand and

contract their structures in one dimension. When water is adsorbed in the interlamellar

space then the interlayer space increases, forcing the clay to swell. Depending on the

layer charge and the type of interlayer cations, the ability to swell can change. Due to

the presence of exchangeable cations that like to be hydrated, water is attracted to the

interlayer region. The swelling mechanisms and the interplay between the

hydrophobic and hydrophilic character of clay minerals is of great interest. One of the

important factor which can affect smectite hydration is the location of isomorphous

substitution in the layer (i.e., whether the layer charge derives from substitution in the

tetrahedral or octahedral sheet). The basal oxygen atoms act as a weak Lewis base

(electron donor) in electrically neutral layers, forming weak hydrogen bonds with

water molecules. Due to isomorphous substitution reaction, the basal oxygen atoms

have an excess of negative charge, and consequently their electron-donating capacity

increases. The properties of cation exchange capacity, intercalation, layer structure

and swelling make the clay particles as the ideal host materials to incorporate the

organic molecules onto the clay surfaces as well as onto the interlayer space of clay

structure.

2.2.2. Influence of clay on FRET

Negatively charged clay particles have layered structure with a cation

exchange capacity [21]. However, if the dye molecules are positively charged, they

will adsorbed onto the negatively charged clay layers as shown in figure 2.6(b). On

the other hand, FRET process depends on the distance between the energy donor and

acceptor and takes place only when the distance is within 1-10 nm. Thus, clay

particles take a vital role to determine the concentration of the dyes on their surfaces

or to make possible close interaction between energy donor and acceptor in contrast to

the aqueous solution as shown in figure 2.6.

As the process FRET varies significantly in presence of clay, the fact can be

utilised for the enhancement of sensing efficiency in some particular cases. If the

variation of FRET is very less between a particular FRET pair in case of sensing of

any particular external analyte, then the variation can be made remarkable by the use

of clay as mentioned above.



Fig. 2.6. Influence of clay on FRET.

2.3. FRET as molecular spy The unique feature of FRET is its capability to inform us whenever two

molecules (donor and acceptor) are close to one another on a molecular scale (usually

within 1-10 nm), and whether they are moving relative to each other. It is also

possible to detect how the donor and acceptor transition moments are oriented relative

to each other. This is because the FRET efficiency depends on donor-acceptor

distance as well as on the relative orientations of the two dipoles. It is possible to

couple FRET pair with other physical and biological methods, and this greatly

extends the usefulness of the process. Therefore flurophore involving FRET can be

considered as analogous to roaming molecular spies with radio transmitters, radiating

information to the experimenter about the state of affairs on the molecular scale, and

informing us where the spies are located and how they are oriented.

Generally FRET (spectroscopic experiments) can be carried out in most

laboratories, whether the ‘‘samples’’ are large (such as in cuvettes, or even on whole

mammalian bodies) or small (such as in the fluorescence microscope, and on the



single molecule level). Therefore, irrespective of the scale of the sample, the

information on the molecular scale derivable from FRET remains accessible.

Accordingly, FRET can be considered as like a spectroscopic microscope, providing

us information about the distance and orientation on the molecular scale regardless of

the size of the sample. Also it is possible to follow the dynamics of changes in

molecular dimensions and proximities by monitoring FRET with time.

2.4. Sensing technology using FRET Typical FRET sensor consists of a recognition element (sensing material)

fused to a pair of fluorophores (FRET pair) capable of FRET or a system containing

the FRET pair and the recognition element. A conformational change in the

recognition element can be exploited to bring the changes in FRET efficiency when

fused to an appropriate FRET pair. Also analyte dependent changes in the spectra of

FRET pair can change the FRET efficiency [16-18]. Therefore, FRET is a unique

phenomenon in generating fluorescence signals sensitive to molecular conformation,

association and separation in the 1–10 nm range [17].

FRET is a nonradiative quantum mechanical process where energy transfer

occurs between two fluorophores in close proximity (less than 10 nm apart) through

long-range dipole−dipole interactions [19, 20]. The rate of energy transfer is highly

dependent on many factors, such as the extent of spectral overlap, the relative

orientation of the transition dipoles and most importantly, the distance between the

donor and acceptor molecules [22, 23]. FRET usually occurs over distances

comparable to the dimensions of most biological macromolecules, that is, about 10 to

100 Å. This makes FRET a spectroscopic ruler to study biological systems [24, 25].

Since FRET is extremely sensitive to changes in the relative positions of two

fluorophores or their orientation, even a subtle change in the conformation of the

recognition element, when attached to a FRET pair, can be visualized as a change in

FRET efficiency. Also presence of minute amount of recognition element may affect

the FRET efficiency.

FRET based sensing system is very appealing because of its simpleness of

building ratiometric fluorescent systems. Unlike those of single-signal sensors, the

ratiometric sensors contain two different fluorophores and use the ratio of the two

fluorescence intensities to quantitatively detect the analytes. They can eliminate most

ambiguities in the detection process by self-calibration of two emission bands. The



external factors, such as excitation source fluctuations and concentration, will not

affect the ratio between the two fluorescence intensities [25, 26].

Conventionally, the FRET-based sensing systems have been designed in the

form of small molecular dyads, which contain two fluorophores connected by a spacer

through covalent links [27] or a system containing the FRET pair and the recognition

element in a controlled environment [27].

References 1. T. H. Förster, Discuss. Faraday Soc. 27 (1959) 7.

2. T. H. Förster, Naturwissenschaften 33 (1946) 166.

3. B. V. Lotsch, G. A. Ozin, Adv. Mater. 20 (2008) 4079.

4. P. R. Selvin, Methods Enzymol. 246 (1995) 300.

5. R. M. Clegg, Methods Enzymol. 211 (1992) 353.

6. T. Förster, Ann. Physik 437 (1948) 55.

7. D. Bhattacharjee, D. Dey, S. Chakraborty, S. A. Hussain, S. Sinha, J. Bio. Phys. 39

(2013) 387.

8. K. E. Sapsford, L. Berti, I. L. Medintz, Minerva Biotecnologica 16 (2004) 247.

9. P. Wu, L. Brand, Anal. Biochem. 218 (1994) 1.

10. D. Seth, D. Chakrabarty, A. Chakraborty, N. Sarkar, Chem. Phys. Lett. 401 (2005)

546.

11. J. M. Drake, J. Klafter, P. Levitz, Science 251 (1991) 1574.

12. Y. Yilmaz, A. Erzan, Ö. Pekcan, Phys. Rev. E 58 (1998) 7487.

13. P. Tinnefeld, M. Sauer, Angew. Chem. Int. Ed. 44 (2005) 2642.

14. J. Perrin, Academie des Sciences 184 (1927) 1097.

15. T. H. Förster, Z. Naturforsch., 4A (1949) 321.

16. T. H. Förster, in Modern quantum chemistry, Istanbul lectures, Part III: Action of

light and organic crystals, Academic Press, ed. O. Sinanoglu, New York, 1965.

17. T. Förster, Modern Quantum Chemistry. Part II. Action of Light and Organic

Molecules, Academic, New York, (1965).

18. D. Dey, D. Bhattacharjee, S. Chakraborty, S. A. Hussain, J. Photochem.

Photobiol. A: Chem. 252 (2013) 174.

19. M. Pope, C. E. Swenberg, Electronic Processes in Organic Crystals, Oxford

University Press, New York, (1982).



20. D. Seth, D. Chakrabarty, A. Chakraborty, N. S. Sarkar, Chem. Phys. Lett. 401

(2005) 546.

21. N. L. Rosi, C. A. Mirkin, Chem. Rev. 105 (2005) 1547.

22. D. Ghosh, D. Bose, D. Sarkar, N. Chattopadhyay, J. Phys. Chem. A 113 (2009)

10460.

23. S. A. Hussain, S. Chakraborty, D. Bhattacharjee, R. A. Schoonheydt, Spectrochim

Acta Part A 75 (2010) 664.

24. V. K. Sharma, P. D. Sahare, R. C. Rastogi, S. K. Ghoshal, D. Mohan,

Spectrochim. Acta Part A 59 (2003) 1799.

25. V. Misra, H. Mishra, H. C. Joshi, T. C. Pant, Sens. Actuators, B 63 (2000) 18.

26. Y. H. Chan, C. Wu, F. Ye, Y. Jin, P. B. Smith, D. T. Chiu, Anal. Chem. 83 (2011)

1448.

27. C. Egami, Y. Suzuki, O. Sugihora, H. Fujimura, N. Okamoto, Jpn. J. Appl. Phys.

36 (1997) 2902


Chapter3: International and National status 17

Chapter-3

International and National status

This chapter contains the International and National status of

the recent research activities related to the topic of the

present thesis work.



Chapter-3

International and National status FRET has been widely used as a spectroscopic technique in all applications of

fluorescence, including medical diagnostics, DNA analysis, optical imaging [1] and

for designing various sensors [2-7]. FRET was first described over 50 years ago, that

is being used more and more in biomedical research and drug discovery today. This

type of nonradiative transfer of electronic excited energy was first proposed by J.

Perrin in 1920. Later Th. Förster [8, 9] in 1946 demonstrated that FRET could occur

over distances up to 100Å. Galanin advanced the possibility of “inductive resonance”

between molecules spaced at such a distance [10]. Forster and Galanin observed a

nontrivial fluorescence quenching in solutions containing millimolar levels of

acceptor molecules [8-10]. Recently, FRET phenomenon have been employed for the

conformation of proteins and knowing their structure [11], for the detection of spatial

distribution and assembly of proteins [12], for designing biosensor [7], for nucleic

acid hybridization [13], distribution and transport of lipids [14].

3.1. International status FRET based sensors have the potential to create time dependent concentration

or activity maps of ions, small ligands, or macromolecules in living cells. In order to

meet the challenge of multidimensional visualization, the dynamic range and response

kinetics of the biosensors are critical attributes, since they directly affect the sensor’s

spatial and temporal resolution. Time-resolved microfluidic flow cytometer capable of

characterizing the FRET based dynamic response of metal-ion sensors in mammalian

cells has been designed [15]. The instrument can be used to examine the cellular

heterogeneity of Zn2+ and Ca2+ sensor FRET response signals. Almost 30 fold

difference between the extracellular and intracellular sensors has been reported [15].

FRET based Cd2+ indicator containing a Cd2+ binding protein obtained from

pseudomonas putida as the Cd2+ sensing key has been reported capable of live cell

dynamic sensing of Cd2+ [16]. Liu et. al. [17] reported FRET based ratiometric sensor

for the detection of Hg2+ ions. Silica nanoparticles were labeled with a hydrophobic

fluorescent nitrobenzoxadiazolyl dye which acts as a FRET donor. Rhodamine was

then covalently linked to the surface of the silica particles which acts as acceptor.



Nanoparticles are then exposed to Hg2+ in water. FRET based system with control

over the location of both donor and acceptor and their separation distance within the

nanoparticles has been developed for ratiometric sensing of Hg2+ in water [17, 18]. A

novel calyx(4) arene derivative locked in the 1,3-alternate conformation bearing two

pyrene and rhodamine fluorophores was synthesized as a selective sensor for the Hg+2

ion [19]. The principle of sensing was based on FRET from pyrene excimer emissions

to ring opened rhodamine absorption upon complexation of the Hg2+ ion. Chao et. al.

reported FRET based ratiometric detection system for mercury ions in water with

polymeric particles as scaffolds [20]. A flexible 8-hydroxyquinoline benzoate linked

bodipy-porphyrin dyad has been designed and synthesized which can be used for

selectively sensing of Fe2+ and Hg2+ ions [21].

FRET-based ratiometric sensing platform based on β-cyclodextrin has also

been demonstrated [22]. β-cyclodextrin provides the hydrophilicity and

biocompatibility; thus, the sensing platform can be used in aqueous medium and in

some biological fluids as well as in live cells. Cyclodextrin based supramolecular

complex has also been used for ratiometric sensing of ferric ion [23]. Cr3+ ion is an

essential trace element in human nutrition and has great impacts on the metabolism of

carbohydrates, fats, proteins and nucleic acids by activating certain enzymes and

stabilizing proteins and nucleic acids [24]. Based on the FRET from naphthalimide

and rhodamine, Cr3+-selective fluorescent probe for monitoring Cr3+ in living cells

with ratiometric fluorescent methods has been developed [25]. Rhodamine based

reversible chemosensor capable of undergoing excimer-Fluorescent Resonance

Energy Transfer (Em-FRET) was designed to sense carboxylate anions using a ditopic

receptor strategy [26]. Intramolecular FRET from the naphthalene emission to the

coumarin absorption has been used to design ion sensor, which affords high

fluorescence selectivity toward F- and Cs+ ions [27]. Sensor containing

guanidiniocarbonylpyrrole and a 9-(aminomethyl) anthracene moiety has been

synthesized, which exhibits ratiometric fluorescence changes for SO3-2 over other

anions. The change in fluorescence is attributed to the FRET and the SO3-2 complex

induced photochemical reaction [28]. Yousaf et. al. [29] reported a FRET-based

biosensor to study the dynamics of RhoA GTPase activation in cells on patterned

substrates. Stable expression of FRET biosensors will accelerate current trends in

cancer research, that is, from cells on a plastic dish to 3-D and/or living tissues and

from biochemistry to live imaging. A sensitive and specific FRET biosensor was



developed by Shaoying et. al. [30] and applied to detect the activity of BCR-ABL

kinase in live cells. Schifferer et. al. [31] demonstrated a genetically encoded dynamic

RNA reporter using intramolecular FRET between mutants of GFP. This may be

useful in several types of application, for example, as reporter in vitro for real time

studies on transcription or stability of RNA, to image very dynamic aspects of gene

expression in vivo or to study relationships between RNA levels and protein

expression in single living cells. FRET based biosensor has also been used for

monitoring the σ1 receptor activation switch in living cells [32]. Mirkin et. al. have

developed AuNPs sensors, which are designed to detect and quantify intracellular

analytes, for example, mRNA in cells [33].

Hsieh et. al. [34] demonstrated that multiple cysteines is a member of the

periplasmic binding protein family can be selectively labeled with two different thiol-

reactive reagents. This technique exploits a protein conformational change upon

binding of a ligand, thus blocking one of the cysteine sites from the reaction

chemistry. Using this technique for sequential labeling of glucose/galactose binding

protein with the two dyes nitrobenzoxadiazole and texas red, two functional FRET

sensors were prepared, and a glucose-dependent FRET signal was demonstrated for

each of these. The ligand protection strategy may be valuable for many further

applications where dual-labeling of proteins is desired.

Chan et. al. demonstrated FRET-based ratiometric pH nanoprobes where they

used semiconducting polymer dots as a platform. The linear range for pH sensing of

the fluorescein-coupled polymer dots was between pH 5.0 and 8.0 [35]. Egami et al.

has introduced a fiber optic pH sensor, using polymer doped with either congo red

(pH range 3 to 5) or methyl red (pH range from 5 to 7) [36]. pH sensor based on the

measurement of absorption of phenol red has also been reported [37], which can sense

a pH range of 7–7.4. Intracellular pH is an important indicator for cellular metabolism

and pathogenesis [38, 39]. pH sensing in living cells has been achieved using a

number of synthetic organic dyes and genetically expressible sensor proteins, even

allowing the specific targeting of intracellular organelles. Esposito et. al. [40] reported

a FRET-based pH sensor platform, based on the pH modulation of YFP acceptor

fluorophores in a fusion construct with ECFP. Quantum dot-fluorescent protein FRET

probes for the sensing intracellular pH has been demonstrated [41] having high

sensitivity and wide dynamic range, ratiometric measurements for internal calibration,

dramatic reduction of photobleaching, and the ability to tailor the probe design for



different pH ranges. These probes are well suited to a wide range of intracellular pH-

dependent imaging applications that are not feasible with fluorescent proteins or

organic fluorophores alone.

3.2. National status In India there are several groups working on different aspects of FRET

phenomenon (Jadavpur University, Kolkata, IISc, Bangalore, I.A.C.S., Kolkata,

N.P.L., New Delhi, N.C.L., Pune, I.I.T., Mumbai, B.H.U., Varanasi, IIT Guwahati,

Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, Department of

Physics, Tripura University, Tripura) and demonstrating sensors for different

chemical and biological analytes using FRET [42-53]. P. P. Nampi and his group used

sol–gel nanoporous silica as substrate for immobilization of conjugated biomolecules

for application as FRET based biosensor [49]. Anamika Aneja and her group

demonstrated a triple-FRET technique for energy transfer between conjugated

polymer and TAMRA dye with possible applications in medical diagnostics [42]. Rati

Ranjan Nayak and his group demonstrated a FRET based DNA sensor using Water-

soluble conjugated polyelectrolytes [48]. A. Mallik and his group studied FRET from

tryptophan in human serum albumin to a bioactive indoloquinolizine system. The

study suggests that the donor and the acceptor are bound to the same protein at

different locations but within the quenching distance [43]. S. L. Bhattar and his group

studied FRET between Anthracene and Proflavine Hemisulphate in micellar solution

and demonstrated its application on determination of Proflavine Hemisulphate [44].

Our research group in the Department of Physics, Tripura University, is

working on designing of different chemical and biological sensors based on FRET

[50-53]. It was observed that the FRET efficiency can be increased by introducing

nano-clay platelets [53].

In the light of above reviews it is obvious that even in the international level

the effort to prepare new FRET based sensing technology is an emerging field of

research. This is due to the wide range of technological application of FRET. It is,

therefore, highly appropriate to make a great stride in these important and promising

areas of research, which can provide us a conceptual understanding with wide

opportunity of technological applications.



References

1. J. R. Lakowicz, Principles of Fluorescence Spectroscopy, 3rd edn, Springer, New

York, 2006.

2. C. Ma, F. Zeng, L. Huang, S. Wu, J. Phys. Chem. B 115 (2011) 874.

3. A. R. Clap, I. L. Medintz, H. Mattoussi, Phys. Chem. Chem. Phys. 7 (2006) 47.

4. T. Pons, I. L. Medintz, K. E. Sapsford, S. Higashiya, A. F. Grimes, D. S. English,

H. Mattoussi, Nano Lett. 7 (2007) 3157.

5. T. Pons, I. L. Medintz, X. Wang, H. Mattoussi, J. Am. Chem. Soc. 128 (2006)

15324.

6. X. L. Zhang, Y. Xiao, X. H. Qian, Angew. Chem., Int. Ed. 47 (2008) 8025.

7. H. Ueyama, M. Takagi, S. Takenaka, J. Am. Chem. Soc. 124 (2002) 14286.

8. T. H. Förster, Disc. Faraday Soc. 27 (1959) 7.

9. T. H. Förster, Naturwissenschaften, 33 (1946) 166.

10. B. V. Lotsch, G. A. Ozin, Adv. Mater. 20 (2008) 4079.

11. T. Jonsson, C. D. Waldburger, R. T. Sauer, Biochem. 35 (1996) 4795.

12. B. S. Watson, T. L. Hazlett, J. F. Eccleston, C. Davis, D. M. Jameson, A. E.

Johnson, Biochem. 34 (1995) 7904.

13. K. M. Parkhurst, L. J. Parkhurst, Biochem. 34 (1995) 285.

14. J. W. Nichols, R.E. Pagano, J. Biol. Chem. 258 (1983) 5368.

15. B. Zagrovic, C. D. Snow, S. Khaliq, M. R. Shirts, V. S. Pande, J. Mol. Biol. 323

(2002) 153.

16. R. J. H. Clark, R. E. Hester (eds.), Advances in Spectroscopy, Wiley, New York,

(1996).

17. L. Baoyu, Z. Fang, W. Shuizhu, Microchimica Acta 180 (2013) 845.

18. L. Baoyu, Z. Fang, W. Guangfei, W. Shuizhu, Chem. Commun. 47 (2011) 8913.

19. R. Correia, P. DeShong, J. Org. Chem. 75 (2010) 7159.

20. M. Chao, Z. Fang, H. Lifang W. Shuizhu, J. Phys. Chem. B 115 (2011) 874.

21. C. Y. Dyad, W. Liang, Y. Xiang, L. Wenjun, B. Yongzhong, J. Jianzhuang, Org.

Lett., 13 (2011) 5774.

22. V. G. Kozlov, V. Bulovic, P. E. Burrows, Forrest, S. R. Nature 389 (1997) 362.

23. C. R. Cantor, P. R. Schimmel, Biophys. Chem. Part-II, Freeman, San Francisco,

1980.

24. K. E. Sapsford, L. Berti, I. L. Medintz, Angew. Chem. Int. Ed. Engl. 45 (2006)

4562.



25. S. A. Hussain, S. Chakraborty, D. Bhattacharjee, R. A. Schoonheydt,

Spectrochim. Acta, Part A 75 (2010) 664.

26. M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayal, D.

Parsons, P. M. French, M. J. Lever, L. O. Sucharov, M. A. Neil, R. Juskaitis, T.

Wilson, J. Microsco. 203 (2001) 246.

27. G. Haran, J. Phys.: Condens. Matter, 15 (2003) R1291.

28. R. B. Best, S. B. Flower, J. L. T. Herrera, J. Clark, Proceedings of the National

Academy of Sciences of the United States of America 99 (2002) 12143.

29. L. Hodgson, E. W. L. Chan, K. M. Hahn, M. N. Yousaf, J. Am. Chem. Soc. 129

(2007) 9264.

30. L. Shaoying, W. Yingxiao, Clin Cancer Res 16 (2010) 3822.

31. M. Schifferer, O. Griesbeck, J. Am. Chem. Soc. 134 (2012) 15185.

32. M. Gómez-Soler, V. Fernández-Dueñas, E. Portillo-Salido, P. Pérez, D.

Zamanillo, J. Miguel Vela, J. Burgueño, F. Ciruela, J. Med. Chem. 57 (2014) 238.

33. A. E. Prigodich, D. S. Seferos, M. D. Massich, D. A. Giljohann , B. C. Lane, C.

A. Mirkin, ACS Nano 3 (2009) 2147.

34. H. V. Hsieh, D. B. Sherman, S. A. Andaluz, T. J. Amiss, J. B. Pitner, J Diabetes

Sci Technol. 6 (2012) 1286.

35. Y. H. Chan, W. Changfeng , Y. Fangmao, J. Yuhui, S. B. Polina, C. T. Daniel,

Anal. Chem. 83 (2011) 1448.

36. C. Egami, Y. Suzuki, O. Sugihara, H. Fujimura, N. Okamoto, Jpn. J. Appl. Phys

36 (1997) 2902

37. J. I. Peterson, S. R. Goldstein, R. V. Fitzgerald, Anal. Chem. 52 (1980) 864.

38. R. Freeman, J. Girsh, I. Willner, ACS Appl. Mater. Interfaces 5 (2013) 2815.

39. A. Ullah, M. I. Lopes, S. Brul, G. J. Smits, Microbiology 159 (2013) 803.

40. A. Esposito, M. Gralle, M. A. C. Dani, D. Lange, F. S. Wouters, Biochem. 47

(2008) 13115.

41. A. M. Dennis, W. J. Rhee, D. Sotto, S. N. Dublin, G. Bao, ACS Nano 6 (2012)

2917.

42. A. Aneja, N. Mathur, P. K. Bhatnagar, P. C. Mathur, J Biol Phys 34 (2008) 487.

43. P. Das, A. Mallick, B. Haldar, A. Chakraborty, N. Chattopadhyay, J. Chem. Sci.

119 (2007) 77.

44. S. L. Bhattar, G. B. Kolekar, S. R. Patil, J. Dispersion Sci. Technol. 32 (2011) 23.



45. S. Ghosh, S. Dey, A. Adhikari, U. Mandal, K. Bhattacharyya, J. Phys. Chem. B

111 (2007) 7085.

46. C. Banerjee, N. Kundu, S. Ghosh, S. Mandal, J. Kuchlyan, N. Sarkar, Journal of

Physical Chemistry B (communicated)

47. S. Chatterjee, S. Nandi, S. Chandra Bhattacharya, J. Photochem. Photobiol., A:

Chemistry 173 (2005) 221.

48. R. R. Nayak, N. Pradhan, L. B. Sukla, B. K. Mishra, Int. J. Plast. Technol. 14

(2010) S1.

49. P. P. Nampi, C. C. Kartha, G. Jose, P. R. Anil Kumar, T. Anilkumara, H. Varma,

Sens. Actuators, B 185 (2013) 252.

50. D. Bhattacharjee, D. Dey, S. Chakraborty, S. A. Hussain, S. Sinha, J. Biol. Phys. 3

(2013) 387.

51. D. Dey, D. Bhattacharjee, S. Chakraborty, S. A. Hussain, J. Photochem.

Photobiol. A: Chemistry 252 (2013) 174.

52. D. Dey, D. Bhattacharjee, S. Chakraborty, S. A. Hussain, Sens. Actuators, B:

Chem. 184 (2013) 268.

53. D. Dey, J. Saha, A. Datta Roy, D. Bhattacharjee, S. A. Hussain, Sens. Actuators,

B: Chem. 195 (2014) 382.


Chapter 4: Experimental techniques 25

Chapter-4


This chapter deals with the details about the materials and

different experimental techniques used for sample preparation

as well as characterizations throughout the present thesis

work.



Chapter-4


4.1. Introduction to the experimental techniques It is very important to study FRET in solution phase as well as in thin films for

many device applications. During the present thesis work Layer-by-Layer (LbL) self

assembled technique and Langmuir-Blodgett (LB) technique have been used for thin

film preparation. Due to its cost effectiveness and simplicity in fabrication, LbL self

assembled technique is a unique tool used for fabrication of mono and multilayered

ultra-thin films. The LbL multilayer is simply formed by the alternate deposition of

oppositely charged poly-electrolytes such that the charge oscillates between positive

and negative with each layer deposition. On the other hand, the Langmuir-Blodgett

(LB) technique offers the possibility to obtain highly ordered well organized mono

and multilayer films which allow the study of physical phenomenon on a molecular

level [1-3]. Of late, LB technique has been used enormously for the preparation of

hybrid organo-clay mono and multilayer films in order to explore various properties

for various device fabrications [4, 5]. To characterize such thin films, spectroscopic

techniques viz. UV-Vis absorption and steady state fluorescence have been employed.

To investigate the morphology of these films, Atomic Force Microscopy (AFM) has

been used.

The details of the experimental techniques employed for the preparation of

mono and multi layered films and their characterizations have been discussed in this

chapter. This chapter also contains various terminology related to Langmuir-Blodgett

film deposition and Layer-by-Layer technique. Preliminary information about the

different instruments used for the formation of thin films and their spectroscopic as

well as morphological characterizations have also been discussed here.

4.2. Materials used 4.2.1. Dyes used as FRET pair

As a FRET pair the organic dye molecules used during this thesis work are

Acriflavine (Acf, 99%, Molecular Probe) and Rhodamine B (RhB, 99%, Molecular

Probe) as energy donor and energy acceptor respectively. Both are water soluble



cationic laser dyes and show very intense fluorescence. The structures of the

molecules are shown below.

Acriflavine (Acf)

Rhodamine B (RhB)

Fig. 4.1. Structure of the organic compounds used as FRET pair in this thesis.

4.2.2. Clay mineral

Smectite clay laponite is used as clay mineral in this study. The laponite clay

mineral was obtained from Laponite Inorganics, UK. The sizes of the laponite were

less than 0.05 µm and its cation exchange capacity (CEC) was 0.74 meq/g. The clay

minerals were stored as freeze-dried powders. These smectites clay particles are 2:1

or T-O-T type of clay with three sheets, silica – alumina – silica in a sandwich like

form. These layered minerals are consists of stacks of negatively charged two-

dimensional aluminosilicate layers. Highly dispersed clay suspension consisting of

single clay layers can be made by stirring the aqueous clay dispersion for more than

24 hour with a magnetic stirrer and followed by ultrasonication for about half an hour

before use.

4.2.3. Double stranded DNA

The DNA used is sheared Salmon sperm DNA having a size of nearly about

2,000 bp with approximate GC content 41.2%, purchased from SRL India and was

used as received. The purity of DNA was checked by UV-Vis absorption and

fluorescence spectroscopy before use.

4.2.4. Matrix materials for film preparation

For thin film preparation we use the supporting materials which are inert with

respect to the investigating property. Water soluble PAH (Poly alyle amine

hydrochloride) and PAA (Polyacrylic acid) have been used for LbL film preparation

which are cationic and anionic in nature respectively. However, in case of LB film

preparation, stearic acid (SA) (Sigma Aldrich, USA, purity>99%) has been used as

amphiphilic matrix material.



4.2.5. Other materials

NaOH and HCl (Thermo Fisher Scientific India Pvt. Ltd.) were used to change

the pH of the solvent. For the sensing of different ions, some salts like NaCl, KCl,

MgCl2, CaCl2, AlCl3, FeCl3 and FeSO4 (Thermo Fisher Scientific India Pvt. Ltd.)

were used.

4.3. Solution preparation Ultra pure Milli-Q water, HPLC grade chloroform (99.9% Aldrich, stabilized

by 0.5-1% ethanol) or HPLC grade methanol [Acros Organics, USA] are used to

prepare the solutions of the materials. To maintain fixed concentrations of the

solutions, these are kept in air-tight condition.

The clay dispersion used for the preparation of organo-clay hybrid film was

prepared in ultrapure Milli-Q water (2 mg/L) and stirred for 24 h by a magnetic stirrer

followed by ultrasonication for 30 minute before use.

4.4. Film preparation 4.4.1. Langmuir-Blodgett technique

In order to generate high degree of organization of molecules onto solid

substrate, the Langmuir-Blodgett (LB) technique is used. The precise control and

uniformity of film thickness in the submicron level employed by LB technique makes

it a unique tool for several technological applications [1-3]. Nowadays, LB technique

has also been used extensively for the preparation of organo-clay hybrid films. A

highly well organized mono molecular Langmuir film at air-liquid interface can be

transferred onto a solid substrate to form LB films. LB technique provides a new

scope for the formation of ultra thin film with controlled structure [6].

The LB technique mainly consists of two parts:

(i) Formation of stable Langmuir (L) monolayer of suitable materials at the air-water

interface and

(ii) Transfer of this floating Langmuir monolayer onto solid substrate to form mono

and multilayer LB films.

LB Film deposition instrument (Model Apex 2000C, figure 4.2) was used for

the formation of stable Langmuir monolayer and LB films onto solid substrate. It is a

highly sophisticated, solely computer controlled instrument. The essential part of this

instrument is a teflon coated trough with a dipper well for dipping the substrate filled

up with distilled and deionised water (as shown in figure 4.3). The working area of



the trough may be varied by a teflon-coated moveable barrier controlled by a

computer and can be moved very slowly just on the water surface. The surface

pressure measurements have been carried out and are monitored by a Wilhelmy plate

arrangement attached to an electronic microbalance.

The stable Langmuir monolayer at air-water interface were deposited or

transferred onto solid substrate by moving the substrate vertically through the floating

Langmuir monolayer by means of stepper motor whose speed can be controlled

precisely by computer.

Hydrophobic as well as hydrophilic substrate can be used for the preparation

of the LB film. Deposition on a hydrophobic surface always occurs while the

substrate is dipping downward and for hydrophilic substrate, it occurs when it is

moving in upward direction. If a layer is transferred on every upward and downward

pass through the interface, then the transfer is known as Y-type. It can also be the case

that a layer is transferred only on the downward passes (X-type) or only on the

upward passes (Z-type).

Fig. 4.2. Langmuir-Blodgett (LB) film deposition instrument.

The successful transfer of monolayer from the air-water interface onto a solid

substrate highly depends on the various factors like (i) nature of the material forming

the monolayer, (ii) surface pressure of lifting, (iii) barrier compression rate, (iv)

temperature, (v) pH of the subphase, (vi) preparation of the substrate, (vii) dipping



speed etc. Depending on the transfer ratio (the decrease in area of the monolayer

divided by the area of the substrate covered by the film) the character of the film can

change. Transfer ratios below unity indicate partial transfer where as those much

greater than unity suggest significant monolayer collapse, structural relaxation, or

dissolution during the process of transfer. Generally, a transfer ratio within the range

of 0.95 to 1.05 is an indication of good deposition. For consistent film quality, the

monolayer must be kept at a constant surface pressure during film transfer. Y-type

deposition technique (figure 4.3) has been used for LB film deposition in the present

thesis work.

Fig. 4.3. Langmuir-Blodgett (LB) deposition technique.

Langmuir-Blodgett film deposition instrument (APEX 2000C) has the

following technical specification:

Technical specification

Overall dimension (LBH): 80 cm80 cm100 cm.

Trough Material: Teflon.

Working area: 30 cm 15 cm = 450 cm2.

Depth of dipping area: 10 cm.

Barrier material: Teflon.

Barrier speed: 0 to 30 cm/min.

Range of surface pressure: 0 to 100 mN/m.

Sensitivity of the surface pressure: Better than 0.05 mN/m.



Accuracy for surface pressure: 0.1 mN/m.

Dipper head minimum speed: 0.03 cm/min.

Dipper head maximum speed: 12 cm/min.

Software: Turbo BASIC and Assembler.

4.4.2. Layer-by-Layer (LbL) self assembled technique

Chemical self-assembly [7-10] and LbL self assembly are the two most

commonly used techniques among the different ways to build organic or

organic/inorganic hybrid multi-layers. But due to versatility, simplicity and

environmental friendliness LbL self-assembly (also known as electrostatic self-

assembly (ESA)), is the most promising technique. [11-22]. However, by contrast

with the other older methods, LbL method provides essentially amorphous films, with

profuse interpenetration of neighboring “layers”. This may certainly be seen as one of

the advantages of the technique, since it avoids defect propagation from layer to layer,

in contrast with other multilayer construction techniques [23-30]. In addition, it is a

simpler technique involving simple equipment for film preparation and can be applied

to larger substrate dimensions. The films prepared in LbL technique are formed by the

alternating deposition of oppositely charged polyelectrolyte and samples on to solid

substrate. The multilayers are particularly simple systems where nanoscale control of

the structure in one direction is easily attainable. In the field of bio-molecular device

fabrication, the use of the LbL method for immobilization of biological components

such as proteins, enzymes, DNA, cell membranes and viruses [31-40] is also very

important.

Fig. 4.4. Single dip coating unit: Model no. : SDC 2007c.



The instrument “Single dip coating unit: Model no. : SDC 2007c” used for

LbL film fabrication is shown in figure 4.4 and its specifications are given below.

Technical specification

1. Deposition speed can be programmed from 1 to 100 mm/min.

2. Deposition arm movement: 150 mm.

3. Unlimited deposition cycles, unlimited delay times.

4. Automatic computer controlled unit

Fig. 4.5. Layer-by-Layer deposition process and formation of multilayer.

Layer-by-Layer (LbL) technique is mainly based on electrostatic attraction

between positively and negatively charged species (shown in figure 4.5). A stepwise

methodology of LbL film fabrication is shown in figure 4.5. It comprises of the

following steps:

1) First of all, electrolyte deposition baths are prepared in aqueous solution using

triple distilled de-ionized millipore water.

2) The fluorescence grade quartz slide is dipped into a beaker containing polycation

or polyanion solution depending upon the requirement to make the substrate surface

either positively charged or negatively charged.

3) The slide is dried completely and then rinsed into a water bath which causes a wash

away of excessive ions from the surface of the slide.

4) Then the film is again dried and is dipped into a beaker containing the sample

solution.



5) The film is again dried and washed. Multilayer of films may be obtained by

repetition of the above mentioned procedure.

6) For a positively charged sample polyanion and for a negatively charged sample

polycation is used in step 2 of the film preparation process.

During consecutive adsorption steps, the substrate is deposited with the

oppositely charged polymer solutions and then between these two steps, the growing

films are normally rinsed with pure solvent, to remove adhering polymer solution.

This washing step removes the excess charge from the surface of the film. At the

equilibrium, it is clear that electro-neutralization is the dominant mechanism for the

fabrication of LbL film, but an analysis of the overall driving force must include

consideration of all charged species. The situation cannot be described simply by only

electrostatic between isolated charges, which would yield enormous free energy

changes. The driving force is ion pairing between polyelectrolyte segments, driven by

release of counter ions and water. Additional salt ions modify the overall interaction

by competing for polymer charge. The control of specific properties at the molecular

level has been achieved for a number of supra-molecular systems through self-

organization of thin film by LbL technique which is evident from some of the results

of our studies.

4.5. Molecular Spectroscopy Molecular spectroscopy is used extensively for the qualitative as well as

quantitative analysis of inorganic and organic compounds. Information about the

identity, structure and environment of chemical species can be obtained based on the

interaction of light with matter.

In this present work, UV-Vis absorption and steady state fluorescence

spectroscopy have been employed for spectroscopic characterisation of materials in

solutions or in ultrathin films.

4.5.1. Ultraviolet-Visible (UV-Vis) absorption spectroscopy

One of the oldest and still most useful as well as important instrumental

methods applied in the study of spectroscopic characteristic is investigation of

absorption property of light by different materials. UV-Vis absorption spectroscopy

can be utilised to sense the factors that influence the electronic distribution. These

factors include changes in molecular environment such as solvation, adsorption on

surfaces and dimer formation of the absorbing species.



Fig. 4.6. Schematic representation of transmission spectroscopy. Light of intensity I0

enters the sample. Photons are absorbed and light of intensity I is transmitted and

travels to the detector.

When light – either visible or ultraviolet – is absorbed by valence (outer)

electrons these electrons are promoted from their normal (ground) states to higher

energy (excited) states. The energies of the orbitals involved in electronic transitions

have fixed values. As light absorption can occur over a wide range, light from 190 nm

to 900 nm is usually used in this UV-Vis absorption spectroscopy.

The absorbance AT of a medium in transmission mode is defined as

AT = log10(Io/I)

The optical setup used in UV-Vis absorption spectroscopy was transmission of

light through the sample (figure 4.6).

Where I0 is the incident intensity and I is the intensity after passage through the

absorbing medium. I and I0 are measured during transmission of the sample and the

reference respectively. For samples like dye-clay suspensions, the reference is pure

water.



Fig. 4.7. Schematic representation showing that light of initial intensity, Io, passing

through an absorbing medium in a cuvette with light path, l, will emerge with a final

intensity, It.

It has been found that absorbance is directly proportional to the path length through

the absorbing medium and the concentration c of the absorbing compound. This is

called the law of Beer-Lambert (figure 4.7) and is given by

AT = εcl

Where ε [dm3 mol-1cm-1] is proportionality constant called the molar extinction

coefficient. When the concentration of the absorbing compound and the path length

l are known, it is possible to determine the molar extinction coefficient ε. Or when the

path length and the molar extinction coefficient ε are known, the concentration can be

determined. Solutions and suspensions were measured in 10 mm quartz cuvettes with

path length 1 cm. Films were deposited on glass slides for measuring their UV-Vis

absorption spectra.

In our work the UV-Vis absorption spectra of all the samples in solution,

ultrathin films and in microcrystal were studied by using a UV-Vis Absorption

Spectrophotometer from Perkin Elmer (Model Lambda-25) at room temperature. A

schematic diagram of the UV-Vis Absorption Spectrophotometer is shown in figure

4.8.



Fig. 4.8. Schematic diagram of the working of theUV-Vis absorption spectrophotometer.

Fig. 4.9. UV-Vis absorption spectrophotometer (Model: lambda-25, Perkin Elmer).

The specification of the UV-Vis Absorption Spectrophotometer (Model: lambda-

25, Perkin Elmer):

Type: Scanning double-beam spectrophotometer for the UV-Vis range; operation by

PC

Dimensions:

Width: 650 mm Height: 260 mm Depth: 560 mm



Mass: 26 kg. Approx

Power requirements: 100V to 240 V AC, 50/60 Hz; 250 VA.

Ambient operating temperature: 150C to 350C

Humidity range: 20% to 80% relative humidity without condensation

Technical standard: In complience with the legal requirements of the EMC directive

89.336/EEC (EN 61326)

Radio interference suppression: In complience with the legal requirements of EMC

directive 89.336/EEC (EN 61326)

Beam center height: 15 mm above cell holder bottom

Beam cross section: 1 mm slit ca. 0.6 mm 9 mm (width height) at focal point of

sample and reference beam in sample compartment.

Optical pathlength in sample compartment: 121 mm

Grating (Monochromator): Holographic concave grating with 1053 lines/mm in the

entre.

Radiation Sources: Pre-aligned deuterium and halogen lamps.

Detector: Photodiodes

(One for the sample beam and one for the reference beam)

Data Output:

Digital port: One RS 232 C interface (serial), for connecting a PC

4.5.2. Fluorescence spectroscopy

Emission of light by a substance that has absorbed light or other

electromagnetic radiation is often called as ‘fluorescence’. In this process, emission of

light caused by the radiative relaxation of an excited molecule from the singlet excited

state to the ground state. For studying the structure and dynamics of complex systems,

fluorescence is the most sensitive molecular property that is used. The fluorescence

photons have the information about energy (wavelength), time, polarization and

intensity (number of photons) at a given wavelength which are important parameters

for the characterization of unknown materials.

Figure 4.10 shows a Jablonski diagram illustrating the electronic energy states

of a molecule and the transitions between them. Energy levels with the same spin as

the ground state are called singlet states and are indicated by the letter S [41] and

different spin to the ground state are called triplet states and are indicated by the letter



T. Non-radiative and radiative transitions are indicated in the diagram by different

colour and also by dash and straight arrows respectively.

Fig. 4.10. Jablonski diagram showing different pathways of a molecule during

absorption and emission of light.

When an electron in a molecule has been promoted to the excited state through

the absorption of EM radiation, it returns to the ground state through radiative and

non-radiative pathways. The radiative pathways involve photon emission and non-

radiative pathways include energy transfer through collisions, resonance energy

transfer through near field dipole-dipole interactions, photochemical decomposition

etc.

A change in the vibrational and rotational states of the molecule can also cause

a loss of energy via a non-radiative route. In case of fluorescence the excited electron

stays in excited singlet states (S1) for 10-9-10-7 seconds and returns to the ground state

S0 very quickly. On the other hand, in phosphorescence, the excited electron in state

S1 changes its spin and therefore its energy and relaxes into a triplet state T1 known as

intersystem crossing. De-excitation from this triplet state to the ground state results in

the emission of a photon.



Fig. 4.11. Figure showing the difference in wavelength between the absorption and

emission bands of a molecule (Stokes shift).

It should be noted that the absorption and emission of energy are unique

characteristics of a particular molecular structure. The difference in energy between

the absorbed photon and the emitted photon is known as the Stokes shift, as shown in

figure 4.11. A large Stokes shift is often highly desirable as it reduces the need for

optical filters, which are used to separate exciting light and fluorescence emission.

The Fluorescence Spectrophotometer (Model No. LS 55) used in our work

was purchased from Perkin Elmer, USA. 1cm quartz cell has been used for measuring

fluorescence emission of different sample solutions. Thin films deposited onto

fluorescence grade quartz substrates were mounted in the sample holder at an angle

45o to the excitation beam of the instrument. Excitation monochromator wavelength

was set at a particular desired value for getting the fluorescence spectra. The working

principle and operation of the instrument is shown in figure 4.12.



Fig. 4.12. The basic internal structure of a fluorescence spectrophotometer.

Fig. 4.13. Fluorescence Spectrophotometer (Model LS-55, Perkin Elmer).

Technical Specification of Fluorescence Spectrophotometer (Model LS-55,

Perkin Elmer):

Principle: Computer controlled rationing luminescence spectrophotometer with the

capability of measuring fluorescence, phosphorescence, chemiluminescence and

bioluminescence.



Source: Xenon discharge lamp, equivalent to 20 kW of 8 ms duration. Pulse width at

half height <10 ms.

Sample detector: Gated photomultiplier with modified S5 response for operation up to

around 650 nm. Red-sensitive R928 photomultiplier can be optionally filtered for

operation up to 900 nm.

Reference detector: Photodiode for operation up to around 900 nm.

Monochromators: Monk-Gillieson type monochromators cover the following ranges:

Excitation 200-800 nm with zero order selectable.

Emission 200-650 nm with standard photomultiplier with zero order selectable, 200-

900 nm with optional R928 photomultiplier.

Synchronous scanning is available with constant wavelength or constant energy

difference.

Wavelength accuracy: ± 1.0 nm

Wavelength reproducibility: ± 0.5 nm

Spectral bandpass: The excitation slits (2.5-15.0 nm) and emission slits (2.5-20.0 nm)

can be varied and selected in 0.1 nm increments.

Phosphorescence mode: Delay and gate times can be varied to measure

phosphorescence. Minimum total period 13.0 ms (50Hz).

Scanning Speed: Scanning speed can be selected in increments of 1 nm for 10-

1500nm/min. Time dependant data can also be collected.

Emission filters: Computer selectable cut-off (high pass) filters at 290, 350, 390, 430

and 515 nm, a blank (to act as shutter), a 1% T attenuator and clear beam position.

Sensitivity: Signal to noise is 500:1 r.m.s., using the Raman band of water with

excitation at 350 nm, excitation and emission bandpass 10 nm.

4.5.3. Atomic Force Microscopy (AFM)

Atomic force microscopy (AFM) is a very high-resolution type of scanning

probe microscopy, with demonstrated resolution of the order of fractions of a

nanometer, more than 1000 times better than the optical diffraction limit. For

imaging, measuring and manipulating matter at nanoscale, AFM is widely used [42].

The process of gathering information is done by sensing the surface with a

mechanical probe. For precise scanning of the instrument, the piezoelectric elements

are used. Schematic diagram of AFM representing the working principle is shown in

figure 4.14.



Fig. 4.14. Schematic diagram of working principle of AFM.

The AFM consists of a cantilever with a sharp tip (probe) at its end that is used

to scan the specimen surface. The cantilever is typically made of silicon nitride with a

tip radius of curvature of the order of nanometers. The force between tip and sample

deflect the cantilever according to Hooke’s law. In general the force acting between

the cantilever and the sample is the sum of Vander Waals, electrostatic, magnetic,

electrodynamics and capillary forces. There are many different operating modes for

an AFM. One is the "contact mode", where the tip is simply moved across the surface

and the cantilever deflections are measured. Another mode is called "tapping mode",

because the tip is tapped against the surface as it travels along. By controlling how

hard the tip is tapped, the AFM can move away from the surface when the needle

feels a ridge, so that it will not hit against the surface when it moves across.

During the present thesis work, AFM images of the films were taken in air

with a commercial AFM system Autoprobe M5 (Veeco Instr.) using silicon

cantilevers with a sharp, high apex ratio tip (UltraLeversTM, Veeco Instr.). All the

AFM images presented here were obtained in intermittent-contact (“tapping”) mode.

The monolayers on Si wafer substrates were used for the AFM measurements.

Scanning was performed at a typical line speed of 0.5 Hz, with a 512 pixel resolution.



Images are leveled by horizontal background subtraction, and the colour scales are

adjusted for optimal representation.

Technical Specification of AFM Instrument (Autoprobe M5 (Veeco Instr.))

Sample Size 45mm x 45mm x 18mm

Motorized Z stage Z travel: 18mm, with pitch and tilt capability

Closed-Loop, Large Area Scanner XY > 90µm, Z > 7.5µm

Open-Loop, Small Area Scanner XY > 5µm, Z > 1.5µm

Noise Floor, Z <50 pm RMS, typical imaging bandwidth

Closed-loop XY noise <1.2 nm RMS, typical imaging bandwidth

Z-Linearizer noise <200 pm RMS, typical imaging bandwidth

Open-loop XY drift <1nm/ min

Closed-loop XY drift <3nm/ min

Warm-up Time Open-loop 15min

System Software NanoDrive v8 real-time control & NanoScope Analysis included

Weights and Measures Microscope:

14” x 14” x 10” or 35.5cm x 35.5cm x 25.5cm (HxWxD); 26.5Lb or 12Kg

Add 4” or 10cm in rear for cables

Controller:

23” x 13.5” x 21” or 58.5cm x 34.5cm x 53.5cm (HxWxD); 73Lb or 33Kg

Add 4” or 10cm in rear for cable



References 1. A. Ulman, An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett

Films of Self assemblies, Academic Press, New York, (1991).

2. G. L. Gaines, Insoluble Monolayer at Liquid-Gas Interface Wiley, New York,

(1966).

3. M. C. Petty, Langmuir-Blodgett Films: An Introduction Cambridge University

Press, Cambridge, UK, (1996).

4. R. H. A. Ras, C. T. Johnston, E. I. Franses, R. Ramaekers, G. Maes, P. Foubert, F.

C. D. Schryver, R. A. Schoonheydt, Langmuir 19 (2003) 4295.

5. Y. Umemura, A. Yamagishi, R. Schoonheydt, A. Persoons, F. C. De Schryver, J.

Am. Chem. Soc. 124 (2002) 992.

6. O. N. Oliveira Jr., Brazjljan Journal of Physks 22 (1992) 60.

7. S. S. Shiratori, M. F. Rubner, Macromolecules 33 (2000) 4213.

8. D. Yoo, S. Shiratori, M. F. Rubner, Macromolecules 31 (1998) 4309.

9. B. Schoeler, G. Kumaraswamy, F. Caruso, Macromolecules 35 (2002) 889.

10. R. Steitz, W. Jaeger, R. Klitzing, Langmuir 17 (2001) 4471.

11. P. E. Laibinis, R. G. Nuzzo, J. Am. Chem. Soc. 113 (1991) 7152.

12. N. Tillman, A. Ulman, T. L. Penner, Langmuir 5 (1989) 101.

13. G. Decher, Fuzzy Nano assemblies: science 277 (1997) 1232.

14. H. Finklea, J. Am. Chem. Soc. 114 (1992) 3173.

15. J. H. Fendler, Chem. Mater. 8 (1996) 1616.

16. I. Ichinose, H. Tagawa, S. Mizuki, Y. Lvov, T. Kunitake, Langmuir 14 (1998)

187.

17. J. Gunn, G. Sagiv, J. Colloid Interface Sci. 112 (1986) 457.

18. L. Y. Wang, Z. Q. Wang, X. Zhang, J. C. Shen, Macromol. Rapid Commun. 18

(1997) 509.

19. W. B. Stockton, M. Rubner, Macromolecules 30 (1997) 2717.

20. J. Rebek, Chem. Rev. 97 (1997) 1647.

21. F. Xu, M. Gao, L. Wang, T. Zhou, L. Jin, J. Jin. Talanta 58 (2002) 427.

22. G. Decher, Science 29 (1997) 277.

23. G. Decher, J. D. Hong, J. Schmitt, Thin solid films 831 (1992) 210.

24. M. Schütte, D. G. Kurth, M. R. Linford, H. Cölfen, H. Möhwald, Angew. Chem.

Int. Ed. Engl 37 (1998) 2891.



25. Y. Shimazaki, M. Mitsuishi, S. Ito, M. Yamamoto, Langmuir 13 (1997) 1385.

26. G. Decher, Macromol. Chem. Macormol. Symp. 46 (1991) 321.

27. H. Antoniadis, B. R. Hsieh, M. A. Abkowitz, S. A. Jenekhe, M. Stolka, Synth.

Met. 62 (1994) 265.

28. S. A. Jenekhe, S. Yi, Appl. Phys. Lett. 77 (2000) 2635.

29. S. A. Sapp, G. A. Sotzing, J. R. Reynolds, Chem. Mater. 10 (1998) 2101.

30. A. Kumar, D. M. Welsh, M. C. Morvant, F. Piroux, K. A. Abboud, J. R.

Reynolds, Chem. Mater. 10 (1998) 896.

31. C. Adachi, T. Tsutsui, S. Saito, Appl. Phys. Lett. 55 (1989) 1489.

32. C. Adachi, S. Tokito, T. Tsutsui, S. Saito, Jpn. J. Appl. Phys. 27 (1988) L269.

33. A. J. Khopade, F. Caruso, Biomacromolecules 3 (2002) 1154.

34. C. X. Cai, H. X. Ju, H. Y. Chen, J. Electroanal. Chem. 397 (1995) 185.

35. Z. Xun, C. Cai, W. Xing, T. Lu, J. Electroanal. Chem. 545 (2003) 19.

36. S. A. Hussain, P. K. Paul, D. Dey, D. Bhattacharjee, S. Sinha, Chem. Phys. Lett.

450 (2007) 49.

37. D. Fretelde, Molecular biology, 2nd edition chap. 4 (1993).

38. H. Su, K. M. R. Kallury, M. Thompson, A. Roach, Anal. Chem. 66 (1994) 769.

39. X. H. Xu, H. C. Yang, T. E. Mallouk, A. J. Bard, J. Am. Chem. Soc. 116 (1994)

8386.

40. J. J. Storhatf, C. A. Mirkin, Chem. Rev. 99 (1999) 1849.

41. G. Shenk, Absorption of Light and Ultraviolet Radiation: fluorescence and

phosporescence emission. Allyn and Bacon chemistry series. Allyn and Bacon, Inc,

Boston, 1973.

42. G. Binnig, C. F. Quate, Ch. Gerber, Phys. Rev. Lett. 56 (1986) 930.


Chapter 5: FRET between Acf and RhB 46

Chapter-5

Comparative study of FRET between two laser dyes Acriflavine and Rhodamine B in solution phase and in hybrid ultrathin films prepared by LbL and LB techniques. FRET between two dyes Acriflavine (Acf) and Rhodamine B (RhB) were investigated in solution and ultrathin films in presence and absence of clay mineral laponite. UV–Vis absorption and fluorescence spectroscopy studies suggest that both the dyes are present mainly as monomer in solution and films. Energy transfer occurred from Acf to RhB in solution and films. The energy transfer efficiency increases in presence of clay laponite and the maximum efficiency were 92.50% and 55.71% in clay dispersion and in films respectively. Presence of laponite particles onto film was confirmed by atomic force microscopy investigations with a surface coverage of more than 75%. Based on the experimental results of this chapter one paper have been published in a peer reviewed reputed journal and the other one is under preparation.

1. Effect of nanoclay laponite and pH on the energy transfer between fluorescent dyes. Dibyendu Dey, D. Bhattacharjee, S. Chakraborty, S.A. Hussain Journal of Photochemistry and Photobiology A-Chem 252 (2013) 174–182

2. A comparative study of FRET between Acriflavine and Rhodamine B on to thin film. Dibyendu Dey, D. Bhattacharjee, Syed Arshad Hussain [Paper based on this work is under preparation.]



Chapter-5

Comparative study of FRET between two laser dyes Acriflavine and Rhodamine B in solution phase and in hybrid ultrathin films prepared by LbL and LB techniques.

5.1. Introduction FRET is an electrodynamic phenomenon that can occur through the transfer of

excited state energy from donor to acceptor. The theoretical analysis was well

developed by Förster [1, 2]. The rate of energy transfer depends upon the extent of

spectral overlapping area of the fluorescence spectrum of donor with the absorption

spectrum of the acceptor, the relative orientation of the donor and acceptor transition

dipole moments and the distance between these molecules [1–3]. Due to its sensitivity

to distance, FRET has been used to investigate molecular level interactions [3–10].

Fluorescence emission rate of energy transfer has wide applications in biomedical,

protein folding, RNA/DNA identification and their energy transfer process [4–10].

FRET mechanisms are also important to other phenomena, such as photosynthesis

kinetics, chemical reactions and Brownian dynamics [11, 12]. Recently, FRET

phenomenon have been employed for the conformation of proteins and knowing their

structure [13], for the detection of spatial distribution and assembly of proteins [14],

for the designing of biosensor [15], for nucleic acid hybridization [16], distribution

and transport of lipids [17]. On the other hand clay platelets are natural nanoparticles

with layered structure. Due to the cation exchangability of clay, the cationic dye

molecules are adsorbed onto the clay surfaces [18, 19]. Dye adsorption enhances the

concentration of the dye molecules, which may promote their intermolecular physical

and chemical interactions. For example, if two molecules are in close proximity,

FRET may occur. Probably the first record on efficient energy transfer in clay mineral

is with cyanine and rhodamine dyes, which are simultaneously adsorbed on clay

mineral surfaces [19, 20]. Further examples of energy transfer in clay mineral systems

are triplet–triplet energy transfer from bound sensitizers to mircene [21] and to

aromatic hydrocarbons adsorbed in hydrophobic organo-clay [22]. It was observed



that the clay/porphyrin complexes are promising and prospective candidates to be

used for construction of efficient artificial light-harvesting system [23]. Czímerová et.

al. [24] reported prominent energy transfer among laser dyes in saponite dispersion.

The FRET between cationic polypeptide polylysine and cyanine dyes was

reported in LbL films of clay minerals [25]. Bujdák et al. studied the FRET between

two rhodamines Rh123 (donor) and Rh610 (acceptor) in both solution and in presence

of nanoclay saponite (SAP) [26]. It was observed that the FRET efficiency was higher

in presence of SAP. The clay mineral works as templates for concentrating the dyes,

accordingly the intermolecular separation between them decreases. To avoid the

aggregation and the fluorescence self quenching of the dyes, a hydrophobic surfactant

was introduced which suppressed the aggregation of the dyes [26]. In another work by

the same group, FRET phenomenon between laser dyes rhodamine 123 (R123),

rhodamine 610 (R610), and oxazine 4 (Ox4) has been reported. It was observed that

the FRET efficiency increases in presence of laponite [27].

In this chapter of the present thesis we represent our results on the

investigations of FRET between two laser dyes Acf and RhB in solution and ultrathin

films in presence and absence of nanoclay particle laponite and different spacer

molecules.

5.2. Experimental 5.2.1. Solution preparation

Molecular structures of the dyes Acf and RhB are shown in the inset of figure

5.1. The dye solution was prepared by using Millipore water. The concentration of

both the dyes was 10-6 M. The clay mineral used in the present work was Laponite.

The clay dispersion was prepared in Millipore water and stirred for 24 h with a

magnetic stirrer followed by 30 minute ultrasonication before use. In dispersion the

clay concentration was 2 ppm.

5.2.2. Film preparation

Electrolyte deposition bath of cationic dye RhB and Acf were prepared with

10-4M aqueous solution separately using triple distilled deionized (resistivity 18.2

MΩ-cm) Millipore water. The anionic electrolytic bath of poly acrylic acid (PAA)

and cationic electrolytic bath of poly allylamine hydrochloride (PAH) were prepared

also with triple distilled deionized Millipore water (0.25 mg/ml). The solution of SA



was prepared in chloroform. The concentration of SA solution was 0.5 mg/ml. The

spectroscopic properties of the dye molecules are largely affected by the way of their

organisation onto ultrathin films. For example the energy transfer between dyes

mainly depends on their association as well as close proximity. Accordingly we have

assembled the dyes Acf and RhB onto ultrathin films in different ways as follows.

(1) Firstly we prepare Acf-RhB mixed films onto solid support by LbL technique both

in presence and absence of clay. A clean fluorescence grade quartz substrate is dipped

alternately in solutions of anionic PAA and oppositely charged Acf+RhB dye

mixtures (1:1 volume ratio). LbL method utilizes the Vander Waals interactions

between the quartz slide and PAA as well as electrostatic interactions between PAA

and cationic dyes. The quartz slide was dipped in the aqueous solution of PAA for 30

mins. Then it was taken out and sufficient time was allowed for drying and then

rinsing in water bath for 2 minutes so that the surplus anion attached to the surface

washed off. The dried substrate was then immersed in cationic dye mixture

(RhB+Acf) followed by same rinsing procedure. To prepare the film in presence of

clay, one bi-layer of PAH was prepared first and then it was dipped into the clay

solution followed by (Acf+RhB) mixture solution.

(2) Secondly we prepare individual layers of Acf and RhB onto solid support

seperated by some spacer layer. Here we have used PAA as spacer. Here first of all

one bi-layer self assembled film of PAA and Acf is prepared in the same procedure

mentioned above. This film is then again dipped sequentially in solution of anionic

PAA and then into oppositely charged RhB solution. This results in an Acf and RhB

layer onto quartz slide seperated by a PAA layer (PAA-Acf-PAA-RhB).

(3) Now we prepare the LbL thin film where clay is introduced as a spacer. This step

is similar as second step but the difference is that negatively charged clay templates

play the role of spacer instead of PAA. Here one bi-layer of PAA-Acf film was

dipped successivelly into the solution of clay and then RhB. This results in PAA-Acf-

clay-RhB LbL film onto quartz substrate.

(4) Now we prepare the LbL thin film where SA is introduced as a spacer in between

two successive Acf and RhB layer. In this step firstly a one bi-layer self assembled

film of PAA and Acf is prepared in the same procedure mentioned as previously. The

SA molecules are introduced on the PAA-Acf film by LB film deposition technique. The



film was prepared at surface pressure 15 mN/m. The deposition speed was maintained at 5

mm/min. Then the film was deposited into the solution of RhB. Here the resultant film is

PAA-Acf-SA-RhB.

5.3. Results and discussion 5.3.1. The UV–Vis absorption and steady state fluorescence spectroscopy

Normalized UV–Vis absorption and steady state fluorescence spectra of pure

Acf and RhB in aqueous solutions are shown in Fig. 5.1. Both absorption and

fluorescence spectra are characteristics of the presence of monomers. The

fluorescence spectra were recorded by exciting the corresponding absorption maxima

of Acf and RhB. The absorption and fluorescence maxima of Acf are centered at 449

and 502 nm respectively which is assigned due to the Acf monomers [28]. Acf

monomer absorption band within 444–453 nm depending on the concentration has

been reported [29]. For Acf dimer it has been reported that instead of a single

monomer band two bands at around 437 and 470 nm are observed with the intensity

of the blue band higher than the other [29]. On the other hand RhB absorption

spectrum possess prominent intense 0–0 band at 553 nm along with a weak hump at

520 nm which is assigned due to the 0–1 vibronic transition [30].

Fig. 5.1. Normalized UV–Vis absorption and fluorescence spectra of Acf and RhB in

aqueous solution. The overlap between Acf fluorescence and RhB absorption spectra

is shown by shaded region. Inset show molecular structure of (a) RhB and (b) Acf.

Similar reports with RhB monomer bands at 553 nm and 0–1 vibronic

components of monomer at 525 nm have been reported. [31]. For J-dimer of RhB the



absorption bands are found to be red shifted to 569 and 531 nm [31]. However, for H-

dimer the dominance of 531 nm band intensity with respect to the intensity of 553 nm

band have been reported [31, 32]. The RhB fluorescence spectrum shows prominent

band at 571 nm which is assigned due to the RhB monomeric emission [30]. A close

look in Fig. 5.1 reveals that there exists sufficient overlapping of Acf fluorescence

spectrum and RhB absorption spectrum. This justifies the selection of these two dyes

in order to study energy transfer from Acf to RhB. Here Acf acts as a donor and RhB

acts as an acceptor. Also both the dyes are highly fluorescent, which are the

prerequisite for FRET to occur [1–3].

5.3.2. FRET between Acf and RhB in solution in presence and absence of nanoclay

laponite

Fig. 5.2 shows the fluorescence spectra of pure Acf & RhB and their mixture

(1:1 volume ratio) in aqueous solution and clay dispersion. The fluorescence spectra

were recorded with excitation wavelength at 420 nm (close to the Acf absorption

maximum). This excitation wavelength has been choosen in order to excite Acf

molecules directly where as to avoid direct excitation of RhB molecules. Figure

reveals strong prominent Acf fluorescence band where as the RhB fluorescence band

is very less in intensity in case of pure dye solution. The less intensity of pure RhB

fluorescence band indicates very small contribution of direct excitation of the RhB

molecules. The fluorescence spectrum of Acf-RhB mixture is very interesting. Here

the Acf fluorescence intensity decreases in favor of RhB fluorescence band. In this

case the Acf emission decreases due to the transfer of energy from Acf to RhB. This

transferred energy excites more RhB molecules followed by light emission from RhB,

which is added to the original RhB fluorescence. As a result the RhB fluorescence

intensity gets sensitized. In order to confirm the origin of sensetized RhB

fluorescence, we have measured excitation spectra. Inset of fig. 5.2 shows the

excitation spectra measured with excitation wavelength fixed at Acf (500 nm) and

RhB (577 nm) fluorescence maximum in case of Acf-RhB mixed aqueous solution.

Interestingly both the excitation spectra are almost similar and possess characteristic

absorption bands of Acf monomers. This confirms that the RhB fluorescence is

mainly due to the light absorption by Acf and corresponding transfer to RhB

monomer. Thus FRET between Acf to RhB has been confirmed.



Fig. 5.2. Fluorescence spectra of Acf, RhB and Acf+RhB (1:1 volume ratio) mixture

in aqueous solution and in clay dispersion. Exciting wavelength was 420 nm. Inset

shows the exitation spectra for Acf+RhB mixture with excitation wavelength at 500

(I) and 577 (II) nm.

It was observed that the Acf fluorescence intensity decreases further in favor

of RhB fluorescence intensity in presence of nanoclay platelets, resulting an increase

in FRET efficiency. It is worthwhile to mention in this context that clay particles are

negatively charged and have layered structure with a cation exchange capacity (CEC).

Both the dyes Acf and RhB under investigation are positively charged. Accordingly

they are adsorbed on to the clay layers. On the other hand FRET process is very

sensitive to distances between the energy donor and acceptor and occurs only when

the distance is of the order of 1-10 nm [1, 2]. Therefore, it can be concluded that clay

particles play an important role in determining the concentration of the dyes on their



surfaces or to make possible close interaction between energy donor and acceptor in

contrast to the aqueous solution. In order to quantify FRET between Acf and RhB,

different FRET parametres have been calculated from the spectral charactaristics

using Förster theory. Details of the calculation procedure have been explained in the

section 2.1.1 of chapter 2 of this thesis. The values are listed in the table 5.1. It has

been observed there is an increase in spectral overlap intigral J() which results in a

change in orientation of the dyes when they are adsorbed onto clay surfaces. Also Acf

and RhB molecules come closer when adsorbed onto laponite surface. The increase in

value of J() and decrease in distance between the dyes in presence of clay surface

results an increase in FRET efficiency up to 78.17% which was 11.37% in absence of

laponite.

Sample J()X1015M-1cm-1nm4 R0 (nm) r (nm) E (%)

Acf+RhB

(without clay)

32.17 6.60 8.07 11.37

Acf+RhB

(with clay)

53.71 8.43 5.33 78.17

Table 5.1 Values of spectral overlap integral ( )(J ), energy transfer efficiency (E),

Förster radius (R0), and donor-acceptor distance (r).

5.3.3. FRET between Acf and RhB in ultrathin films in presence and absence of

nanoclay laponite

Fig. 5.3 shows the fluorescence spectra of pure Acf, RhB and their mixture

(1:1 volume ratios) in LbL (fig. 5.3a) and LB (fig. 5.3b) films. Here also pure Acf

shows strong fluorescence with monomer band at 523 nm which is red shifted with

respect to solution. This shift may be due to the change in microenvironment when

Acf molecules are incorporated into the polymer (PAA) backbone in the restricted

geometry of solid surface during film formation. For RhB the trend is very similar to

solution and shows very weak fluorescence with peak at around 575 nm. Energy

transfer is observed for mixed dye system in both LbL and LB films. However,

energy transfer efficiency increases in presence of clay in both the cases. The

corresponding FRET parameters are listed in table 5.2. It has been observed that the

energy transfer efficiency increases up to 32.54% in ultrathin films in presence of

clay. The increase in efficiency is mainly due to the close proximity of the Acf and



RhB when adsorbed onto clay surface as well as slight change in the orientation of the

dyes.

Fig. 5.3. Fluorescence spectra of Acf, RhB and Acf+RhB (1:1 volume ratio) mixture

in ultrathin films ((a) LbL film and (b) LB film) in presence and absence of clay.

Exciting wavelength was 420 nm.

Sample J()X1015M-1cm-1nm4 R0 (nm) r (nm) E (%)

Acf+RhB (LbL film) 3.82 5.93 7.29 22.52

Acf+RhB (LB film) 2.85 5.65 7.48 15.60

Acf+RhB (with clay) 4.12 6.63 7.19 32.54

Table 5.2 Values of spectral overlap integral ( )(J ), energy transfer efficiency (E),

Förster radius (R0), and donor-acceptor distance (r).

In order to check the effect of donor/acceptor concentration on FRET,

fluorescence spectra of Acf-RhB mixture in presence of clay platelet laponite were

measured with fixed volume of Acf and varying volume of the acceptor (shown in

figure 5.4) in both solution and LbL film. Interestingly it was observed that the FRET

efficiency increases with the increase in acceptor concentration in the Acf-RhB

mixture. The maximum FRET efficiencies were 92.5% and 55.71% in clay dispersion

and clay film respectively (datas are shown in table 5.3 and 5.4).From table 5.3 and



5.4 it has been seen that the diatance between Acf and RhB decreases with increase in

acceptor concentration. This results increase in FRET efficiency with increasing

acceptor concentration.

Fig. 5.4. Fluorescence spectra of Acf+RhB mixture for fixed amount of donor (Acf) and varying amount of acceptor in (a) clay dispersion and (b) LbL films in presence of clay. The inset shows the variation of FRET efficiency as a function of acceptor concentration.

% of acceptor (RhB) J()X1015M-1cm-1nm4 R0 (nm) r (nm) E (%)

20 43.25 5.87 6.63 32.41

30 46.87 6.10 6.56 39.15

40 50.52 6.38 6.16 55.02

50 53.71 6.60 5.33 78.17

60 56.72 6.87 5.20 84.10

70 60.10 7.20 5.23 87.20

80 63.50 7.53 5.18 90.40

90 67.20 7.82 5.14 92.50

Table 5.3 Values of spectral overlap integral (J(λ)), energy transfer efficiency (E%), Förster radius (R0) and D–A distance (r), calculated from the spectral characteristics of Fig. 5.4a.



% of acceptor (RhB) J()X1015M-1cm-1nm4 R0 (nm) r (nm) E (%)

20 15.33 2.78 4.10 8.82

30 18.87 2.95 3.81 17.50

40 22.25 3.10 3.68 26.10

50 25.17 3.23 3.44 32.54

60 28.20 3.51 3.75 40.20

70 31.50 3.82 3.89 47.10

80 34.70 4.13 4.09 51.50

90 37.27 4.50 4.33 55.71

Table 5.4 Values of spectral overlap integral (J(λ)), energy transfer efficiency (E%), Förster radius (R0) and D–A distance (r), calculated from the spectral characteristics of Fig. 5.4b.

5.3.4. Atomic Force Microscopy study

To confirm the incorporation of clay particles onto LbL films and to have idea

about the structure of the film, LbL film was studied by Atomic Force Microscope

(AFM). Fig. 5.5a and b show typical AFM image of hybrid LbL film deposited on a

Si substrate along with the line analysis spectrum. In the figure, the laponite particles

are clearly visible. The hybrid film consists of a close-packed array of hybridized

laponite particles. The surface coverage is more than 75%. Few overlapping of

laponite particles are also observed. White spots are indication of aggregates of

laponite particles; while some uncovered regions are also observed. From the height

profile analysis, it is seen that the height of the monolayer varies between −2 nm to +2

nm. This includes the height of the PAH layer on substrate plus the height of the

laponite layer, and Acf-RhB molecules adsorbed onto the clay surfaces. It is

worthwhile to mention in this context that AFM image of Acf–RhB LbL film without

clay shows a smooth surface indicating the uniform deposition of dyes without any

aggregates (figure not shown). Since the dimension of the individual dye molecules

are beyond the scope of resolution, hence its not possible to distinguish individual Acf

or RhB molecules. Therefore, as a whole the AFM investigation gives compelling

visual evidence of incorporation of laponite particles onto the LbL films.



Fig. 5.5. (a and b) AFM image of Acf–RhB mixed LbL film in presence of clay

laponite.

5.3.5. Effect of spacer molecules on FRET between Acf and RhB in ultrathin films

In the previous section the mixture of Acf and RhB were adsorbed onto the

polymer backbone (figure5.6a) onto LbL films. FRET is very much dependent on the

intermolecular seperation between the D-A pair. The variation of intermolecular

seperation between the D-A pair can change the FRET efficiency between a particular

FRET pair. So it is very important to observe the change in FRET efficiency between

a particular FRET pair with varying intermolecular seperation between the D-A pair.

In this section we have inserted different spacers (PAA, SA, Clay) between Acf and

RhB layers and measured the energy transfer efficiency between Acf and RhB. All the

values of corresponding FRET parameters are calculated by using equations of

theoretical consideration and have been tabulated in table 5.5. From table it is

observed that the FRET efficiency is 22.52% for Acf and RhB mixed film without

any spacer. When we introduce spacer (PAA and SA) it is observed that the FRET

efficiencies have decreased. FRET process is very sensitive to distances between the



energy donor and acceptor and occurs only when the distance is of the order of 1-10

nm. Now due to the introduction of spacer molecules between Acf and RhB, the

intermolecular separation between them increases (from 7.21 nm to 7.32 and 7.48 nm)

and FRET efficiency decreases (from 22.52% to 19.67% and 15.60%) as shown in

table 5.5. Now when we increase the SA layer farther the FRET efficiency decreases

significantly (from 22.52% to 7.33%).

Sample J()X1015M-1cm-1nm4 R0(nm) r(nm) FRET efficiency

(E%)

PAA-(Acf+RhB) 3.82 5.93 7.21 22.52

PAH-Clay-(Acf+RhB) 4.12 6.63 7.19 32.54

PAA-Acf-(PAA)-RhB 3.27 5.78 7.32 19.67

PAA-Acf-(1 layer SA)-RhB 2.85 5.65 7.48 15.60

PAA-Acf-(3 layer SA)-RhB 1.82 5.24 8.00 07.33

PAA-Acf-(clay)-RhB 4.12 6.63 7.13 25.71

Table 5.5 Variation of FRET parametres between Acf and RhB in presence of PAA,

clay and SA of different layers.

On the other hand in case of clay the FRET efficiency is larger (25.71%). Clay

particles are negatively charged and have layered structure with a cation exchange

capacity. Acf and RhB under investigation are positively charged and they are

adsorbed on to the clay layers. Therefore, clay particles play an important role in

determining the concentration of the dyes on their surfaces or to make possible close

interaction between energy donor and acceptor. The variation of intermolecular

separation between Acf and RhB has been shown schematically in figure 5.6.



Fig. 5.6. Schematic representation of FRET between Acf and RhB in presence and

absence of PAA, clay and SA.

5.4. Conclusion FRET between two fluorescent dyes Acf and RhB were investigated

successfully in solution and thin films in presence and absence of clay mineral

particle laponite. UV–Vis absorption and fluorescence spectroscopy studies reveal

that both the dyes present mainly as monomer in solution and films and there exist

sufficient overlap between the fluorescence spectrum of Acf and absorption spectrum

of RhB, which is a prerequisite for the FRET to occur from Acf to RhB. Energy

transfer occurred from Acf to RhB in both solution and films in presence and absence



of laponite. The energy transfer efficiency increases in presence of clay laponite in

both solution and in LbL films. The maximum efficiencies were found to be 92.50%

and 55.71% in clay dispersion and clay films respectively. Atomic force microscopy

investigations confirmed the presence of laponite particle in films with a surface

coverage of more than 75%. Due to the introduction of different spacer molecules

between Acf and RhB layer, the FRET efficiency can be modulated for a particular

FRET pair.

References 1. T. H. Förster, Zeitschrift für Naturforschung 4A (1949) 321.

2. T.H. Förster, Modern Quantum Chemistry, Istanbul Lectures, Part III: Action of

Light and Organic Crystals, Academic Press, New York, 1965.

3. S. A. Hussain, S. Chakraborty, D. Bhattacharjee, R. A. Schoonheydt, Spectrochim.

Acta, Part A 75 (2010) 664.

4. P. M. W. French, Biosensors 12 (1999) 41.

5. M. J. Cole, J. Siegel, S. E. D. Webb, R. Jones, K. Dowling, M. J. Dayal, D.

Parsons, P. M. French, M. J. Lever, L. O. Sucharov, M. A. Neil, R. Juskaitis, T.

Wilson, J. Microsc 203 (2001) 246.

6. G. Haran, J. Phys.: Condens. Matter 15 (2003) R1291.

7. R. B. Best, S. B. Flower, J. L. T. Herrera, J. Clark, Proceedings of the National



(2002) 153.

9. R. J. H. Clark, R. E. Hester (Eds.), Advances in Spectroscopy, Wiley, New York,

1996.

10. M. S. Csele, P. Engs, Fundamentals of Light and Lasers, Wiley, New York, 2004.

11. J. M. Drake, J. Klafter, P. Levitz, Science 251 (1991) 1574.

12. Y. Yilmaz, A. Erzan, Ö. Pekcan, Physical Review 58 (1998) 7487.

13. T. Jonsson, C. D. Waldburger, R. T. Sauer, Biochemistry 35 (1996) 4795.

14. B. S. Watson, T. L. Hazlett, J. F. Eccleston, C. Davis, D. M. Jameson, A. E.

Johnson, Biochemistry 34 (1995) 7904.

15. D. Bhattacharjee, D. Dey, S. Chakraborty, S. A. Hussain,•S. Sinha, J. Biol. Phys.

39 (2013) 387.

16. K. M. Parkhurst, L. J. Parkhurst, Biochemistry 34 (1995) 285.



17. J. W. Nichols, R.E. Pagano, J. Biol. Chem. 258 (1983) 5368.

18. S. A. Hussain, R.A. Schoonheydt, Langmuir 26 (2010) 11870.

19. R. A. Schoonheydt, Clays Clay Miner. 50 (2002) 411.

20. R. H. A. Ras, B. van Duffel, M. Van der Auweraer, F.C. De Schryver, R.A.

Schoonheydt, Proceedings of the 12th International Clay Conference, Bahia

Blanca, Argentina, 2001.

21. D. Madhavan, K. Pitchumani, Tetrahedron 58 (2002) 9041.

22. M. G. Neumann, H. P. M. Oliveira, A. P. P. Cione, Adsorption 8 (2002) 141.

23. Y. Ishida, T. Shimada, D. Masui, H. Tachibana, H. Inoue, S. Takagi, J. Am.

Chem. Soc. 133 (2011) 14280.

24. A. Czímerová, J. Bujdak, N. Iyi, J. Photochem. Photobiol., A 187 (2007) 160.

25. A. Czímerová, N. Iyi, J. Bujdák, J. Colloid Interface Sci 306 (2007) 316.

26. J. Bujdák, D. Chorvát, N. Iyi, J. Phys. Chem. C 114 (2010) 1246.

27. J. Bujdák, A. Czímerová, F. López Arbeloa, J. Colloid Interface Sci. 364 (2011)

497.

28. P. D. Sahare, V. K. Sharma, D. Mohan, A. A. Rupasov, Spectrochim. Acta, Part A

69 (2008) 1257.

29. J. L. Rosenberg, F. S. Humphries, J. Phys. Chem. 71 (1967) 330.

30. S. A. Hussain, S. Banik, S. Chakraborty, D. Bhattacharjee, Spectrochim. Acta,

Part A 79 (2011) 1642.

31 T. Fujii, H. Nishikiori, T. Tamura, Chem. Phys. Lett. 233 (1995) 424.

32. K. Kemnitz, N. Tamai, I. Yamazaki, N. Nakashima, K. Yohsihara, J. of Phys.

Chem. 90 (1986) 5094.


Chapter 6: Design of pH sensor 62

Chapter-6

Development of pH sensor using

fluorescence resonance energy transfer

This chapter reports the change in FRET efficiency between

two laser dyes with the variation of solvent pH. Energy

transfer efficiency was pH sensitive and it varies from 4.5% to

44.45% in mixed dye solution for a change in pH from 3.0 to

12.0. With proper calibration it is possible to use the present

system under investigation to sense pH over a wide range from

3.0 to 12.0.

Based on the experimental results of this chapter one paper has been published in a peer reviewed reputed journal.

1. Effect of nanoclay laponite and pH on the energy transfer between fluorescent dyes


Journal of Photochemistry and Photobiology A-Chem 252 (2013) 174– 182



Chapter-6

Development of a pH sensor using fluorescence resonance energy transfer

6.1. Introduction The sensing of pH is one of the most powerful techniques which are essential

in many fields of application ranging from agriculture and environment to industry,

medicine and food. In medical science, abnormal pH values inside the cell indicate

inappropriate cell function, growth and division. It is also helpful to diagnose some

common disease like cancer and Alzheimer’s etc. For the sensing of pH there are two

very well known methods namely, (1) Optical chemical sensors (also termed

optrodes) and (2) FRET based pH sensors. In case of optrodes the change in

absorbance or fluorescence intensity of the pH sensitive dyes indicate a change in pH

of the environment. On the other hand FRET based pH sensors are indicated by the

ratiometric changes of the fluorescence of both donor and acceptor with pH of the

environment. Fluorescent sensors are one of the important methods for sensing of

different chemical and biological materials but for this type of sensors change in

fluorescent intensity could very well be perturbed by environmental factors. The

introduction of FRET sensors can minimize this environmental perturbation, because

it measures the ratio of two emissions in different environment. Accordingly energy

transfer has been used as a very powerfull tool for pH measurement [1].

In the present chapter we have investigated the effect of pH on the FRET

between two laser dyes Acf and RhB. The spectra of Acf are highly pH sensitive due

to the presence of electron donor type functional group [2, 3]. Therefore it is very

interesting to study the FRET parameters at different pH using Acf as donor in order

to explore their possible application as pH sensor [2].


The dye solution was prepared by using Millipore water. The concentration of

both the dyes was 10-6 M. For the increase and decrease of pH of the solvent, NaOH

and HCl were used respectively.



6.3. Results and discussion 6.3.1. Effect of pH on FRET

FRET between Acf and RhB has already been studied and the results are

shown in details in chapter 5 of this present thesis. Here we shall discuss the effect of

solvent pH on FRET between Acf and RhB. Among the molecules under current

investigation Acf is pH sensitive because of its basic nature of the central nitrogen

atom [4]. The fluorescence spectra of Acf are affected with change in pH [5]. This

may in turn cause a change in spectral overlapping of the donor fluorescence and

acceptor absorbance resulting a change in FRET efficiency. In order to check the

effect of pH on FRET process, fluorescence spectra of Acf+RhB mixture in aqueous

solution prepared at different pH were measured (Fig. 6.1). It was observed that the

Acf fluorescence was red shifted with decrease in pH.

Fig. 6.1. Fluorescence spectra of Acf–RhB (1:1 volume ratio) mixture in aqueous

solution at different solvent pH. Dye concentration was 10-6 M. Excitation wavelength

is 420 nm.



It is interesting to mention in this context that proflavine molecule is very

similar to Acf with regards to protonation and deprotonation. Proflavine has been

found to exist as single protonated, double protonated as well as neutral molecules in

aqueous solution with pKa=9.5 for single protonated and 0.2 for double protonated

form [6,7]. The excited state dissociation constants are 12.5 for single protonated and

1.5 for double protonated species. It has been observed that Acf mainly remain as

double protonated form in nafion (a perfluorosulfonate cation exchange membrane)

due to the high local proton concentration [7]. Larger red shift in Acf fluorescence in

nafion has been observed and explained due to change in the dipole moments in the

excited state of the double protonated Acf [6] and due to the broad distribution of pKa

in nafion matrix [6]. In the present case at lower pH red shift of Acf fluorescence is

observed. At lower pH Acf molecules mainly remain as double protonated form due

to the increase in local proton concentration with decreasing pH. Accordingly the

dipole moments of the excited state of double protonated Acf have been changed. This

change in dipole moments may be responsible for the observed large stoke shift/red

shift of the Acf fluorescence.

pH of the solution J(λ)×1015 M−1 cm−1 nm4 E (%)

3.0 18.00 04.50

4.5 23.80 07.20

6.0 30.10 14.11

7.5 34.90 21.70

9.0 39.40 28.50

10.5 43.20 35.20

12.0 47.70 44.45

Table 6.1 Values of spectral overlap integral (J(λ)) and energy transfer efficiency

(E%), calculated from the fluorescence spectra of aqueous solution of Acf-RhB

mixture measured at different solvent pH.

The values of J(λ) and FRET efficiencies calculated (using the Förster theory)

from the spectra measured at different pH are listed in table 6.1. Interestingly it was

observed that the FRET efficiency increases with increase in pH. It was found that for

the same donor acceptor concentration and excitation wavelength, the value of



spectral overlap integral J(λ) changes a lot with change in pH. But the shape of the

fluorescence spectra remains almost similar. It is worthwhile to mention in this

context that RhB contains COOH group, which can dissociate in certain conditions to

form cations–anions (zwitterions). In basic medium RhB shows the zwitterionic form

which could be responsible for the close interaction between cationic acriflavine

(Acf+) and COO− group of zwitterionic RhB. This will increase the possibility of

closer approach of Acf and RhB at higher pH resulting an increase in FRET

efficiency. In acidic medium (lower pH) RhB generally remains in cationic form with

lower pKa value [8]. Also the shift of Acf fluorescence with pH results a change in

spectral overlap between Acf fluorescence and RhB absorbance, i.e. J(λ) value. This

will in turn affect the FRET efficiency. The electron donor type functional group of

Acf become more basic with increase in pH in the excited state, consequently the

fluorescence spectra shifts toward shorter wavelength providing a larger value of

spectral overlap integral (table 6.1) with increasing pH. This increase in J(λ) in turn

causes an increase in FRET efficiency. The value of J(λ) changes from 18×1015 M−1

cm−1 nm4 to 47.7×1015 M−1 cm−1 nm4 for change in pH from 3.0 to 12.0. Accordingly

the energy transfer efficiency varies from 4.5% to 44.45%. Therefore in the present

system under investigation the FRET process between Acf and RhB is very pH

sensitive.

6.3.2. Sensing of pH of aqueous solution by FRET

Figure 6.2a and b show the plot of spectral overlap integral (J(λ)) and energy

transfer efficiency (E%) as a function of pH. Interestingly it was observed that both

J(λ) and E% increases almost linearly with increasing pH. Therefore, pH dependence

of the energy transfer between the present D–A pair Acf and RhB under investigation

makes the system a suitable candidate for sensing of pH. Any of the data from Table

6.1 may be used to sense the pH with appropriate calibration. It is interesting to

mention in this context that energy transfer has already been used for pH

measurement [4]. Chan et al. demonstrated FRET-based ratiometric pH nanoprobes

where they used semiconducting polymer dots as a platform. The linear range for pH

sensing of the fluorescein-coupled polymer dots was between pH 5.0 and 8.0 [9].

Egami et al. has introduced a fiber optic pH sensor, using polymer doped with either

congo red (pH range 3–5) or methyl red (pH range from 5 to 7) [10]. pH sensor based

on the measurement of absorption of phenol red has also been reported [11], which



can sense a pH range of 7–7.4. In the present system of pH measurement using the

change in FRET parameter with pH is capable of measuring over a wide range of pH

3.0–12.0. This is one advantage with respect to previous systems.

Fig. 6.2. Variation of (a) spectral overlap integral (J(λ)) and (b) energy transfer

efficiency (E%) with increasing pH of the solution.

6.4. Conclusion Energy transfer efficiency between Acf and RhB was found to be pH sensitive

and varies from 4.5% to 44.45% in mixed dye solution for a change in pH from 3.0 to

12. With proper calibration it is possible to use the present system under investigation

to sense pH over a wide range of pH from 3.0 to 12.0. The advantage of the present

system is that it can be used to sence a wide range of pH (3.0 to 12.0) campared to the

reported results.

References 1. A. M. Dennis, W. J. Rhee, D. Sotto, S. N. Dublin, G. Bao, ACS nano, 6 (2012)

2917.

2. V. Misra, H. Mishra, H. C. Joshi, T. C. Pant, Sens. Actuators, B 63 (2000) 18.

3 J. E. L. Corry, G. D. W. Curtis, R. M. Baird, Handbook of Culture Media for Food

Microbiology, Second Edition, progress in industrial microbiology 37 (2003).



4 V. K. Sharma, P. D. Sahare, R. C. Rastogi, S. K. Ghoshal, D. Mohan, Spectrochim.

Acta, Part A 59 (2003) 1799.

5. R. R. Beumera, M. C. te Giffela, S. V. R. Anthoniea, L. J. Cox, Int. J. Food

Microbiol. 13 (1996) 137.

6. M. P. Pileni, M. Grätzel, J. Phys. Chem. 84 (1980) 2402.

7 C. B. Airaudo, A. G. Sorbier (Eds.), Marseille, France, (Translated from Labo-

Pharma, Nos. 248, 250, 251), (1980) 77.

8. I. L. Arbeloa, K. K. R. Mukherjee, Chem. Phys. Lett. 128 (1986) 474.

9. Y. H. Chan, C. Wu, F. Ye, Y. Jin, P. B. Smith, D. T. Chiu, Anal. Chem. 83 (2011)

1448.

10. C. Egami, Y. Suzuki, O. Sugihora, H. Fujimura, N. Okamoto, Jpn. J. Appl. Phys.

36 (1997) 2902.

11. J. I. Peterson, S. R. Goldstein, R. V. Fitzgerald, D. K. Buckhold, Anal. Chem. 52

(1980) 864.


Chapter 7: Design of Hard water sensor 69

Chapter-7

Development of hard water sensor using


This chapter mainly concern with a method for the sensing of

water hardness by determining the concentration of calcium

and magnesium in water, based on fluorescence resonance

energy transfer (FRET) process. The principle of the proposed

sensor is based on the change of FRET efficiency between two

laser dyes Acriflavine (Acf) and Rhodamine B (RhB) in presence

of permanent hard water components (CaCl2 and MgCl2).

Nanodimensional clay platelet laponite was used to enhance the

efficiency of the sensor.


1. Development of hard water sensor using fluorescence resonance energy transfer.

Dibyendu Dey, D. Bhattacharjee, S. Chakraborty, S.A.

Sensors and Actuators B: Chemical 184 (2013) 268-273.



Chapter-7

Development of hard water sensor using fluorescence resonance energy transfer 7.1. Introduction

“Hard water” has high mineral content in compare to “soft water”.

Generally the hard water is not harmful to one’s health, but can cause serious

problems in industrial settings, where water hardness should be monitored to avoid

breakdowns of the costly equipments that handle water. The hardness of water is

determined by the concentration of multivalent cations in water. The most common

cations found in hard water include Ca2+ and Mg2+. The presence of dissolved

carbonate minerals (CaCO3 and MgCO3) provide a temporary hardness in water,

which can be reduced either by boiling the water or by addition of lime (calcium

hydroxide) [1, 2]. On the other hand the dissolved chloride minerals (CaCl2 and

MgCl2) cause the permanent hardness of water that can not be removed easily, as it

becomes more soluble as the temperature increases [3]. In that sense it is very

important to identify the permanent hardness of water before use.

The FRET phenomenon may be very effective tool for the designing of hard

water sensors. Based on the FRET between two laser dyes here we demonstrated a

hard water sensor. To the best of our knowledge this could be the first attempt, where

FRET process has been used for the detection of the hardness of water. FRET

between two molecules is an important physical phenomenon, where transfer of

energy from an excited fluorophore to a suitable acceptor fluorophore occurred [4, 5].

This technique is very important for the understanding of some biological systems and

has potential applications in optoelectronic and thin film devices [6–10]. Combining

FRET with optical microscopy, it is possible to determine the approach between two

molecules within nanometers. The main requirements for the FRET to occur are (i)

sufficient overlap between the absorption band of acceptor fluorophore and the

fluorescence band of donor fluorophore and (ii) both the donor and acceptor molecule

must be in close proximity of the order of 1–10 nm [4, 5]. The intervening of solvent

or other macromolecules has little effect on the FRET efficiency. If the distance

between the donor and acceptor changes then FRET efficiency also changes.



In this chapter of the present thesis we tried to investigate the effect of Mg2+ or

Ca2+ or both on the FRET efficiency between two fluorophores, Acf and RhB in

presence of nanoclay sheet laponite. Here we have chosen Mg2+ or Ca2+ because the

presence of these two cations mainly determines the extent of hardness of the water.

Our investigation showed that FRET efficiency decreases with increasing salt

concentration. It has also been demonstrated that with proper calibration, FRET

between Acf and RhB can be used to sense the hardness of water.


Both the dyes Acf and RhB were used in our studies are cationic in nature.

The clay mineral used in the present work was Laponite. Dye solutions were prepared

in Milli-Q water. For spectroscopic measurement the solution concentration was

optimized at 10−6M. In order to check the effect of hard water components (Ca2+ and

Mg2+ ions) on spectral charecteristics, MgCl2 and CaCl2 were added to the dye

solution. The clay dispersion was prepared using Millipore water and stirred for 24 h

with a magnetic stirrer followed by 30 min ultrasonication before use. The

concentration of clay was kept fixed at 2 ppm throughout the experiment. To check

the effect of clay on the spectral characteristics the dye solutions (Acf and RhB) were

prepared in the clay suspensions (2 ppm). In order to check the effect of salt on

spectral characteristics in presence of clay, first of all the salts were added in the clay

dispersion at different concentration. Then the dyes were added in the salt mixed clay

dispersions. In all cases the clay concentration was 2 ppm and the dye concentration

was 10−6M.

7. 3. Results and discussion 7.3.1. Sensing of Hard Water by FRET


shown in details in chapter 5 of this thesis. Here in this chapter we have used this

same FRET pair for the sensing of water hardness. Our main purpose is to detect

CaCl2 and MgCl2 or their mixture as a permanent hard water component in aqueous

solution by using the FRET process between Acf and RhB. Fluorescence spectra of

aqueous solution of Acf-RhB mixture in presence of MgCl2, CaCl2 and their mixture

(CaCl2+MgCl2) have been studied (figure 7.1). For all the cases, the concentration of



CaCl2/MgCl2/their mixture was kept at 0.06 mg/ml, which is the initializing

concentration of hard water known as moderately hard water.

Fig. 7.1. Fluorescence spectra of Acf+RhB (1:1 volume ratio) in water solution (1),

with MgCl2 (2), CaCl2 (3), and CaCl2+MgCl2 (4), pure Acf (5), pure RhB (6). Dye

concentration was 10-6M and salt concentration was 0.06 mg/ml.

It was observed that the transfer of energy from Acf to RhB decreased in

presence of salt (shown in table 7.1). The FRET efficiencies are calculated by using

the Förster theory. The introduction of cationic Ca2+ and Mg2+ ions in the solution

may cause an increase in the electrostatic repulsion between cationic Acf and RhB

molecules which can result in a large intermolecular separation. Accordingly, the

FRET efficiency decreases.



Samples E%

Acf+RhB 11.37

Acf+RhB+ CaCl2 1.7

Acf+RhB+ MgCl2 5.2

Acf+RhB+ MgCl2 +CaCl2 4.38

Acf+RhB+clay 78.17

Acf+RhB+ CaCl2 with clay 37.78

Acf+RhB+ MgCl2 with clay 51.59

Acf+RhB+ MgCl2 +CaCl2 with clay 48.18

Table 7.1 Values of energy transfer efficiency (E %) for Acf and RhB mixture (1:1

volume ratio) in different conditions. The salt concentration was 0.06 mg/ml

(moderately hard water).

Fig. 7.2. Fluorescence spectra of Acf+RhB (1:1 volume ratio) in clay suspension (1), with MgCl2 (2), CaCl2 (3), and CaCl2+MgCl2 (4) pure Acf with clay (5), pure RhB with clay (6). Dye concentration was 10-6M and clay concentration was 2 ppm and salt concentration was 0.06 mg/ml.



In the present study our aim is to design a sensor which can sense the presence

of Ca2+ or Mg2+ or both by observing the change in FRET efficiency. Accordingly it

is very important to have large FRET efficiency between Acf and RhB as well as

noticeable change in FRET efficiency between Acf and RhB due to the introduction

of hard water components (Ca2+ or Mg2+), so that we can use it as a hard water sensor

with minimum error level. Accordingly in order to enhance the FRET efficiency, we

have incorporated nano clay laponite in Acf-RhB mixture (figure 7.2). It has been

observed that the FRET efficiency increases in presence of laponite particle. The

reason of increase in FRET efficiecy in presence of clay has been explained in details

in chapter 5. It was also observed that the transfer of energy from Acf to RhB

decreases quite remarkably due to the introduction of CaCl2/MgCl2/their mixture for

the concentration of 0.06 mg/ml in presence of clay. It was observed that the transfer

of energy is much smaller due to the presence of CaCl2 in compare to MgCl2. (Table

7.1 summarize the calculated efficiencies).

Fig. 7.3. Schematic representation of FRET between Acf and RhB in presence of

clay and salt.

The decrease in FRET between Acf and RhB in presence of hard water

components, must involve the reaction of the cations (Ca2+ and Mg2+) present in hard

water with the clay minerals through cation exchange reaction. In general, this

bonding energy is of the order: Ca > Mg > K > H > Na. Thus the probability of



adsorption of Ca2+ in clay suspension is more than Mg2+. The tendency of Ca2+ ion to

interact with the negatively charged clay layers is more compared to Mg2+ ion of same

concentration of both clay suspension and salt solution. Accordingly, most of the

negative charges in the clay surfaces are neutralized by Ca2+ ion compared to Mg2+

ion and there exists very few unoccupied negative charges on the clay surface for the

cationic dye molecules to be adsorbed. As a result the separation between the cationic

dye molecules increases more in CaCl2 solution rather than MgCl2 leading to a less

FRET in presence of CaCl2 compare to MgCl2 (shown schemetically in figure 7.3).

7.3.2. Variation of salt concentration

In order to check the extent of hardness on the FRET efficiency, we have

measured the fluorescence spectra of Acf+RhB mixture with different salt (MgCl2,

CaCl2, and MgCl2+CaCl2 mixture) concentration in presence of clay laponite and the

FRET efficiency have been calculated. It has been observed that the FRET efficiency

decreases with increasing salt concentration of either MgCl2 or CaCl2, or their mixture

(figure 7.4). This result suggests that it is possible to sense the hardness of water by

observing the change in FRET efficiency with salt concentration.

Fig. 7.4. The fluorescence spectra of Acf–RhB mixture in presence of clay with

varying amount of salt (MgCl2+CaCl2 mixture) concentration viz. 0.05 mg/ml (1),

0.06 mg/ml (2) and 0.12 mg/ml (3). Inset shows the variation of FRET efficiency as a

function of salt concentration of either MgCl2 or CaCl2, or CaCl2+ MgCl2 mixture

from 0.03 mg/ml to 0.20 mg/ml.



Salt concentration (mg/ml)

FRET efficiency (E%) in presence of MgCl2 CaCl2 MgCl2+CaCl2

mixture 0.03 78.52 64.27 73.73 0.05 71.55 55.32 68.34 0.06 51.59 37.78 48.18 0.08 32.47 18.75 21.57 0.12 19.67 10.54 13.48 0.20 11.34 5.67 07.38

Table 7.2 Values of energy transfer efficiency (E %) for Acf-RhB mixture (1:1

volume ratio) at different salt (MgCl2, CaCl2, MgCl2+CaCl2 mixture) concentration in

presence of clay. MgCl2 and CaCl2 mixture were prepared by adding 1:1 volume ratio

of individual salts of same concentration.

7.3.3. Design of sensor

In the process of hard water sensing first of all clay (laponite) dispersion will

be prepared using the sample water followed by addition of dyes (Acf and RhB). By

observing the FRET efficiency between Acf and RhB it would be possible to sense

the hardness of the test water.

Fig. 7.5. FRET efficiency of Acf-RhB mixture for the different concentration of

CaCl2 + MgCl2 in presence of clay (values of FRET efficiencies were calculated from

spectra of Fig. 7.4).

From figure 7.5 it has been observed that the FRET efficiency for 0.06 mg/ml

and 0.12 mg/ml concentration are 48.2% and 13.5% respectively. If the FRET

efficiency is observed to be higher than 48.2%, then the water will be recognized as



soft water whereas, if the efficiency lies in between 13.5% and 48.2% then the water

will be recognized as moderately hard. On the other hand if the observed FRET

efficiency is less than 13.5% then the water will be recognized as very hard. Therefore

with proper calibration it is possible to design a hard water sensor which can sense

hard water very easily.

7.4. Conclusion In order to demonstrate hard water sensor based on FRET, we have

investigated the FRET between Acf and RhB in presence of salts CaCl2 or MgCl2 or

both. It was observed that the presence of hard water components Ca2+ or Mg2+ or

both affected the FRET efficiency to a large extent. In presence of CaCl2 or MgCl2 the

FRET efficiency is decreased to 37.78% and 51.59%, respectively. With suitable

calibration of these results it is possible to design a hard water sensor that can sense

the water hardness within the range 0.03–0.2 mg/ml. Here the incorporation of clay

platelate laponite enhances the sensing efficiency.

References 1. H. Weingärtner, Water in Ullmann’s Encyclopedia of Industrial Chemistry, Wiley-

VCH, Weinheim, (2006).

2. T. J. Sorg, R. M. Schock, D. A. Lytle, J. Am. Water Works Assoc. 91 (1999) 85.

3. P. B. Sweetser, C. E. Bncker, Anal. Chem. 26 (1954) 195.

4. T. H. Förster, Zeitschrift fur Naturforschung 4A (1949) 321.

5. T. H. Förster, Discuss. Faraday Soc. 27 (1959) 7.

6. G. Haran, J. Phys.: Condens. Matter 15 (2003) R1291.

7. R. B. Best, S. B. Fowler, J. L. Toca Herrera, J. Clarke, Proceedings of the National



(2002) 153.

9. R. J. H. Clark, R. E. Hester, Spectroscopy of Inorganic based Materials Advances

in Spectroscopy, Wiley, New York, (1996).

10. M. S. Csele, P. Engs, Fundamentals of Light and Lasers, Wiley, New York,

(2004).


Chapter 8: Design of Ion sensor 78

Chapter-8

Development of an ion sensor using


This chapter mainly concern with a method for the sensing of

different ions by determining the concentration of

corresponding salts (KCl, NaCl, MgCl2, CaCl2, FeCl3, FeSO4, and

AlCl3) in water, based on FRET process. The principle of the

propose sensor is based on the change of FRET efficiency

between two laser dyes Acf and RhB in presence of different

ions (K+, Na+, Mg2+, Ca2+, Fe2+, Fe3+, Al3+). Nanodimensional clay

platelet laponite was used to enhance the efficiency of the

sensor.


1. Development of an Ion-Sensor using Fluorescence Resonance Energy Transfer.


Sensors and Actuators B: Chemical 195 (2014) 382-388.



Chapter-8

Design of an ion sensor using fluorescence resonance energy transfer 8.1. Introduction

The development of ion sensor technology has emerged as a dynamic

approach for identifying and quantitating specific analytes of environment. Now a

days the growing need for multicomponent analyses and shorter sample preparation

methods, new sensing techniques with decreasing costs are very important. There are

several sensing techniques which include ion selective or gas sensitive electrodes,

thermistors, chemically and biologically modified metal or semiconductors [1–3].

Chalcogemide glass sensors are used for the detection of millimole levels of copper,

iron, chromium, lead, cadmium and mercury in natural waste waters [4]. The method

is also used for the detection of heavy metal ions in solutions [4]. But the sensing of

ions present in micromole amount could be much interesting and useful. It will be

very much interesting to sense K+ and Na+ ions in blood samples. In blood the K+ and

Na+ concentrations are 4.5 and 120 mM respectively [5]. Increase in concentration of

these ions in blood can cause serious health problems. Another type of sensor is

fluorescent ion sensors. There are few reports where the detection of heavy metal ions

such as Hg2+, Pb2+ and Cd2+ have been done by fluorescent ion sensors with high

sensitivity and simplicity [6–9]. But in this method the change in fluorescent intensity

could be perturbed by environmental factors [10, 11]. It is interesting to mention in

this context that the introduction of ratiometric sensors can minimize this

environmental perturbation, because it measures the ratio of two emissions in

different environment. The design of ratiometric sensors can be done by two methods

(i) ICT (intermolecular charge transfer) and (ii) FRET (Fluorescence Resonance

Energy Transfer). For many ICT based ion sensors it is difficult to determine the ratio

between two relatively broad signal emissions. Recently FRET based sensing has

become most effective method for the detection of ions in environment. FRET based

sensors have been widely used in metal ion detection [12, 13], sensing of the

fluorophores [14–18], silica [19, 20], and polymer particles [21–23].



Here in the process of designing ion sensor based on FRET process, we have

used two dyes Acf and RhB as energy donor and acceptor respectively. In principle

both the dyes are suitable for FRET. Both the dyes are highly fluorescent and the

fluorescence spectrum of Acf sufficiently overlaps with the absorption spectrum of

RhB. P.D. Sahare et. al. [24] reported the FRET in binary solution mixture of these

two dyes.

In this chapter of the present thesis we investigated the effect of K+, Na+,

Mg2+, Ca2+, Fe2+, Fe3+, Al3+ on the FRET efficiency between two fluorophores, Acf

and RhB in presence of nanoclay sheet laponite. Our investigation showed that FRET

efficiency decreases with increasing salt concentration. It has also been demonstrated

that with proper calibration, FRET between Acf and RhB can be used to sense

different ions on the basis of their size and charge up to a concentration 10µM.


The dyes Acf and RhB used in our studies are cationic in nature. The clay

mineral used in the present work was Laponite. The salts NaCl, KCl, MgCl2, CaCl2,

FeSO4, FeCl3 and AlCl3 were mixed seperately with the Acf+RhB mixed dye solution

and the fluorescence spectra of the mixture has been measured. Dye solutions were

prepared in Milli-Q water. For spectroscopic measurement the solution concentration

was optimized at 10−6M. The clay dispersion was prepared using Millipore water and

stirred for 24 h with a magnetic stirrer followed by 30 min ultrasonication before use.

The concentration of clay was kept fixed at 2 ppm through out the experiment. To

check the effect of clay on the spectral characteristics the dye solutions (Acf and RhB)

were prepared in the clay suspensions (2 ppm). The dye concentration was maintained

at 10−6M. In order to check the effect of salt on spectral characteristics in presence of

clay, first of all the salts were added in the clay dispersion at different concentration.

Then the dyes were added in the salt mixed clay dispersions. In all cases the clay

concentration was 2 ppm and the dye concentration was 10−6M.

8.3. Results and discussion 8.3.1. Sensing of ions by FRET


shown in details in the previous chapters of the thesis. Here in this chapter we have



used this same FRET pair for the sensing of different ions. In order to sense different

ions we have introduced different salts in the Acf-RhB mixed aqueous solution and

the FRET between Acf to RhB has been measured (Fig. 8.1). It has been observed that

in all the cases the FRET efficiency decreased. However, the change in FRET

efficiency is very small. The FRET efficiency changes from 11.37% (in absence of

salt) to 9.2% (in presence of KCl) or 7.4% (in presence of MgCl2) or 5.2% (in

presence of FeCl3).

Fig. 8.1. (a) Fluorescence spectra of pure Acf (1), pure RhB (2) and Acf+RhB (1:1 volume ratio) in water solution (3), with KCl (4), MgCl2 (5), FeCl3 (6). (b) Fluorescence spectra of pure Acf with clay (1), pure RhB with clay (2) and Acf+RhB (1:1 volume ratio) in clay suspension (3), with KCl (4), MgCl2 (5), FeCl3 (6). Salt concentration was 10 µM.

It has been observed from the previous chapters that FRET efficiency

increases in presence of nanoclay platelets. Again the presence of salts causes a

decrease in FRET efficiencies. In presence of clay the change or variation of FRET

efficiency due to presence of salts is more compared to that in absence of clay

platelet. Therefore incorporation of clay laponite in the present system lower the error

level in sensing different ions or increases the ion sensing efficiency/sensitivity.



8.3.2. Ions with variable size

In order to have an idea about the effect of ion size on FRET efficiency, we

have selected three different sets of salts – (i) NaCl and KCl (both are monovalent),

(ii) MgCl2 and CaCl2 (both are divalent), (iii) FeCl3 and AlCl3 (both are trivalent), and

measured the FRET efficiency between Acf to RhB in presence of these salts in clay

dispersion (Fig. 8.2).

Fig. 8.2. (a) Fluorescence spectra of pure Acf with clay (1), pure RhB with clay (2) and Acf+RhB (1:1 volume ratio) in clay suspension (3), with KCl (4), NaCl (5) and (b) fluorescence spectra of pure Acf with clay (1), pure RhB with clay (2) and Acf+RhB (1:1 volume ratio) in clay suspension (3), with CaCl2(4), MgCl2(5). (c) Fluorescence spectra of pure Acf with clay (1), pure RhB with clay (2) and Acf+RhB (1:1 volume ratio) in clay suspension (3), with FeCl3(4), AlCl3(5). Salt concentration was 10 µM.

8.3.3. Ions with variable valency

In order to check the effect of valency of ions on the energy transfer efficiency

we have measured the fluorescence spectra of Acf+RhB mixture (1:1 volume ratio) in



aqueous clay dispersion in absence and presence of salt FeSO4 (divalent) and FeCl3

(trivalent). The extent of decrease in FRET efficiency is more in presence of FeCl3

compared to that in presence of FeSO4. In this study we have selected the salts in such

a way that their molecular size remains same but their charge changes. Here FeSO4

provides a Fe2+ ion and FeCl3 provides a Fe3+ ion in aqueous medium. So the larger

electrostatic repulsion of the Fe3+ ion provides a larger intermolecular separation

between the Acf and RhB in compared to Fe2+ ion on to the clay templates when the

ions and dyes were adsorbed on to clay surface. As a result the FRET efficiency

decreases more in case of FeCl3 (shown in table 8.1). This has been explained with

schematic diagram (figure 8.4).

Fig. 8.3. Fluorescence spectra of pure Acf with clay (1), pure RhB with clay (2) and

Acf+RhB (1:1 volume ratio) in clay suspension (3), with FeCl3(4), FeSO4(5). Salt

concentration was 10 µM.

8.3.4. Effect of variation of salt concentration on FRET efficiency

Now in order to check the effect of variation of salt concentration on the

FRET efficiency, we have measured the fluorescence spectra of Acf and RhB mixture

with different salt concentration (10, 100 and 1000 µM) in clay dispersion and the



FRET efficiency has been calculated and tabulated in table 8.1. It has been observed

that the FRET efficiency decreases with increasing salt concentration. The increase in

salt concentration basically increases the amount of cations in the solvent and as a

result a larger area of the clay layers is occupied by the salt cations. Accordingly a

comparatively smaller amount of dye molecules are attached to the clay templates

resulting in a less probability of occurrence of FRET between Acf and RhB. As a

whole our investigations suggest that it is possible to sense the ions by observing the

change in FRET efficiency with ion size, valency and varying salt concentration.

Table 8.1 Values of energy transfer efficiency (E %) for Acf and RhB mixture (1:1

volume ratio) with different salt concentration in presence of clay.

Salts

FRET efficiency(E%) with clay

salt con=

10 µM

salt con=

100 µM

salt con=

1000 µM

KCl 56.8 48.7 41.2

NaCl 62.15 55.7 51.2

MgCl2 47.4 42.5 36.8

CaCl2 41.1 36.5 31.2

FeSO4 46.8 41.7 35.8

FeCl3 38.5 32.7 27.8

AlCl3 45.6 39.8 33.5



Fig. 8.4. Schematic representation of anionic clay sheet (a) and FRET between Acf

and RhB in presence of (b) clay, (c) clay and salt with larger size ions, (d) clay and

salt with smaller size ions, (e) clay and salt with trivalent ions, (f) clay and salt with

divalent ions.

8.3.5. Design of ion sensor

In the process of ion sensing first of all clay (laponite) dispersion will be

prepared using the sample water (in presence of ions) followed by addition of dyes

(Acf and RhB). Then the fluorescence spectra of the solution will be measured. By

observing the FRET efficiency calculated from the observed fluorescence spectra it

would be possible to sense different ions present in the sample water. With proper



calibration it is possible to design an ion sensor which can sense ions on the basis of

their size (figure 8.5a-8.5c) and similar observation can be done for the sensing of

ions of different valency but same size (as shown in figure 8.5d).

Fig. 8.5. FRET efficiency of Acf and RhB mixture for (a) KCl (1), NaCl (2) and

without salt (3) (b) CaCl2 (1), MgCl2 (2) and without salt (3) (c) FeCl3 (1), AlCl3 (2)

and without salt (3) (d) FeSO4 (1), FeCl3 (2) and without salt (3) in presence of clay.

(Values of FRET efficiencies were calculated from the spectra of Figs. 8.2 and 8.3.)

8.4. Conclusion In order to design FRET based ion sensor, we have investigated the FRET

between Acf and RhB in presence of different salts, viz. NaCl, KCl, MgCl2, CaCl2,

FeSO4, FeCl3 and AlCl3. The salts were choosen in such a way that they produce ions

in the solution with different size and valency. It has been observed that in presence of

KCl, NaCl, MgCl2, CaCl2, FeSO4, FeCl3, and AlCl3 the FRET efficiency is

decreased to 56.8%, 62.15%, 47.4%, 41.1%, 46.8%, 38.5% and 45.6% respectively

from 78.17%. With suitable calibration of these results it is possible to design an ion

sensor that can sense the presence of different ions in water up to a concentration of

10 µM or more. Here also the incorporation of clay platelate laponite enhances the

sensing efficiency.



References 1. C. R. Lowe, Curr. Opin. Chem. Biol. 3 (1999) 106.

2. K. R. Rogers, M. Mascini, Field Anal. Chem. Technol. 2 (1998) 317.

3. J. Janata, M. Josowicz, P. Vanysek, V. De, Anal. Chem. 70 (1998) 179.

4. Y. G. Vlasov, E. A. Bychkov, A. V. Lizgin, Chalcogenide, Talanta 41 (1994) 1059.

5. H. He, H. Li, G. Mohr, B. Kovacs, T. Werner, O. S. Wolfbeis, Anal. Chem. 65

(1993) 123.

6. J. Han, K. Burgess, Chem. Rev. 110 (2010) 2709.

7. H. Wang, D. Wang, Q. Wang, X. Li, C. A. Schalley, Org. Biomol. Chem. 8 (2010)

1017.

8. J. Wang, X. Qian, Chem. Commun. (2006) 109.

9. Q. He, E. W. Miller, A. P. Wong, C. J. Chang, J. Am. Chem. Soc. 128 (2006) 9316.

10. S. C. Dodani, Q. He, C. J. Chang, J. Am. Chem. Soc. 131 (2009) 18020.

11. H. Takakusa, K. Kikuchi, Y. Urano, S. Sakamoto, K. Yamaguchi, T. Nagano, J.

Am. Chem. Soc. 124 (2002) 1653.

12. G. Chen, Y. Jin, L. Wang, J. Deng, C. Zhang, Chem. Commun. 47 (2011) 12500.

13. Z. Zhou, M. Yu, H. Yang, K. Huang, F. Li, T. Yi, C. Huang, Chem. Commun.

(2008) 3387.

14. K. Wang, Z. Tang, C. J. Yang, Y. Kim, X. Fang, W. Li, Y. Wu, C. D. Medley, Z.

Cao, J. Li, P. Colon, H. Lin, W. Tan, Angew. Chem. Int. Ed. 48 (2009) 856.

15. U. H. F. Bunz, V. M. Rotello, Chem. Int. Ed. 49 (2010) 3268.

16. B. Tang, N. Zhang, Z. Chen, K. Xu, L. Zhuo, L. An, G. Yang, Chemistry 14

(2008) 522.

17. B. Y. Wu, H. F. Wang, J. T. Chen, X. P. Yan, J. Am. Chem. Soc. 133 (2011) 686.

18. Y. Chen, M. B. O’Donoghue, Y. F. Huang, H. Kang, J. A. Phillips, X. Chen, M.

C. Estevez, C. J. Yang, W. Tan, J. Am. Chem. Soc. 132 (2010) 16559.

19. S. H. Kim, M. Jeyakumar, J. A. Katzenellenbogen, J. Am. Chem. Soc. 129 (2007)

13254.

20. J. Lei, L. Wang, J. Zhang, J. Chem. Commun. 46 (2010) 8445.

21. J. Chen, F. Zeng, S. Wu, J. Zhao, Q. Chen, Z. Tong, Chem. Commun. (2008)

5580.

22. M. Q. Zhu, L. Zhu, J. J. Han, W. Wu, J. K. Hurst, A. D. Q. Li, J. Am. Chem. Soc.

128 (2006) 4303.



23. E. S. Childress, C. A. Roberts, D. Y. Sherwood, C. L. M. LeGuyader, E. J.

Harbron, Anal. Chem. 84 (2012) 1235.

24. P. D. Sahare, V. K. Sharma, D. Mohan, A. A. Rupasov, Spectrochim. Acta Part A

69 (2008) 1257.


Chapter 9: Design of DNA sensor 89

Chapter-9

Development of a DNA sensor using a

molecular logic gate

This chapter reports the increase in FRET efficiency between

two laser dyes in the presence of deoxyribonucleic acid (DNA).

Two types of molecular logic gates have been designed where

DNA acts as input signal and fluorescence intensity of

different bands are taken as output signal. Use of these logic

gates as a DNA sensor has been demonstrated.

Based on the experimental results of this chapter two papers have been published in peer reviewed reputed journal.

1. Development of a DNA sensor using a molecular logic gate.

D. Bhattacharjee, Dibyendu Dey, S. Chakraborty, S.A. Hussain, S. Sinha

Journal of Biological Physics 39 (2013) 387-394.

2. Molecular Logic Gates using FRET Phenomenon.

Dibyendu Dey, D. Bhattacharjee, S. Chakraborty and Syed Arshad Hussain

Proceedings of the Conference on Recent Trends of Research in Physics (CRTRP-

2012) (ISBN: 978-81-904362-9-8 (2013))



Chapter-9

Development of a DNA sensor using a molecular logic gate 9.1. Introduction The research on molecular logic is initialized by the recognition of a

pioneering work by de Silva et. al. [1]. The change in fluorescence property of a dye

due to the introduction of some external agent could be considered to be analogous to

the digital responses in electronic logic gates. Molecules can undergo changes in

ground or exited state due to the interference of some external chemical or biological

material [2]. This kind of change can be realized by the change in fluorescence

intensity, and the output can be related to the operation of some well known digital

logic gates. Using some basic logic, the molecules can process and manipulate

information as like electronic computers and human brain. Using this concept some

simple logic gates [3] as well as few complex circuitry [4-6] can be constructed, such

as adder/subtractors [7-9], encoders/decoders [10, 11], multiplexers/ demultiplexers

[12-14], and keypad locks [15-19]. There are many molecular logic gates where

chemicals are used as inputs and optical signals are the outputs [20, 21]. Now a days

for the sensing of different organic [22], inorganic [23] and biological [24-26]

materials these molecular logic gates are being used. For the easier understanding of

the outputs of the sensors they are compared with some well known digital logic gates

and from different outputs of those logic gates we can have some idea about the

different structural features and properties of the test sample.

In the present chapter we have investigated FRET between two laser dyes Acf

and RhB. It was observed that the FRET efficiency changes in presence of dsDNA.

Based on this effect we have demonstrated two photoregulated molecular logic gates

viz. NOT and YES-NOT gate. Use of these logic gates as dsDAN sensor has also

been demonstrated.


The DNA used is sheared Salmon sperm DNA having a size of nearly about

2,000 bp with approximate GC content 41.2%, purchased from SRL India and was



used as received. The purity of DNA was checked by UV-Vis absorption and

fluorescence spectroscopy before use. The concentration of DNA aqueous solution

was 1 μg/ml.

9.3. Results and discussion 9.3.1. FRET between Acf and RhB in the presence of DNA


shown in details in the previous chapters of the thesis. Here in this chapter we have

used this same FRET pair for the sensing of DNA. Figure 9.1 shows the fluorescence

spectra of Acf–RhB mixed aqueous solution (1:1 volume ratio) in the presence (curve

2) and in absence (curve 1) of DNA. The DNA concentration was 1 μg/ml. It is

interesting to observe that in the presence of DNA, the RhB fluorescence intensity

increases and the Acf fluorescence intensity decreases further compared to that in the

absence of DNA. This indicates that the presence of DNA influences the extent of

energy transfer.

Fig. 9.1. Fluorescence spectra of Acf–RhB (1:1 volume ratio) mixed aqueous solution

in absence of DNA (1) and in presence of DNA (2). Excitation wavelength was 420

nm (Acf absorption maximum) and concentration of individual dye (pure Acf and

RhB) 10−6 M. DNA concentration was 1 μg/ml.



It has been observed that the FRET efficiency (calculated from Förster theory)

of the dye pair increases from 11.37% (absence of DNA) to 79.1% (presence of

DNA). These data support the increase in energy transfer between Acf and RhB in the

presence of DNA. It is interesting to mention in this context that the FRET process is

distance-dependent and if the intermolecular distance between donor and acceptor

decreases, then the transfer of energy from donor to acceptor becomes very efficient.

FRET is effective over a distance ranging between 1 and 10 nm [27, 28]. Also, an

increase in spectral overlap integral enhances the energy transfer [29, 30]. In DNA,

the nucleotide bases lie horizontally between the two spiraling polymer strands with

negatively charged phosphate backbones attached on either side of the base pair [31,

32]. The distance between two consecutive base pairs is 0.34 nm [33]. In the present

case, both of the dyes Acf and RhB used are cationic. In the presence of DNA, they

are attached with the DNA strands through the electrostatic attraction with the

negatively charged phosphate backbone of DNA. As a result, both of the dyes come

close to each other, resulting in a favorable condition for energy transfer.

Accordingly, the energy transfer efficiency increases in the presence of DNA.

Attachment of the dyes onto the phosphate backbone of DNA has been shown

schematically in Fig. 9.2. It may be mentioned in this context that Shu Wang et al.

reported that the negatively charged DNA bring a close electrostatic interaction with

the cationic water soluble conjugated polymer backbone referring to an efficient

FRET [34]. DNA strands have also been used in FRET-based biosensors, where they

are used as spacers between FRET dye pairs.

Fig. 9.2. (a) Molecular structure of RhB, (b) molecular structure of Acf, (c) structure of DNA showing the negatively charged phosphate deoxyribose backbone, (d) schematic diagram showing the attachment of Acf & RhB onto phosphate backbone of DNA.



9.3.2. Design of molecular logic gates

Based on the efficiency of FRET between Acf and RhB in the presence and

absence of DNA, two types of molecular logic gates have been proposed, namely

NOT and YES/NOT gates. These molecular logic gates, unlike digital counterparts,

sense the presence of a biological material DNA, which acts as an input signal. The

output signal is the fluorescence intensity of a particular band (500 and 578 nm).

Using these logic gates, it is possible to detect the DNA in aqueous solution up to a

very low concentration of 1 μg/ml.

9.3.3. Design of NOT gate as DNA sensor

Based on the spectral characteristic in Fig. 9.1, we have designed the logic

gates. Here we consider the fluorescence intensity of the 500 nm band during FRET

between Acf and RhB as the output signal and presence of DNA as input.

Fluorescence intensity of 400 units (shown in Fig. 9.1) has been chosen as the

reference level. Table 1 shows the logic of the NOT gate. In the absence of DNA

(input = 0), fluorescence intensity at the 500-nm band is greater than the reference

level (output = 1). In the presence of DNA (input = 1), the 500-nm fluorescence band

intensity is less than the reference level (output = 0). Thus, an effective NOT gate can

be developed that can sense the presence of DNA in aqueous solution having a

concentration as low as 1 μg/ml. Thus, by observing the fluorescence intensity of the

500-nm band, it is possible to detect the presence of DNA.

9.3.4. Design of YES-NOT gate as DNA sensor

In this case, the input is similar to that of the NOT gate, where the output

signals are the fluorescence intensities of 500 and 578 nm bands. When the input

signal is zero (absence of DNA), the intensity of the 500 nm band is greater than the

reference level (output = 1) and for the 578 nm band, the fluorescence intensity is less

than the reference level (output = 0). When the input signal is 1 (presence of DNA),

the output of the 500 nm band is 0 whereas the 578 nm band is 1. In this case, the

YES-NOT gate strongly confirms the presence and absence of DNA in the aqueous

solution. Table 9.2 shows the logic of the YES-NOT gate. Here, by comparing the

intensity at 500 nm with the reference level it is possible to detect the presence or

absence of DNA. It is worthwhile to mention in this context that in the present

manuscript the experiments have been done with sheared salmon sperm DNA having

a size of nearly about 2,000 bp with approximate GC content 41.2%. The actual size

of the genomic DNA is approximately 3 × 109 bp, which is sheared to 2,000 bp.



Therefore, the sheared DNA in solution contains a huge number of different kinds of

sequences. In order to check the dependence of experimental results on the specific

sequences of DNA, we have also tested all of the experiments with isolated human

DNA (GC content 40%) and found similar results (result not shown). Therefore, the

working principle of the designed logic gate depends on the interaction of Acf-RhB

with phosphate moiety of DNA and is independent of any specific sequences of DNA.

Using this designed logic gate, only the presence or absence of DNA can be detected.

Input DNA Output (Fluorescence intensity of 500-

nm band)

0 (Absence of DNA) 1 (Fluorescence intensity greater than

reference level)

1 (Presence of DNA) 0 (Fluorescence intensity less than

reference level)

Table 9.1 Function table of NOT gate using fluorescence intensity

Input DNA Output (Fluorescence

intensityof 500-nm band)

Output (Fluorescence

intensityof 578-nm band)

0 (Absence of DNA) 1 (Fluorescence intensity

greaterthan reference

level)

0 (Fluorescence intensity

lessthan reference level)

1 (Presence of DNA) 0 (Fluorescence intensity

lessthan reference level)

1 (Fluorescence intensity

greaterthan reference

level)

Table 9.2 Function table of YES-NOT gate using fluorescence intensity

9.4. Conclusion Based on the experimental observation that the presence of DNA increases the

FRET between the two laser dyes, Acf and RhB, two types of molecular logic gates,

namely a NOT and a YES-NOT gate, have been designed. These two molecular logic

gates have been found to be efficient in detecting the presence of DNA in aqueous

solution having concentrations as low as 1 μg/ml.



References 1. A. P. de Silva, H. Q. N. Gunaratne, C. P. McCoy, Nature 364 (1993) 42.

2. A. M. Dennis, W. J. Rhee, D. Sotto, S. N. Dublin, G. Bao, ACS nano, 6 (2012)

2917.

3. J. P. Sauvage, Acc. Chem. Res. 31 (1998) 611.

4. A. P. de Silva, S. Uchiyama, Nat. Nanotechnol. 2 (2007) 399.

5. K. Szacizowski, Chem. Rev.108 (2008) 3481.

6. J. Andreasson, U. Pischel, Chem. Soc. Rev. 39 (2010) 174.

7. D. Margulies, G. Melman, A. Shanzer, J. Am. Chem. Soc. 128 (2006) 4865.

8. U. Pischel, Angew. Chem. Int. Ed. 46 (2006) 4026.

9. O. A. Bozdemir, R. Guliyev, O. Buyukcakir, S. Selcuk, S. Kolemen, G. Gulseren,

T. Nalbantoglu, H. Boyaci, E. U. Akkaya, J. Am. Chem. Soc. 132 (2010) 8029.

10. J. Andreasson, S. D. Straight, T. A. Moore, A. L. Moore, D. J. Gust, J. Am. Chem.

Soc. 130 (2008) 11122.

11. P. Ceroni, G. Bergamini, V. Balzani, Angew. Chem. Int. Ed. 48 (2009) 8516.

12. P. Inestrosa, E. Montenegro, J.M. Collado, D. Suau, R. Chem. Commun. 11

(2008) 1085.

13. J. Andreasson, S. D. Straight, S. Bandyopadhyay, R. H. Mitchell, T. A. Moore, A.

L. Moore, D. Gust, Angew. Chem. Int. Ed. 46 (2007) 958.

14. M. Amelia, M. Baroncini, A. Credi, Angew. Chem. Int. Ed. 47 (2008) 6240.

15. D. Margulies, C. E. Felder, G. Melman, A. Shanzer, J. Am. Chem. Soc. 129

(2007) 347.

16. G. Strack, M. Ornatska, M. Pita, E. Katz, J. Am. Chem. Soc. 130 (2007) 4234.

17. W. Sun, C. Zhou, C. H. Xu, C. J. Fang, C. Zhang, Z. X. Li, C. H. Yan, Chem. Eur.

J. 14 (2008) 6342.

18. M. Suresh, A. Ghosh, A. Das, Chem. Commun. 48 (2008) 3906.

19. J. Andreasson, S. D. Straight, T. A Moore, A. L. Moore, D. Gust, Chem. Eur. J.

15 (2009) 3936.

20. S. A. Hussain, P. K. Paul, D. Dey, D. Bhattacharjee, S. Sinha, Chem. Phys. Lett.

450 (2007) 49.

21. K. Fujimoto, H. Shimizu, M. Inouye J. Org. Chem. 69 (2004) 3271.

22. H. Liu, Y. Zhou, Y. Yang, W. Wang, L. Qu, C. Chen, D. Liu, D. Zhang, D. Zhu,

J. Phys. Chem. B 112 (2008) 6893.



23. C. T. Clelland, V. Risca, C. Bancroft, Nature 399 (1999) 533.

24. C. Mao, T. H. LaBean, J. H. Reif, N. C. Seeman, Nature 407 (2000) 493.

25. K. Tanaka, A. Okamotoa, I. Saito, BioSystem 81 (2005) 25.

26. P. D. Sahare, V. K. Sharma, D. Mohan, A. A. Rupasov, Spectrochim. Acta, Part A

69 (2008) 1257

27. D. Seth, D. Chakrabarty, A. Chakraborty, N. S. Sarkar, Chem. Phys. Lett. 401

(2005) 546.

28. T. H. Förster, Z. Naturforsch. 4A (1949)321.

29. S. A. Hussain, S. Chakraborty, D. Bhattacharjee, R. A. Schoonheydt,

Spectrochim. Acta, Part A 75 (2010) 664.

30. T. H. Förster, Action of Light and Organic Crystals, Academic Press, New York

(1965).

31. J. Malicka, I. Gryczynski, J. R. Lakowicz, Biochem. Biophys. Res. Commun. 306

(2003) 213.

32. N. Mathur, A. Aneja, P. K. Bhatnagar, P. C. Mathur, J. Sens. 2008 (2008) 1.

33. J. D. Watson, F. H. C. Crick, Nature 171 (1953) 737.

34. S. Wang, B. S. Gaylord, G. C. Bazan, J. Am. Chem. Soc. 126 (2004) 5446.


Chapter10: Overall conclusion and future prospect 97

Chapter-10

Overall conclusion and future prospect

This chapter contains overall conclusion and future prospect of my research.



Chapter-10

Overall conclusion and future prospect

The demand for highly sensitive nonisotopic and noninvasive bioanalysis

systems for biotechnological applications, such as those needed in clinical

diagnostics, food quality control, and drug delivery, has driven research in the use of

FRET for biological and chemical applications. Development of FRET based sensing

system for practical applications is a challenge, requiring an interdisciplinary outlook.

In the present thesis work, I have investigated the FRET between two laser dyes Acf

and RhB in absence and presence of nanoclay laponite in solution and in ultrathin

films. It has been observed that the energy transfer occurred between Acf and RhB in

solution and ultrathin films. Presence of nanoclay laponite increases the efficiency of

energy transfer. In certain cases the energy transfer efficiency increases from 11.37%

to 78.17% in presence of nanoclay laponite. Based on the FRET studies between Acf

and RhB we have demonstrated several sensors: pH sensor, ion sensor, hard water

sensor and DNA sensor.

The FRET efficiency between Acf and RhB was found to be pH sensitive and

vary from 4.5% to 44.45% in mixed dye solution for a change in pH from 3.0 to 12.

With proper calibration it is possible to use the present system to sense pH over a

wide range of pH from 3.0 to 12.0. The advantage of the present system is that it can

be used to sence a wide range of pH compared to the earlier reported FRET based pH

sensors.

It has been demonstrated that presence of hard water components, Ca2+ and

Mg2+ ions affected the FRET efficiency between Acf and RhB. FRET efficiency

decreased in presence of hard water components. We have demonstrated that with

suitable calibration of these results it is possible to design a hard water sensor that can

sense the water hardness of the range 0.03–0.2 mg/ml. It has also been demonstrated

that the incorporation of nano clay laponite enhances the sensing efficiency.

In presence of KCl, NaCl, MgCl2, CaCl2, FeSO4, FeCl3, and AlCl3 the FRET

efficiency between Acf and RhB decreases to 56.8%, 62.15%, 47.4%, 41.1%, 46.8%,

38.5% and 45.6% respectively from 78.17%. With suitable calibration of these results



it is possible to design an ion sensor that can sense the presence of different ions in

water up to a concentration of 10 µM.

It has also been observed that, in presence of DNA the FRET between Acf and

RhB increases. Based on the fact, two types of molecular logic gates, namely a NOT

and a YES-NOT gate, have been designed. These two molecular logic gates have

been found to be efficient in detecting the presence of DNA in aqueous solution

having concentrations as low as 1 μg/ml.

In future I have a plan to extend my research on FRET to design different

biosensors and chemical sensors based on the energy transfer between different new

FRET pairs. Recently I am working on designing of Arsenic sensor and sensing of

DNA sequence. This may lead to a better understanding of the mechanism of drug

delivery systems and conformational study of different biological systems.

Reprints of publications

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Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 174– 182

Contents lists available at SciVerse ScienceDirect

Journal of Photochemistry and Photobiology A:Chemistry

journa l h o me pag e: www.elsev ier .com/ locate / jphotochem

ffect of nanoclay laponite and pH on the energy transfer betweenuorescent dyes

ibyendu Dey, D. Bhattacharjee, S. Chakraborty, Syed Arshad Hussain ∗

epartment of Physics, Tripura University, Suryamaninagar 799022, Tripura, India

r t i c l e i n f o

rticle history:eceived 19 September 2012eceived in revised form4 November 2012ccepted 3 December 2012vailable online 17 December 2012

a b s t r a c t

Fluorescence resonance energy transfer (FRET) between two dyes acriflavine (Acf) and rhodamine B (RhB)were investigated in solution and layer-by-layer (LbL) self assembled films in presence and absence ofclay mineral laponite. UV–Vis absorption and fluorescence spectroscopy studies suggest both the dyespresent mainly as monomer in solution and films. Energy transfer occurred from Acf to RhB in solutionand LbL films. The energy transfer efficiency increases in presence of clay laponite and the maximumefficiency were 92.50% and 55.71% in clay dispersion and in LbL films respectively. Presence of laponite

eywords:luorescence resonance energy transferyeslayaponite

particles onto LbL film was confirmed by atomic force microscopy investigations with a surface coverageof more than 75%. Energy transfer efficiency was pH sensitive and the energy transfer efficiency variesfrom 4.5% to 44.45% in mixed dye solution for a change in pH from 3.0 to 12.0. With proper calibration itis possible to use the present system under investigation to sense pH over a wide range of pH from 3.0to 12.0.

ayer-by-layer

. Introduction

Fluorescence resonance energy transfer (FRET) is an electrody-amic phenomenon that can occur through the transfer of excitedtate energy from donor to acceptor. The theoretical analysis wasell developed by Förster [1,2]. The rate of energy transfer dependspon the extent of spectral overlapping area of the fluorescencepectrum of donor with the absorption spectrum of the acceptor,he relative orientation of the donor and acceptor transition dipolesnd the distance between these molecules [1–3]. Due to its sensi-ivity to distance, FRET has been used to investigate molecular levelnteraction [3–10]. Fluorescence emission rate of energy trans-er has wide applications in biomedical, protein folding, RNA/DNAdentification and their energy transfer process [4–10].

FRET mechanisms are also important to other phenomena,uch as photosynthesis kinetics, chemical reactions and Brown-an dynamics [11,12]. Recently, FRET phenomenon have beenmployed for the conformation of proteins and knowing theirtructure [13], for the detection of spatial distribution and assem-ly of proteins [14], for the designing biosensor [15], for nucleic
cid hybridization [16], distribution and transport of lipids [17].
On the other hand clay platelets are natural nanoparticles withayered structure. Due to the cation exchangability of clay, the

∗ Corresponding author. Tel.: +91 9862804849/381 2375317;ax: +91 3812374802.

E-mail addresses: sa [email protected], [email protected] (S.A. Hussain).

010-6030/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jphotochem.2012.12.003

© 2012 Elsevier B.V. All rights reserved.

cationic dye molecules are adsorbed onto the clay surfaces [18,19].Dye adsorption enhances the concentration of the dye molecules,which may promote their intermolecular physical and chemicalinteractions. For example, if two molecules are in close proxim-ity, fluorescence resonance energy transfer may occur. Probablyfirst record on efficient energy transfer in clay mineral systems,based on the interaction between two different dyes are clay min-eral dispersions with cyanine and rhodamine dyes simultaneouslyadsorbed on clay mineral surfaces [19,20]. Further examples ofenergy transfer in clay mineral systems are triplet–triplet energytransfer from bound sensitizers to mircene [21] and to aromatichydrocarbons adsorbed in hydrophobic organo-clay [22]. It wasobserved that the clay/porphyrin complexes are promising andprospective candidates to be used for construction of efficient arti-ficial light-harvesting system [23]. Czímerová et al. [24] reportedprominent energy transfer among laser dyes in saponite dispersion.The FRET between cationic polypeptide polylysine and cyaninedyes was reported in LbL films of clay minerals [25]. Bujdák et al.studied the FRET between two rhodamines Rh123 (donor) andRh610 (acceptor) in both solution and in presence of nanoclaysaponite (SAP) [26]. It was observed that the FRET efficiency washigher in presence of SAP. The clay mineral works as templates forconcentrating the dyes, accordingly the intermolecular separationbetween them decreases. To avoid the aggregation and the fluo-
rescence self quenching of the dyes, a hydrophobic surfactant wasintroduced which suppressed the aggregation of the dyes [26]. Inanother work by the same group, FRET phenomenon between laserdyes rhodamine 123 (R123), rhodamine 610 (R610), and oxazine
dx.doi.org/10.1016/j.jphotochem.2012.12.003

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dx.doi.org/10.1016/j.jphotochem.2012.12.003

Photo

4m(iwas

trcATBoStBs[6efiaspda

2

2

cammLcsdM

FaiD

D. Dey et al. / Journal of Photochemistry and

(Ox4) has been reported. The dye molecules played the role ofolecular antennas and energy donors (R123), energy acceptors

Ox4), or both (R610). It was observed that the FRET efficiencyncreases in presence of laponite [27]. In one of our previous work

e have observed that the energy transfer efficiency among dyesssembled in Langmuir–Blodgett films increases in presence of clayheets [3].

In the present communication, the FRET phenomenon betweenwo laser dyes acriflavine (Acf) and rhodamine B (RhB) has beeneported. We investigated this phenomenon in aqueous solution,lay dispersion and in layer-by-layer (LbL) self assembled films.lso the effects of pH on the FRET efficiency have been investigated.hese two dyes Acf and RhB are in principle suitable for FRET [28].oth the dyes are highly fluorescent. The fluorescence spectrumf Acf sufficiently overlaps with the absorption spectrum of RhB.ahare et al. [28] observed energy transfer in binary solution mix-ure of acriflavine and rhodamine 6G and acriflavine and rhodamine

by life time measurement. The spectra of Acf are highly pH sen-itive due to the presence of electron donor type functional group29]. Excitation energy transfer between acriflavine and rhodamineG as pH sensor has already been demonstrated [29]. However,ffect of nanoclay platelet laponite as well as pH on energy trans-er using Acf and RhB has never been studied. Therefore it is verynteresting to study the FRET parameters at different pH using Acfs donor in order to explore their possible application as pH sen-or. The aim of this study was to investigate the effect of nanoclaylatelet laponite and pH on FRET efficiency between these twoyes in solution and in LbL films in order to explore their possiblepplications.

. Experimental

.1. Materials

Both the dyes Acf and RhB were purchased from Sigma Chemi-al Co., USA and used as received. Molecular structures of the dyesre shown in the inset of Fig. 1. Millipore water or HPLC gradeethanol [Acros Organics, USA] were used as solvent. The clayineral used in the present work was laponite, obtained from

aponite Inorganic, UK and used as received. The concentration of
lay dispersion was kept 2 ppm throughout the experiment. Theize of the clay platelet is less than 0.05 m and CEC is 0.739 meq/getermined with CsCl [30]. The clay dispersion was prepared inillipore water and stirred for 24 h with a magnetic stirrer followed
ig. 1. Normalized UV–Vis absorption and fluorescence spectra of Acf and RhB inqueous solution. The overlap between Acf fluorescence and RhB absorption spectras shown by shaded region. Inset show molecular structure of (a) RhB and (b) Acf.ye concentration was 10−6 M.

biology A: Chemistry 252 (2013) 174– 182 175

by 30 min ultrasonication before use. Poly(acrylic acid) (PAA) andpoly(allylamine hydrochloride) (PAH) were used as polyanionand polycation during layer-by-layer (LbL) self assembled filmpreparation. Both PAA and PAH were purchased from AldrichChemical Co., USA and was used without further purification.

2.2. Film preparation

Electrolyte deposition bath of cationic dye RhB and Acf were pre-pared with 10−4 M aqueous solution using triple distilled deionized(resistivity 18.2 M cm) Millipore water. The anionic electrolyticbath of PAA was prepared also with triple distilled deionized Mil-lipore water (0.25 mg/ml). LbL self assembled films were obtainedby dipping thoroughly clean fluorescence grade quartz substratealternately in solutions of anionic PAA and oppositely charged RhBand Acf dye mixtures. LbL method utilizes the Van der Walls inter-actions between the quartz slide and PAA as well as charge transferinteraction between PAA and cationic dyes [31,32]. The quartz slidewas dipped in the aqueous solution of PAA for 30 min. Then it wastaken out and sufficient time was allowed for drying and then rins-ing in water bath for 2 min so that the surplus anion attached tothe surface washed off. The dried substrate was then immersedin cationic dye mixture (RhB + Acf) followed by same rinsing pro-cedure. Deposition of PAA and RhB and Acf layers resulted in onebi-layer of self assemble film. The incorporation of clay in the LbLfilm was done with the help of aqueous PAH solution (0.25 mg/ml).For this the quartz slide was first dipped in electrolytic polycation(PAH) aqueous solution for 30 min followed by same rinsing inwater bath and drying procedure and then dipped into the anionicclay dispersion which is again followed by rinsing action in waterbath. The slide thus prepared was dipped in the cationic electrolyticsolution of RhB and Acf. Due to electrostatic interaction cationic Acfand RhB were adsorbed onto the negative charged surface of theclay in LbL films.

2.3. UV–Vis absorption and fluorescence spectra measurement

UV–Vis absorption and steady state fluorescence spectra wererecorded by a Perkin Elmer UV–Vis Spectrophotometer (Lambda-25) and a Perkin Elmer Fluorescence Spectrophotometer (LS-55)respectively. The fluorescence light was collected from the samplesurface at an angle of 45 (front face geometry) and the excitationwavelength was 420 nm.

2.4. Theoretical considerations

Solving the enigma surrounding fluorescence quenching exper-iments revealed the phenomenon of FRET and led Perrin [33] topropose dipole–dipole interactions as the mechanism via whichmolecules can interact without collisions at distances greater thantheir molecular diameters. Some 20 years later, Förster [1,2] builtupon Perrin’s idea to put forward an elegant theory which provideda quantitative explanation for the non-radiative energy transfer interms of his famous expression given by

kT (r) = 1D

(R0

r

)6(1)

where kT(r) is the rate of energy transfer from donor to acceptor,r is the distance between donor and acceptor and R0 is the well-known Förster radius given by the spectral overlap between thefluorescence spectrum of the donor and the absorption spectrum
of the acceptor. The distance at which resonance energy transferis 50% efficient, is called the Förster distance. At r = R0, the transferefficiency is 50% and at this distance the donor emission would bedecreased to half of its intensity in the absence of acceptor.

1 Photo

[

R

wtwdtb

i

J

i

R

w

E

Td

E

Hw

E

wpdt(

aaoc

dtbnf

(

udN

76 D. Dey et al. / Journal of Photochemistry and

The value of R0 can be defined by the following expression34–37]

60 =

[9000(ln 10)k2D

1285Nn4

]∫ ˛

0

FD()εA()4d (2)

here FD the normalized fluorescence intensity of the donor; εA(),he extinction coefficient of the acceptor (in M−1 cm−1); , theavelength (in nm); D, the fluorescence quantum yield of theonor in the absence of acceptor; n, is the refractive index ofhe medium; k2, orientation factor of transition dipole momentetween donor (D) and acceptor (A); N, Avogadro number.

The integral part of Eq. (2) is known as the spectral overlapntegral J() and is given by

() =∫ ˛

0

FD()εA()4d (3)

Therefore the above definition of R0 in Eq. (2) can be rewrittenn terms of J() with units M−1 cm−1 nm4 as

0 = 0.2108k2n−4˚DJ()1/6(4)

here R0 is in units of A0.The energy transfer efficiency can be termed as [34–36]

= kT (r)

kT (r) + −1D

= DkT (r)1 + DkT (r)

(5)

his is the fraction of the transfer rate to the total decay rate of theonor. Using Eqs. (1) and (5) E can be defined as

= R60

R60 + r6

(6)

ere, in the present work the efficiency of the energy transfer (E)as calculated using the equation

= 1 − FDA

FD(7)

here FDA is the relative fluorescence intensity of the donor in theresence of acceptor and FD is the fluorescence intensity of theonor in the absence of the acceptor. This equation is equivalento Eq. (5) [37]. The values of J(), R0 and r are calculated using Eqs.3), (4) and (6).

The fluorescence quantum yield of the donor in the absence ofcceptor (D) has been calculated by using the standard theory [38]nd the calculated value of D is 0.87 for pure acriflavine in aque-us solution. The reported value of for D Acf is very close to thisalculated value [29,39].

The orientation factor (k2) of transition dipole moment betweenonor (D) and acceptor (A) mainly depends on the angle betweenhe transition dipole moments of D and A molecules and the anglesetween each of these two dipole moments with the vector con-ecting their centers [40]. In this case the values of k2 are taken

rom reference [40] and are as follows:

(i) k2 = 2/3 (in case of solution where the dipole moments of theindividual molecules are orientational and rotate by them-selves).

ii) k2 = 0.47 (in case of solid films where the dipole moments of theindividual molecules are orientational but they do not rotate by
themselves).
The value of refractive index (n) of the medium has also beensed from the references. For water solution it is 4/3 [40], for clayispersion it is 1.39 [3], for LbL film it is 1.59 [3] (quartz slide), foraOH solution it is 1.36 [40].

biology A: Chemistry 252 (2013) 174– 182

3. Results and discussion

3.1. The UV–Vis absorption and steady state fluorescencespectroscopy

Normalized UV–Vis absorption and steady state fluorescencespectra of pure Acf and RhB in aqueous solutions are shown in Fig. 1.Both absorption and fluorescence spectra are characteristics of thepresence of monomers. The fluorescence spectra were recorded byexciting the corresponding absorption maxima of Acf and RhB. Theabsorption and fluorescence maxima of Acf are centered at 449 and502 nm respectively which is assigned due to the Acf monomers[28]. Acf monomer absorption band within 444–453 nm depend-ing on the concentration has been reported [41]. For Acf dimer ithas been reported that instead of a single monomer band two bandsat around 437 and 470 nm are observed with the intensity of theblue band higher than the other [41].

On the other hand RhB absorption spectrum possess prominentintense 0–0 band at 553 nm along with a weak hump at 520 nmwhich is assigned due to the 0–1 vibronic transition [42]. Simi-lar reports with RhB monomer bands at 553 nm and 0–1 vibroniccomponents of monomer at 525 nm have been reported. [43]. ForJ-dimer of RhB the absorption bands are found to be red shiftedto 569 and 531 nm [43]. However, for H-dimer the dominanceof 531 nm band intensity with respect to the intensity of 553 nmband have been reported [43,44]. The RhB fluorescence spectrumshows prominent band at 571 nm which is assigned due to the RhBmonomeric emission [42].

A close look in Fig. 1 reveals that there exists sufficient overlap-ping of Acf fluorescence spectrum and RhB absorption spectrum.This justifies the selection of these two dyes in order to quantifyenergy transfer from Acf to RhB. Here Acf acts as a donor and RhBacts as an acceptor. Also both the dyes are highly fluorescent, whichare the prerequisite for FRET to occur [1–3].

3.2. FRET between Acf and RhB

3.2.1. Solution and clay dispersionIn order to investigate the possible FRET between Acf and RhB,

the fluorescence spectra of Acf, RhB and their mixture in differentconditions are measured with exciting wavelength at 420 nm. Theexcitation (absorption) wavelength was selected approximately toexcite the Acf molecules directly and to avoid or minimize the directexcitation of the RhB molecules.

Fig. 2a shows the fluorescence spectra of pure Acf, RhB and theirmixture (50:50 volume ratios) in aqueous solution as well as in claydispersions. Fig. 2a reveals strong prominent Acf fluorescence bandwhere as the RhB fluorescence band is very less in intensity in caseof pure dye solution. The less intensity of pure RhB fluorescenceband indicates very small contribution of direct excitation of theRhB molecules. The fluorescence spectra of Acf–RhB mixture is veryinteresting, here the Acf fluorescence intensity decreases in favorof RhB fluorescence band. This decrease in Acf fluorescence inten-sity is due to the transfer of energy from Acf to RhB molecules. Thistransferred energy excites more RhB molecules followed by lightemission from RhB, which is added to the original RhB fluorescence.As a result the RhB fluorescence intensity gets sensitized. Insetof Fig. 2a shows the excitation spectra measured with excitationwavelength fixed at Acf (500 nm) and RhB (571 nm) fluorescencemaxima in case of Acf–RhB mixed aqueous solution. Interestinglyboth the excitation spectra are almost similar and possess charac-
teristic absorption bands of Acf monomers. This confirms that theRhB fluorescence is mainly due to the light absorption by Acf andcorresponding transfer to RhB monomer. Thus FRET between Acfto RhB has been confirmed.

D. Dey et al. / Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 174– 182 177

Fig. 2. (a) Fluorescence spectra of RhB (1), Acf (2), and Acf + RhB (50:50) mixture (3) in aqueous solution and RhB (4), Acf (5), and Acf + RhB (50:50) mixture (6) in claydispersion. Inset shows the excitation spectra for Acf + RhB mixture with excitation wavelength at 500 (I) and 571 (II) nm. (b) Fluorescence spectra of Acf and RhB mixturei r (Acs 10−6

i(rt(naaisardtfdtb

uwteep

mAaAStR

Also the scattering of light by the clay templates are not same inboth the cases due to difference in orientation of the dye moleculesadsorbed on to clay surface.

Table 1Fluorescence intensity and band position of Acf and RhB mixtures in aqueous solu-tion and in clay dispersion. Excitation wavelength was 420 nm.

1:1 volume ratio ofAcf and RhB

Acf fluorescence RhB fluorescence

n aqueous laponite dispersion at different concentration for fixed amount of donopectra were measured with excitation wavelength 420 nm. Dye concentration was

It is worthwhile to mention in this context that when a molecules excited can transfer its energy to another identical moleculehomo-transfer) or different molecule (hetero-transfer) throughadiative reabsorption, or through direct charge exchange (Dexter-ype transfer), or through non-radiative dipole–dipole couplingFörster-type transfer) [46]. Dexter-type energy transfer is promi-ent when the donor and acceptor are in sub-nanometric distancepart. Radiative re-absorption energy transfer is a trivial case where

real photon emitted by a donor is absorbed by an acceptor. Its the non-trivial energy transfer phenomenon known as FRET orimply RET in which we are interested. [45]. FRET is more sensitivet shorter distances (1–10 nm) between donor and acceptor andadiative transfer is dominant at longer distances [46]. At very closeistances (<1 nm), Dexter-type energy transfer dominates wherehe wave functions of the two entities start overlapping allowingor electron exchange [47]. To avoid the Dexter-type transfer, theistance between donor and acceptor should be >1 nm and radia-ive reabsorption can be ignored by considering a larger distanceetween the donor acceptor pair.

In our present case we have also studied the energy transfersing very dilute solution and from the spectral characteristicse have calculated the donor–acceptor distance which is within

he range of FRET (1–10 nm). This confirms only the non-radiativenergy transfer (FRET) from Acf to RhB. Sahare et al. also studiednergy transfer using same donor–acceptor pair and confirmed therocess as FRET [28].

In order to check the effect of nanoclay platelets on FRET, weeasured the fluorescence spectrum of pure Acf, RhB as well ascf–RhB mixture in laponite clay dispersion [Fig. 2a]. Intensitiesnd band positions for Acf and RhB fluorescence are listed in Table 1.

red shift of RhB fluorescence band of the order of 8 nm occurred.uch smaller shift in RhB fluorescence in montmorillonite and hec-orite were reported and attributed to monomer fluorescence ofhB adsorbed on the external clay surface or in the interlamellar

f). Inset shows the FRET efficiency as a function of acceptor concentration. All theM. For clay dispersion the dye loading was 0.1% of CEC of laponite.

regions of clay sheets [19]. In the present case we also consider thatclay dispersions mainly contain the RhB monomer and the observedshift is due to the consequent adsorption of dye molecules on theclay surface and in the interlamellar space of the clay sheets. Alsothe intensities of both Acf and RhB fluorescence decrease in clay dis-persion. In order to check whether fluorescence self-quenching isconcerned to this phenomenon we have measured the fluorescenceintensity with higher dye concentration and observed that therewas no decrease in fluorescence intensity due to self-quenching.This confirms that there is no fluorescence self-quenching. Similardecrease in fluorescence intensity in presence of clay platelets hasbeen reported and attributed to be due to the scattering of light bythe clay templates [24]. Therefore in the present case the decreasein Acf and RhB fluorescence in presence of clay may be due to thelight scattering by clay templates. It is interesting to mention that,the absorbance and fluorescence intensity of any dye molecule inpresence of clay is very much dependent on the orientation of thedye molecules when adsorbed on to the clay templates [24]. The dif-ference in orientation of Acf and RhB molecules onto clay surfacemay be responsible for a different degree of fluorescence intensity.

Band position(nm)

Intensity Band position(nm)

Intensity

Aqueous solution 502 897 571 58Clay dispersion 502 756 579 49

178 D. Dey et al. / Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 174– 182

Table 2Values of spectral overlap integral (J()), energy transfer efficiency (E%), Försterradius (R0), and donor–acceptor distance (r) calculated from the spectral charac-teristics of Figs. 2a and 3a.

J() × 1015 M−1

cm−1 nm4E (%) R0 (nm) r (nm)

Aqueous solution 32.17 11.37 8.43 8.07Clay dispersion 53.71 78.17 6.60 5.33

idoe

atAmhdocdeoFl

toan

Table 3Values of spectral overlap integral (J()), energy transfer efficiency (E%), Försterradius (R0) and donor–acceptor distance (r) calculated from the spectral charac-teristics of Fig. 2b.

% of acceptor (RhB) J() × 1015 M−1

cm−1 nm4E (%) R0 (nm) r (nm)

20 43.25 32.41 5.87 6.6330 46.87 39.15 6.10 6.5640 50.52 55.02 6.38 6.1650 53.71 78.17 6.60 5.3360 56.72 84.10 6.87 5.2070 60.10 87.20 7.20 5.2380 63.50 90.40 7.53 5.18

F(sd

LbL film without clay 17.25 07.53 2.53 3.84LbL film with clay 25.17 32.54 3.23 3.44

The most interesting observation in clay dispersion was thatn the Acf and RhB mixed system the Acf fluorescence intensityecreases further in favor of RhB fluorescence intensity in presencef nanoclay platelets (Fig. 2a, curve 6), results an increase in FRETfficiency.

It is worthwhile to mention in this context that clay particlesre negatively charged and have layered structure [19,20]. Bothhe dyes Acf and RhB under investigation are positively charged.ccordingly due to the cation exchangeability of clay the dyeolecules are adsorbed onto the clay layers [19,20]. On the other

and FRET process is very sensitive to distances between the energyonor and acceptor and occurs only when the distance is of therder of 1–10 nm [1–3]. Therefore, in the present case, clay parti-les play an important role in determining the concentration of theyes on their surfaces or to make possible close interaction betweennergy donor and acceptor in contrast to the aqueous solution. Inne of our previous work we demonstrated the enhancement ofRET efficiency between two dyes in presence of nanoclay sheetaponite [3].

Analysis of fluorescence spectra (Fig. 2a) reveal that the spec-
ral overlapping integral J() between the fluorescence spectrumf Acf (donor) and absorption spectra of RhB (acceptor) as wells energy transfer efficiency increases due to incorporation ofanoclay sheets (Table 2). Also due to the presence of nanoclay
ig. 3. (a) Fluorescence spectra of RhB (1), Acf (2), Acf + RhB (50:50) mixture (3) in LbL filmb) Fluorescence spectra of Acf and RhB mixture for fixed amount of donor (Acf) and varyhows the variation of FRET efficiency as a function of acceptor concentration. All the speye deposition bath was prepared with dye concentration 10−4 M in aqueous solution. Cl

90 67.20 92.50 7.82 5.14

sheet laponite, the intermolecular distance between Acf and RhBdecreases from 8.07 nm to 5.33 nm. So clay particles play a vitalrole in concentrating the dyes on their surfaces and thus reducingthe intermolecular distance providing a favorable condition for effi-cient energy transfer. Consequently the energy transfer efficiencyincreases from 11.37% to 78.17% in presence of clay platelets (thecalculation procedure is shown in Supporting information).

In order to check the effect of donor/acceptor concentration onFRET, fluorescence spectra of Acf–RhB mixture in presence of clayplatelet laponite were measured with fixed amount of Acf (donor)and varying amount of RhB (acceptor). Fig. 2b shows the fluores-cence spectra of Acf–RhB mixed dye system with fixed amount ofAcf and varying amount of RhB in presence of clay platelet laponite.The values of spectral overlap integral (J()), energy transfer effi-ciency (E), Förster radius (R0) and the donor acceptor distance(r) calculated from Fig. 2b and listed in Table 3. Interestingly itwas observed that for a fixed amount of donor Acf the FRET effi-
ciency increases with the increase in acceptor concentration in theAcf–RhB mixture. Maximum energy transfer efficiency (Table 3)was 92.50% for acceptor concentration of 90%.
without clay and RhB (4), Acf (5), Acf + RhB (50:50) mixture (6) in LbL film with clay.ing amount of acceptor in LbL films in presence of clay particle laponite. The insetctra were measured with excitation wavelength 420 nm. For LbL film preparation

ay concentration for clay deposition bath was 2 ppm.

Photobiology A: Chemistry 252 (2013) 174– 182 179

3

tbAitcievwdlitH

fii(dficwmt

3

sTTdF

Table 4Values of spectral overlap integral (J()), energy transfer efficiency (E%), Försterradius (R0) and donor–acceptor distance (r), calculated from the spectral charac-teristics of Fig. 3b.

% of acceptor (RhB) J() × 1015 M−1

cm−1 nm4E (%) R0 (nm) r (nm)

20 15.33 8.82 2.78 4.1030 18.87 17.50 2.95 3.8140 22.25 26.10 3.10 3.6850 25.17 32.54 3.23 3.4460 28.20 40.20 3.51 3.7570 31.50 47.10 3.82 3.89

D. Dey et al. / Journal of Photochemistry and

.2.2. Layer-by-layer self assembled filmsFig. 3a shows the fluorescence spectra of pure RhB, Acf and

heir mixture (50:50 volume ratios) in 1 bi-layer LbL self assem-led films in presence and absence of clay particles. Here also purecf shows strong fluorescence with monomer band at 523 nm, both

n presence and absence of clay, which is red shifted with respecto aqueous solution or clay dispersion. This shift may be due to thehange in microenvironment when Acf molecules are incorporatednto the polymer (PAH and PAA) backbone in the restricted geom-try of solid surface during LbL film formation. For RhB the trend isery similar to clay dispersion and shows very weak fluorescenceith peak at around 575 nm. Energy transfer is observed for mixedye system in LbL film. However, the energy transfer efficiency is

ess compared to their solution counterpart. This observed decreasen energy transfer efficiency with respect to solution may be due tohe observed small value of the overlap integral (J()) in LbL films.owever, in presence of clay the efficiency increases in LbL films.

Fluorescence spectra of AcF–RhB mixed LbL films prepared withxed amount of Acf and varying amount of RhB in presence of clay

s shown in Fig. 3b. The corresponding energy transfer efficiencyE), Förster radius (R0), spectral overlapping integral (J()) and theistance between the donor and acceptor (r) for Acf–RhB mixed LbLlms are listed in Table 4. Here the trend is very similar to that oflay dispersion. Here also the energy transfer efficiency increasesith increase in acceptor concentration in the mixed films. Theaximum FRET efficiency was 55.71% for an acceptor concentra-

ion of 90% in the mixed LbL films.

.3. Effect of pH on FRET

Among the molecules under current investigation Acf is pH sen-itive because of its basic nature of the central nitrogen atom [48].
he fluorescence spectra of Acf are affected with change in pH [29].his may in turn cause a change in spectral overlapping of theonor fluorescence and acceptor absorbance resulting a change inRET efficiency. In order to check the effect of pH on FRET process,
Fig. 4. Plot of (a) spectral overlap integral J() and (b) e

80 34.70 51.50 4.13 4.0990 37.27 55.71 4.50 4.33

fluorescence spectra of Acf–RhB mixture in aqueous solution pre-pared at different pH were measured (figure available in Supportinginformation). It was observed that the Acf fluorescence was redshifted with decrease in pH.

It is interesting to mention in this context that proflavinemolecule is very similar to acriflavine with regards to protona-tion and deprotonation. Proflavine has been found to exist assingle protonated, double protonated as well as neutral moleculesin aqueous solution with pKa ∼9.5 for single protonated and 0.2for double protonated form [49,50]. The excited state dissociationconstants are 12.5 for single protonated and 1.5 for double proton-ated species. It has been observed that acriflavine mainly remainas double protonated form in nafion (a perfluorosulfonate cationexchange membrane) due to the high local proton concentration[50]. Larger red shift in Acf fluorescence in nafion has been observedand explained due to change in the dipole moments in the excitedstate of the double protonated Acf [49] and due to the broad distri-bution of pKa in nafion matrix [49]. In the present case at lower pH
red shift of Acf fluorescence is observed. At lower pH Acf moleculesmainly remain as double protonated form due to the increase inlocal proton concentration with decreasing pH. Accordingly thedipole moments of the excited state of double protonated Acf have
nergy transfer efficiency (E%) as a function of pH.

180 D. Dey et al. / Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 174– 182

Table 5Values of spectral overlap integral (J()), energy transfer efficiency (E%), Försterradius (R0) and donor–acceptor distance (r), calculated from the fluorescence spectraof aqueous solution of Acf–RhB mixture measured at different pH.

pH of AcF solution J() × 1015 M−1 cm−1 nm4 E (%)

3.0 18.00 04.504.5 23.80 07.206.0 30.10 14.117.5 34.90 21.709.0 39.40 28.50

bf

twIecs

ctboaawpae

bflaietA4F

eoitmdc

hspppic[n7irs

10.5 43.20 35.2012 47.70 44.45

een changed. This change in dipole moments may be responsibleor the observed large stoke shift/red shift of the Acf fluorescence.

The values of J() and FRET efficiencies calculated from the spec-ra measured at different pH are listed in Table 5. Interestingly itas observed that the FRET efficiency increases with increase in pH.

t was found that for the same donor acceptor concentration andxcitation wavelength, the value of spectral overlap integral J()hanges a lot with change in pH. But the shape of the fluorescencepectra remains almost similar.

It is worthwhile to mention in this context that RhB containsCOOH group, which can dissociate in certain conditions to formations–anions (zwitterions). In basic medium RhB shows the zwit-erionic form which could be responsible for the close interactionetween cationic acriflavine (Acf+) and COO− group of zwitteri-nic RhB. This will increase the possibility of closer approach of Acfnd RhB at higher pH resulting an increase in FRET efficiency. Incidic medium (lower pH) RhB generally remains in cationic formith lower pKa value [51]. Also the shift of Acf fluorescence withH results a change in spectral overlap between Acf fluorescencend RhB absorbance, i.e. J() value. This will in turn effect the FRETfficiency.

The electron donor type functional group of Acf become moreasic with increase in pH in the excited state, consequently theuorescence spectra shifts toward shorter wavelength providing

larger value of spectral overlap integral (Table 5) with increas-ng pH. This increase in J() in turn causes an increase in FRETfficiency. The value of J() changes from 18 × 1015 M−1 cm−1 nm4

o 47.7 × 1015 M−1 cm−1 nm4 for change in pH from 3.0 to 12.0.ccordingly the energy transfer efficiency varies from 4.5% to4.45%. Therefore in the present system under investigation theRET process between Acf and RhB is very pH sensitive.

Fig. 4a and b show the plot of spectral overlap integral J() andnergy transfer efficiency (E) as a function of pH. Interestingly it wasbserved that both J() and E increases almost linearly with increas-ng pH. Therefore, pH dependence of the energy transfer betweenhe present donor–acceptor pair Acf and RhB under investigation

akes the system a suitable candidate for sensing pH. Any of theata from Table 5 may be used to sense the pH with appropriatealibration.

It is interesting to mention in this context that energy transferas already been used for pH measurement [29]. Chan et al. demon-trated Förster resonance energy transfer (FRET)-based ratiometricH nanoprobes where they used semiconducting polymer dots as alatform. The linear range for pH sensing of the fluorescein-coupledolymer dots was between pH 5.0 and 8.0 [52]. Egami et al. has

ntroduced a fiber optic pH sensor, using polymer doped with eitherongo red (pH range 3–5) or methyl red (pH range from 5 to 7)53]. pH sensor based on the measurement of absorption of phe-ol red has also been reported [54], which can sense a pH range of
–7.4. In the present system of pH measurement using the change
n FRET parameter with pH is capable of measuring over a wideange of pH 3.0–12.0. This is one advantage with respect to previousystem.

Fig. 5. (a and b) AFM image of Acf–RhB mixed LbL film in presence of clay laponite.

3.4. Atomic force microscopy

To confirm the incorporation of clay particles onto LbL films andto have idea about the structure of the film, LbL film was studiedby atomic force microscope (AFM). Fig. 5a and b show typical AFM

D. Dey et al. / Journal of Photochemistry and Photobiology A: Chemistry 252 (2013) 174– 182 181

ion of

iStacaptltAwwsdoRco

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Fig. 6. Schematic representat

mage of PAH–laponite–Acf–RhB hybrid LbL film deposited on ai substrate along with the line analysis spectrum. In the figure,he laponite particles are clearly visible. The hybrid film consists of

close-packed array of hybridized laponite particles. The surfaceoverage is more than 75%. Few overlapping of laponite particles arelso observed. White spots are indicative of aggregates of laponitearticles; while some uncovered regions are also observed. Fromhe height profile analysis, it is seen that the height of the mono-ayer varies between −2 nm and +2 nm. This includes the height ofhe PAH layer on substrate plus the height of the laponite layer, andcf and RhB molecules adsorbed onto the clay surfaces. It is worth-hile to mention in this context that AFM image of Acf–RhB LbL filmithout clay shows a smooth surface indicating the uniform depo-

ition of dyes without any aggregates (figure not shown). Since theimension of the individual dye molecules are beyond the scopef resolution, hence its not possible to distinguish individual Acf orhB molecules. Therefore, as a whole the AFM investigation giveompelling visual evidence of incorporation of laponite particlesnto the LbL films.

.5. Schematic representation of FRET in LbL

A schematic diagram showing the FRET process between Acfnd RhB in LbL film in presence and absence of nanoclay platelets shown in Fig. 6a and b. In LbL films without clay the anionicolymer (PAA) was attached to the quartz backbone by Van deralls force and then the cationic sample molecules (Acf and RhB)ere attached to the polymer backbone with electrostatic attrac-

ion (Fig. 6a). On the other hand, in presence of clay the cationicolymer (PAH) was first attached to the quartz backbone by Vaner Walls force and then the anionic clay platelets were attachedo the polymer backbone through electrostatic attraction followedy successive adsorption of the cationic sample molecules (Acf andhB) onto negatively charged clay platelets (Fig. 6b). From the spec-roscopic study no aggregation was observed in the LbL complex

oreover the height profile of the AFM image (Fig. 5) also showshat the distribution of the dye molecules is uniform over the LbLomplex. Uniform distribution of the dye molecules in the LbL com-lex are also shown in the schematic diagram.

. Conclusion

Fluorescence resonance energy transfer (FRET) between twouorescent dyes acriflavine and rhodamine B were investigateduccessfully in solution and layer-by-layer (LbL) self assembledlms in presence and absence of clay mineral particle laponite.

the structure of LbL complex.

UV–Vis absorption and fluorescence spectroscopy studies revealthat both the dyes present mainly as monomer in solution and filmsand there exist sufficient overlap between the fluorescence spec-trum of Acf and absorption spectrum of RhB, which is a prerequisitefor the FRET to occur from Acf to RhB. Energy transfer occurred fromAcf to RhB in both solution and LbL films in presence and absence oflaponite. The energy transfer efficiency increases in presence of claylaponite in both solution and in LbL films. The maximum efficien-cies were found to be 92.50% and 55.71% for the mixed dye system(90% RhB + 10% Acf) in clay dispersion and LbL films respectively.Atomic force microscopy investigations confirmed the presence oflaponite particle in LbL films with a surface coverage of more than75%. Due to the basic nature of the central nitrogen atom Acf is pHsensitive and it was observed that the overlap between Acf fluo-rescence and RhB absorption spectrum changes with change in pH.Consequently energy transfer efficiency was found to be pH sen-sitive and varies from 4.5% to 44.45% in mixed dye solution for achange in pH from 3.0 to 12.0. With proper calibration it is possibleto use the present system under investigation to sense pH over awide range of pH from 3.0 to 12.0.

Acknowledgements

The author SAH is grateful to DST, CSIR and DAE for financialsupport to carry out this research work through DST Fast-Track project Ref. No. SE/FTP/PS-54/2007, CSIR project Ref. No.03(1146)/09/EMR-II and DAE Young Scientist Research Award(No. 2009/20/37/8/BRNS/3328). We are grateful to Prof. Robert A.Schoonheydt, K.U. Leuven, Belgium for providing the clay samples.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jphotochem.2012.12.003.

References

[1] T.H. Förster, Experimentelle und theoretische Untersuchung des Zwis-chenmolekularen übergangs von Elektrinenanregungsenergie, Zeitschrift fürNaturforschung 4A (1949) 321–327.

[2] T.H. Förster, in: O. Sinanoglu (Ed.), Modern Quantum Chemistry, Istanbul Lec-
tures, Part III: Action of Light and Organic Crystals, Academic Press, New York,1965.
[3] S.A. Hussain, S. Chakraborty, D. Bhattacharjee, R.A. Schoonheydt, Fluorescenceresonance energy transfer between organic dyes adsorbed onto nano-clay andLangmuir–Blodgett (LB) films, Spectrochimica Acta Part A 75 (2010) 664–670.

http://dx.doi.org/10.1016/j.jphotochem.2012.12.003

http://dx.doi.org/10.1016/j.jphotochem.2012.12.003

1 Photo

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[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

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[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

82 D. Dey et al. / Journal of Photochemistry and

[4] P.M.W. French, Biomedical optics in the 21th century, Biosensors 12 (1999)41–46.

[5] M.J. Cole, J. Siegel, S.E.D. Webb, R. Jones, K. Dowling, M.J. Dayal, D. Parsons, P.M.French, M.J. Lever, L.O. Sucharov, M.A. Neil, R. Juskaitis, T. Wilson, Time-domainwhole-field fluorescence lifetime imaging with optical sectioning, Journal ofMicroscopy 203 (2001) 246–257.

[6] G. Haran, Single-molecule fluorescence spectroscopy of biomolecular folding,Journal of Physics: Condensed Matter 15 (2003) R1291–R1317.

[7] R.B. Best, S.B. Flower, J.L. Toca Herrera, J. Clark, A simple method for pro-bing the mechanical unfolding pathway of proteins in detail, Proceedings ofthe National Academy of Sciences of the United States of America 99 (2002)12143–12148.

[8] B. Zagrovic, C.D. Snow, S. Khaliq, M.R. Shirts, V.S. Pande, Native-like mean struc-ture in the unfolded ensemble of small proteins, Journal of Molecular Biology323 (2002) 153–164.

[9] R.J.H. Clark, R.E. Hester (Eds.), Advances in Spectroscopy, Wiley, New York,1996.

10] M.S. Csele, P. Engs, Fundamentals of Light and Lasers, Wiley, New York, 2004.11] J.M. Drake, J. Klafter, P. Levitz, Chemical and biological microstructures as

probed by dynamic processes, Science 251 (1991) 1574–1579.12] Y. Yilmaz, A. Erzan, Ö. Pekcan, Critical exponents and fractal dimension at the

sol–gel phase transition via in situ fluorescence experiments, Physical ReviewE 58 (1998) 7487–7491.

13] T. Jonsson, C.D. Waldburger, R.T. Sauer, Nonlinear free energy relationships inArc repressor unfolding imply the existence of unstable, native-like foldingintermediates, Biochemistry 35 (1996) 4795–4802.

14] B.S. Watson, T.L. Hazlett, J.F. Eccleston, C. Davis, D.M. Jameson, A.E. Johnson,Macromolecular arrangement in the aminoacyl-tRNA-elongation factor Tu-GTP ternary complex. A fluorescence energy transfer study, Biochemistry 34(1995) 7904–7912.

15] D. Bhattacharjee, D. Dey, S. Chakraborty, S.A. Hussain, S. Sinha, Developmentof a DNA sensor using molecular logic gate, Journal of Biological Physics,http://dx.doi.org/10.1007/s10867-012-9295-3

16] K.M. Parkhurst, L.J. Parkhurst, Kinetic studies by fluorescence resonance energytransfer employing a double-labeled oligonucleotide: hybridization to theoligonucleotide complement and to single-stranded DNA, Biochemistry 34(1995) 285–292.

17] J.W. Nichols, R.E. Pagano, Resonance energy transfer assay of protein-mediatedlipid transfer between vesicles, Journal of Biological Chemistry 258 (1983)5368–5371.

18] S.A. Hussain, R.A. Schoonheydt, Langmuir–Blodgett monolayers of cationicdyes in the presence and absence of clay mineral layers: N,N′-dioctadecylthiacyanine, octadecyl rhodamine B and laponite, Langmuir 26 (2010)11870–11877.

19] R.A. Schoonheydt, Smectite-type clay minerals as nanomaterials, Clays and ClayMinerals 50 (2002) 411–420.

20] R.H.A. Ras, B. van Duffel, M. Van der Auweraer, F.C. De Schryver, R.A.Schoonheydt, Proceedings of the 12th International Clay Conference, BahiaBlanca, Argentina, 2001.

21] D. Madhavan, K. Pitchumani, Efficient triplet–triplet energy transfer using clay-bound ionic sensitizers, Tetrahedron 58 (2002) 9041–9044.

22] M.G. Neumann, H.P.M. Oliveira, A.P.P. Cione, Energy transfer between aromatichydrocarbons dissolved in a C18-surfactant layer adsorbed on laponite, Adsorp-tion 8 (2002) 141–146.

23] Y. Ishida, T. Shimada, D. Masui, H. Tachibana, H. Inoue, S. Takagi, Efficientexcited energy transfer reaction in clay/porphyrin complex toward an artificiallight harvesting system, Journal of the American Chemical Society 133 (2011)14280–14286.

24] A. Czímerová, J. Bujdak, N. Iyi, Fluorescence resonance energy transfer betweenlaser dyes in saponite dispersions, Journal of Photochemistry and PhotobiologyA 187 (2007) 160–166.

25] A. Czímerová, N. Iyi, J. Bujdák, Energy transfer between rhodamine 3B andoxazine 4 in synthetic-saponite dispersions and films, Journal of Colloid andInterface Science 306 (2007) 316–322.

26] J. Bujdák, D. Chorvát, N. Iyi, Resonance energy transfer between rhodamine
molecules adsorbed on layered silicate particles, Journal of Physical ChemistryC 114 (2010) 1246–1252.
27] J. Bujdák, A. Czímerová, F. López Arbeloa, Two-step resonance energy transferbetween dyes in layered silicate films, Journal of Colloid and Interface Science364 (2011) 497–504.

[

[

biology A: Chemistry 252 (2013) 174– 182

28] P.D. Sahare, V.K. Sharma, D. Mohan, A.A. Rupasov, Energy transfer studiesin binary dye solution mixtures: acriflavine + rhodamine 6G and acri-flavine + rhodamine B, Spectrochimica Acta Part A 69 (2008) 1257–1264.

29] V. Misra, H. Mishra, H.C. Joshi, T.C. Pant, Excitation energy transfer betweenacriflavine and rhodamine 6G as a pH sensor, Sensors and Actuators B 63 (2000)18–23.

30] T. Szabo, R. Mitea, H. Leeman, G.S. Premachandra, C.T. Johnston, I.M. Szekeres,R.A. Schoonheydt, Adsorption of protamine and papine proteins on saponite,Clays and Clay Minerals 56 (2008) 494–504.

31] G. Decher, Fuzzy nanoassemblies: toward layered polymeric multicomposites,Science 277 (1997) 1232–1237.

32] D. Dey, S.A. Hussain, R.K. Nath, D. Bhattacharjee, Preparation and char-acterization of an anionic dye-polycation molecular films by electrostaticlayer-by-layer adsorption process, Spectrochimica Acta Part A 70 (2008)307–312.

33] J. Perrin, Fluorescence et induction moleculaire par resonance, Comptes RendusHebdomadaires des Seances de l Academie des Sciences 184 (1927) 1097–1100.

34] T. Förster, Modern Quantum Chemistry. Part 2. Action of Light and OrganicMolecules, Academic, New York, 1965.

35] J.R. Lakovitz, Principles of Fluorescence Spectroscopy, Kluwer Aca-demic/Plenum, New York, 1999.

36] M. Pope, C.E. Swenberg, Electronic Processes in Organic Crystals, Oxford Uni-versity Press, New York, 1982.

37] D. Seth, D. Chakrabarty, A. Chakraborty, N.S. Sarkar, Study of energy trans-fer from 7-amino coumarin donors to rhodamine 6G acceptor in non-aqueousreverse micelles, Chemical Physics Letters 401 (2005) 546–552.

38] A.T.R. Williams, S.A. Winfield, J.N. Miller, Relative fluorescence quantum yieldsusing a computer controlled luminescence spectrometer, Analyst 108 (1983)1067–1071.

39] K.K. Pandey, T.C. Pant, Diffusion-modulated energy transfer, Chemical PhysicsLetters 170 (1990) 244–252.

40] D. Ghosh, D. Bose, D. Sarkar, N. Chattopadhyay, Excited-state-proton-transfer-triggered fluorescence resonance energy transfer: from 2-naphthylamine tophenosafranin, Journal of Physical Chemistry A 113 (2009) 10460–10465.

41] J.L. Rosenberg, F.S. Humphries, Oxygen quenching of acriflavine phosphores-cence, Journal of Physical Chemistry 71 (1967) 330–338.

42] S.A. Hussain, S. Banik, S. Chakraborty, D. Bhattacharjee, Adsorption kinetics ofa fluorescent dye in a long chain fatty acid matrix, Spectrochimica Acta Part A79 (2011) 1642–1647.

43] T. Fujii, H. Nishikiori, T. Tamura, Absorption spectra of rhodamine B dimers indip-coated thin films prepared by the sol–gel method, Chemical Physics Letters233 (1995) 424–429.

44] K. Kemnitz, N. Tamai, I. Yamazaki, N. Nakashima, K. Yohsihara, Fluorescencedecays and spectral properties of rhodamine B in submono- mono-, and mul-tilayer systems, Journal of Physical Chemistry 90 (1986) 5094–5101.

45] E.J. Bowen, B. Brocklehurst, Energy transfer in hydrocarbon solutions, Transac-tions of the Faraday Society 49 (1953) 1131–1133.

46] S.R. Bobbara, Energy transfer between molecules in the vicinity of metalnanoparticle, PhD Thesis, June 2011.

47] D.L. Dexter, A theory of sensitized luminescence in solids, Journal of ChemicalPhysics 21 (1953) 836.

48] V.K. Sharma, P.D. Sahare, R.C. Rastogi, S.K. Ghoshal, D. Mohan, Excited statecharacteristics of acridine dyes: acriflavine and acridine orange, Spectrochi-mica Acta Part A 59 (2003) 1799–1804.

49] M.P. Pileni, M. Grätzel, Light-induced redox reactions of proflavine in aqueousand micellar solution, Journal of Physical Chemistry 84 (1980) 2402–2406.

50] C.B. Airaudo, A.G. Sorbier (Eds.), Marseille, France, Fluorescence and its analyt-ical Applications in Pharmacy (Translated from Labo-Pharma, Nos. 248, 250,251), 1980, p. 77.

51] I.L. Arbeloa, K.K.R. Mukherjee, Solvent effect on photophysics of the molec-ular forms of rhodamine B. Solvation models and spectroscopic parameters,Chemical Physics Letters 128 (1986) 474–479.

52] Y.H. Chan, C. Wu, F. Ye, Y. Jin, P.B. Smith, D.T. Chiu, Development of ultrabrightsemiconducting polymer dots for ratiometric pH Sensing, Analytical Chemistry83 (2011) 1448–1455.

53] C. Egami, Y. Suzuki, O. Sugihora, H. Fujimura, N. Okamoto, Wide range pHfiber sensor with congo-red and methyl-red-doped poly(methyl methacrylate)cladding, Japanese Journal of Applied Physics 36 (1997) 2902–2905.

54] J.I. Peterson, S.R. Goldstein, R.V. Fitzgerald, D.K. Buckhold, Fiber optic pH probefor physiological use, Analytical Chemistry 52 (1980) 864–869.

http://dx.doi.org/10.1007/s10867-012-9295-3

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Sensors and Actuators B 184 (2013) 268– 273

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators B: Chemical

journa l h om epage: www.elsev ier .com/ locate /snb

evelopment of hard water sensor using fluorescence resonance energy transfer

ibyendu Dey, D. Bhattacharjee, S. Chakraborty, Syed Arshad Hussain ∗

epartment of Physics, Tripura University, Suryamaninagar 799022, Tripura, India

a r t i c l e i n f o

rticle history:eceived 30 December 2012eceived in revised form 17 April 2013ccepted 18 April 2013vailable online 26 April 2013

a b s t r a c t

A method is presented for the sensing of water hardness by determining the concentration of calciumand magnesium in water, based on fluorescence resonance energy transfer (FRET) process. The principleof the proposed sensor is based on the change of FRET efficiency between two laser dyes Acriflavine(Acf) and Rhodamine B (RhB) in presence of permanent hard water components (CaCl2 and MgCl2).Nanodimensional clay platelet laponite was used to enhance the efficiency of the sensor.

eywords:ard waterensorluorescence resonance energy transferyeslay aponite
. Introduction

“Hard water” has high mineral content compared to “soft water”.enerally the hard water is not harmful to one’s health, but canause serious problems in industrial settings, where water hardnesshould be monitored to avoid breakdowns of the costly equip-ents that handle water. The hardness of water is determined

y the concentration of multivalent cations (positively chargedetal complexes with a charge greater than 1+) in water. Theost common cations found in hard water include Ca2+ and Mg2+.

he presence of dissolved carbonate minerals (CaCO3 and MgCO3)rovide a temporary hardness in water, which can be reducedither by boiling the water or by addition of lime (calcium hydrox-de) [1]. On the other hand the dissolved chloride minerals (CaCl2nd MgCl2) cause the permanent hardness of water that cannot beemoved easily, because it becomes more soluble as the tempera-ure increases [2]. In that sense it is very important to identify theermanent hardness of water before use. One of the most usefulteps to water analysis is the determination of the concentrationf calcium and magnesium ions, whether individually or overallardness. In the routine laboratories the volumetric methods arehe most commonly used methods for water analysis. Now a days
he involvement of absorption or fluorescence spectroscopy forater analysis has received particular attention [3]. Sweetser andncker were the first who used the spectroscopic measurements to
∗ Corresponding author. Tel.: +91 3812375317; fax: +91 3812374802;obile: +91 9862804849.

E-mail addresses: sa [email protected], [email protected] (S.A. Hussain).

925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.snb.2013.04.077

determine the concentration of calcium and magnesium ionsin water [3]. Ion chromatography (IC) is another very powerfulmethod for the analyses of anions and cations in aqueous solution[4,5]. Argiiello and Fntz reported a method for the separation of Ca2+

and Mg2+ in hard water samples based on ion-chromatography andspectroscopic method [6]. Gömez et al. reported a method for thesimultaneous spectroscopic determination of calcium and magne-sium using a diode-array detector [7].

The fluorescence resonance energy transfer (FRET) phe-nomenon may be very effective tool for the designing of hard watersensors. Based on the FRET between two laser dyes here we demon-strated a hard water sensor. To the best of our knowledge this couldbe the first attempt, where FRET process has been used for thedetection of the hardness of water. FRET between two molecules isan important physical phenomenon, where transfer of energy froman excited fluorophore to a suitable acceptor fluorophore occurred[8,9]. This technique is very important for the understanding ofsome biological systems and has potential applications in optoelec-tronic and thin film devices [10–14]. Combining FRET with opticalmicroscopy, it is possible to determine the approach between twomolecules within nanometers. The main requirements for the FRETto occur are (i) sufficient overlap between the absorption band ofacceptor fluorophore and the fluorescence band of donor fluoro-phore and (ii) both the donor and acceptor molecule must be inclose proximity of the order of 1–10 nm [8,9]. The intervening ofsolvent or other macromolecules has little effect on the FRET effi-
ciency. If the distance between the donor and acceptor changesthen FRET efficiency also changes.
Here in the process of designing hard water sensor basedon FRET process, we have used two dyes Acriflavine (Acf) and

dx.doi.org/10.1016/j.snb.2013.04.077


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dx.doi.org/10.1016/j.snb.2013.04.077

ctuators B 184 (2013) 268– 273 269

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Fig. 1. Fluorescence spectra of Acf + RhB (1:1 volume ratio) in water solution (1),with MgCl2 (2), CaCl2 (3), and CaCl2 + MgCl2 (4), pure Acf (5), pure RhB (6). Dye con-

−6

D. Dey et al. / Sensors and A

hodamine B (RhB) as energy donor and acceptor. In principle bothhe dyes are suitable for fluorescence resonance energy transfer.oth the dyes are highly fluorescent and the fluorescence spectrumf Acf sufficiently overlaps with the absorption spectrum of RhB.ahare et al. [15] reported the fluorescence resonance energy trans-er in binary solution mixture using these two dyes. In one of ourarlier work we have demonstrated a pH sensor based on the FRETetween Acf and RhB [16]. The energy transfer efficiency has beenffected if the distance between the donor and acceptor pair haseen altered due to the presence of any external agency or changef the microenvironment. It has been observed that when distanceetween fluorophores (dyes) is decreased due to adsorption on toanoclay sheet, the FRET efficiency increases [17,18].

In the present communication we tried to investigate the effectf Mg2+ or Ca2+ or both on the FRET efficiency between two fluo-ophores, Acf and RhB in presence of nanoclay sheet laponite. Heree have chosen Mg2+ or Ca2+ because the presence of these two

ations mainly determines the extent of hardness of the water. Ournvestigation showed that FRET efficiency decreases with increas-ng salt concentration. It has also been demonstrated that withroper calibration, FRET between Acf and RhB can be used to sensehe hardness of water.

. Materials and methods

.1. Material

Both the dyes Acriflavine (Acf) and Rhodamine B (RhB) wereurchased from Sigma Chemical Co., USA and used as received.ltrapure Milli-Q water (resistivity 18.2 M cm) was used as sol-ent. The dyes used in our studies are cationic in nature. The clayineral used in the present work was Laponite, obtained from

aponite Inorganic, UK and used as received. The size of the claylatelet is less than 0.05 m and CEC is 0.739 meq/g determinedith CsCl [19]. Both MgCl2 and CaCl2 were purchased from Thermo

isher Scientific India Pvt. Ltd. and used as received. Dye solutionsere prepared in Milli-Q water. For spectroscopic measurement

he solution concentration was optimized at 10−6 M. The clay dis-ersion was prepared using Millipore water and stirred for 24 hith a magnetic stirrer followed by 30 min ultrasonication beforese. The concentration of clay was kept fixed at 2 ppm through outhe experiment. To check the effect of clay on the spectral char-cteristics the dye solutions (Acf and RhB) were prepared in thelay suspensions (2 ppm). The dye concentration was maintainedt 10−6 M. In order to check the effect of salt on spectral character-stics in presence of clay, first of all the salts were added in the clayispersion at different concentration. Then the dyes were added inhe salt mixed clay dispersions. In all cases the clay concentrationas 2 ppm and the dye concentration was 10−6 M.

.2. UV–vis absorption and fluorescence spectra measurement

UV–vis absorption and fluorescence spectra of the solutionsere recorded by a Perkin-Elmer Lambda-25 Spectrophotometer

nd Perkin-Elmer LS-55 Fluorescence Spectrophotometer, respec-ively. For fluorescence measurement the excitation wavelengthas 420 nm (close to the absorption maxima of Acf).

. Results and discussion

.1. UV–vis absorption and steady state fluorescence spectra
tudy
The absorption and emission maxima of Acf are centered at49 and 502 nm, respectively which is assigned due to the Acf

centration was 10 M and salt concentration was 0.06 mg/ml. (For interpretation ofthe references to color in this figure legend, the reader is referred to the web versionof this article.)

monomers [15]. On the other hand RhB absorption spectrumpossess prominent intense 0–0 band at 553 nm along with a weakhump at 520 nm which is assigned due to the 0–1 vibronic transi-tion [20]. The RhB fluorescence spectrum shows prominent band at571 nm which is assigned due to the RhB monomeric emission [20].The corresponding absorption and emission spectra of the aboveresults are shown in figure 1 of the supporting information.

3.2. FRET between Acf and RhB in aqueous solution with salt

To study the energy transfer between Acf and RhB the fluo-rescence spectra of Acf and RhB mixture (1:1 volume ratio) weremeasured with excitation wavelength fixed at 420 nm (close to theabsorption monomer of Acf). Fig. 1 shows the fluorescence spec-tra of Acf, RhB and their mixture in water solution in presence andabsence of salt. From the figure it was observed that the fluores-cence intensity of pure Acf (curve 5 of Fig. 1) is much higher, on theother hand the fluorescence intensity of pure RhB (curve 6 of Fig. 1)is almost negligible. This is because the excitation wavelength(420 nm) was chosen in order to excite the Acf molecule directlyand to avoid the direct excitation of the RhB molecule. However, theAcf–RhB mixture fluorescence spectrum is very interesting. Herethe Acf emission decreases with respect to pure Acf and on theother side RhB emission increases with respect to pure RhB (curve1 of Fig. 1). This is mainly due to the transfer of energy from Acfmolecule to RhB molecule via fluorescence resonance energy trans-fer. In order to confirm this, excitation spectra were recorded withmonitoring emission wavelength 500 nm (Acf emission maximum)and 571 nm (RhB emission maximum) and observed that both theexcitation spectra are very similar to the absorption spectrum of Acfmonomer (figure 2 of supporting information). This confirms thatthe RhB fluorescence is mainly due to the light absorption by Acf
and corresponding transfer to RhB monomer. Thus FRET betweenAcf and RhB has been confirmed.
Our main purpose is to detect CaCl2 and MgCl2 or their mixtureas a permanent hard water component in aqueous solution using

2 ctuators B 184 (2013) 268– 273

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Fig. 2. Fluorescence spectra of Acf + RhB (1:1 volume ratio) in clay suspension (1),with MgCl2 (2), CaCl2 (3), and CaCl2 + MgCl2 (4) pure Acf with clay (5), pure RhB with

70 D. Dey et al. / Sensors and A

he FRET process between Acf and RhB. There are few studies wherehe effect of some chloride salts in FRET has been studied. Felbert al. studied the effect of NaCl on FRET between CFP and YFP [21].he effect of chloride ions and similar halide ions results a decreasen FRET efficiency at pH close to its pKa value [22,23]. Yoshioka et al.tudied the self assembly of HsRed51 by measuring the FRET fromhe fluorescein-labeled protein to the Rhodamine-labeled proteinhich is dependent strongly on high salt concentration indicat-

ng the ionic interaction between positively and negatively chargedminoacids [24].

Here we have studied the effect of CaCl2 and MgCl2 and theirixture on the transfer of energy from Acf to RhB in their aqueous

olution. Fluorescence spectra of Acf–RhB mixture in presence ofgCl2, CaCl2 and their mixture (CaCl2 + MgCl2) are also shown in

ig. 1 (curves 2–4). For all the spectra shown in Fig. 1 the concentra-ion of CaCl2/MgCl2/their mixture was kept at 0.06 mg/ml, which ishe initializing concentration of hard water known as moderatelyard water [1,2]. From Fig. 1 it was observed that the transfer ofnergy from Acf to RhB was decreased in presence of salt (curves–4). Based on the fluorescence spectra the fluorescence energyransfer efficiency have been calculated using the following equa-ion [25],

= 1 − FDA

FD

here FDA is the fluorescence intensity of the donor in the presencef acceptor and FD is the fluorescence intensity of the donor in thebsence of the acceptor.

It has been observed that the FRET efficiency in aqueous solu-ion is 11.37% which decreases to 5.2% and 1.7% for the presence of

gCl2 and CaCl2, respectively. The aqueous solutions of the saltsenerate the cationic Ca2+ and Mg2+ ions. The laser dyes Acf andhB both are cationic in nature and repeal each other in aque-us solution. The introduction of cationic Ca2+ and Mg2+ ions inhe solution may cause an increase in the electrostatic repulsionetween Acf and RhB molecules which can result in a large inter-olecular separation. Accordingly, the FRET efficiency decreases.n the other hand the diameter of Ca2+ ion is larger in compared

o Mg2+ ion which could be responsible for a small but noticeableariation in FRET between Acf and RhB in presence of Ca2+ and Mg2+

ons. It is also observed that with the increase in salt concentrationhe transfer of energy from Acf to RhB decreases further (figure nothown). In presence of both Ca2+ and Mg2+ ions, the FRET efficiencylso decreases and the efficiency is 4.38% which lies in between thefficiencies for the presence of either Ca2+ or Mg2+.

.3. FRET between Acf and RhB in clay dispersion with salt

Our previous investigations revealed that the energy transferccurred from Acf to RhB in aqueous solution. Also in presence ofalt (Ca2+ or Mg2+) the energy transfer efficiency decreases. How-ver the energy transfer efficiency as well as the change in efficiencyn presence of salt is very small due to the large intermolecular sep-ration between Acf and RhB. In the present study our aim is toesign a sensor which can sense the presence of Ca2+ or Mg2+ oroth by observing the change in FRET efficiency. Accordingly it isery important to have large FRET efficiency between Acf and RhBs well as noticeable change in FRET efficiency between Acf andhB due to the introduction of hard water components (Ca2+ org2+), so that we can use it as a hard water sensor with minimum

rror level. Here in the designed sensor the hardness of the sampleater will be sensed just by observing the change in the FRET effi-

iency. Accordingly in order to enhance the FRET efficiency we havencorporated nano clay laponite in Acf–RhB mixture. It has beenbserved that the FRET efficiency increases in presence of laponitearticle.

clay (6). Dye concentration was 10−6 M and clay concentration was 2 ppm and saltconcentration was 0.06 mg/ml. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

It is important to mention in this context that in one of ourearlier works it has been observed that the presence of nanoclaylaponite increased the FRET efficiency between N,N′-dioctadecylthiacyanine perchlorate (NK) and octadecyl rhodamine B chloride(RhB) [18,17]. Effect of nanoclay laponite on the energy transfer effi-ciency between Acf and RhB has also been studied [16]. It has beenobserved that the presence of clay platelet increases the energytransfer efficiency. Fluorescence spectra of Acf, RhB and their mix-ture in absence and presence of salt in aqueous clay dispersion areshown in Fig. 2. Here in presence of laponite the FRET efficiency hasincreased to 78.17% which was 11.37% in absence of clay. This hasbeen evidenced from the observed decrease of Acf fluorescence infavor of RhB fluorescence intensity in presence of nanoclay platelets(curve-1 of Fig. 2).

It is worthwhile to mention in this context that clay particlesare negatively charged and have layered structure with a cationexchange capacity [26,27]. Both the dyes Acf and RhB under inves-tigation are positively charged. Accordingly they are adsorbed on tothe clay layers [26,27]. On the other hand FRET process is very sensi-tive to distances between the energy donor and acceptor and occursonly when the distance between the D and A pair is of the orderof 1–10 nm [8,9]. Therefore in the present case, clay particles playan important role in determining the concentration of the dyes ontheir surfaces or to make possible close interaction between energydonor and acceptor in contrast to the pure aqueous solution. Nowour main purpose is to observe the change in FRET between Acf andRhB in clay dispersion due to the introduction of CaCl2/MgCl2/theirmixture. From Fig. 2 (curves 2–4) it was observed that the transferof energy from Acf to RhB decreases quite remarkably due to theintroduction of CaCl2/MgCl2/their mixture for the concentration of0.06 mg/ml in presence of clay. It was observed that the transfer ofenergy is much smaller due to the presence of CaCl2 in compare to
MgCl2 (Table 1 summarize the calculated efficiencies).
The decrease in FRET between Acf and RhB in presence ofhard water components, must involve the reaction of the cations(Ca2+ and Mg2+) present in hard water with the clay minerals

D. Dey et al. / Sensors and Actuato

Table 1Values of energy transfer efficiency (E%) for Acf and RhB mixture (1:1 volume ratio)in different conditions. The salt concentration was 0.06 mg/ml (moderately hardwater). Dye concentration was 10−6 M and clay concentration was 2 ppm.

Samples E%

Acf + RhB 11.37Acf + RhB + CaCl2 1.7Acf + RhB + MgCl2 5.2Acf + RhB + MgCl2 + CaCl2 4.38Acf + RhB + clay 78.17Acf + RhB + CaCl2 with clay 37.78

tttotpMccmbcmcM

3

iFAldf(

that FRET efficiency decreases with increasing salt concentration

Acf + RhB + MgCl2 with clay 51.59Acf + RhB + MgCl2 + CaCl2 with clay 48.18

hrough cation exchange reaction. Marshall [28] formulated thathe replacement of cations on a charged clay mineral surface byhose present in a contact solution takes place according to therder of bonding energy of the common metal cations. In general,his bonding energy is of the order: Ca > Mg > K > H > Na. Thus therobability of adsorption of Ca2+ in clay suspension is more thang2+. The tendency of Ca2+ ion to interact with the negatively

harged clay layers is more compared to Mg2+ ion of same con-entration of both clay suspension and salt solution. Accordingly,ost of the negative charges in the clay surfaces are neutralized

y Ca2+ ion compare to Mg2+ ion and there exists very few unoc-upied negative charges on the clay surface for the cationic dyeolecules to be adsorbed. As a result the separation between the

ationic dye molecules increases more in CaCl2 solution rather thangCl2 leading to a less FRET in presence of CaCl2 compare to MgCl2.

.4. Schematic diagram

A schematic diagram showing the organization at Acf and RhBn absence and presence of clay laponite and salt is shown inig. 3. Normally in absence of clay and salt the distance betweencf and RhB molecules in aqueous solution is larger resulting

ower energy transfer efficiency (Fig. 3a). In presence of clay theyes are adsorbed by cation exchange reaction on to the clay sur-ace and accordingly the distance between Acf and RhB decreasesFig. 3b). These results can increase the energy transfer efficiency. In

Fig. 3. Schematic representation of FRET betwee

rs B 184 (2013) 268– 273 271

presence of both clay and salt, the probability of adsorption ofCa2+ and Mg2+ cations are larger in compared to the cationic dyes(Fig. 3c). This is because in the process of dye–clay–salt solutionpreparation initially the salt was added to the clay dispersionfollowed by the dye addition. Accordingly, most of the negativecharges on the clay surface are neutralized by the Ca2+ and Mg2+

cations and there exist very few unoccupied negative charges onthe clay surface to adsorb the cationic dyes. Accordingly the aver-age distance between Acf and RhB become larger. This results adecrease in energy transfer efficiency.

3.5. Effect of variation of salt concentration on FRET efficiency

In the previous sections we have seen that presence of laponiteparticle increases the FRET efficiency between Acf and RhB.Whereas, presence of Mg2+ or Ca2+ or both decreases the FRETefficiency. Now in order to check the effect of variation of salt con-centration (extent of hardness) on the FRET efficiency, we havemeasured the fluorescence spectra of Acf and RhB mixture withdifferent salt (MgCl2, CaCl2, and MgCl2 + CaCl2 mixture) concentra-tion in presence of clay laponite and the FRET efficiency have beencalculated. It has been observed that the FRET efficiency decreaseswith increasing salt concentration of either MgCl2 or CaCl2, or theirmixture.

Fig. 4 shows the fluorescence spectra of Acf–RhB mixturein presence of clay with varying amount of salt (MgCl2 + CaCl2mixture) concentration viz. 0.05 mg/ml (soft water), 0.06 mg/ml(moderately hard water) and 0.12 mg/ml (very hard water). Fromthe figure it has been observed that the RhB fluorescence intensitydecreases with increase in salt concentration. Also the correspond-ing Acf fluorescence intensity increases. This indicates that withincrease in salt concentration FRET efficiency between Acf andRhB decreases. The plot of FRET efficiency as a function of salt(MgCl2 + CaCl2 mixture) concentration (inset of Fig. 4) clearly shows

(ranging from 0.03 mg/ml to 0.2 mg/ml). The values of FRET efficien-cies with salt concentration are listed in Table 2. This result suggeststhat it is possible to sense the hardness of water by observing thechange in FRET efficiency with salt concentration.

n Acf and RhB in presence of clay and salt.

272 D. Dey et al. / Sensors and Actuators B 184 (2013) 268– 273

Fig. 4. Fluorescence spectra of Acf + RhB (1:1 volume ratio) in clay dispersion withCaCl2 + MgCl2 of concentration 0.05 mg/ml (1), 0.06 mg/ml (2) and 0.12 mg/ml (3).Inset shows the variation in FRET efficiency as a function of salt concentration ofeither MgCl2 or CaCl2, or CaCl2 + MgCl2 mixture from 0.03 mg/ml to 0.20 mg/ml. Dyeconcentration was 10−6 M and clay concentration was 2 ppm. MgCl2 + CaCl2 mixturew(r

3

shcfsrflt

(tt

0r4c1aot

TVaeoc

as prepared by adding 1:1 volume ratio of individual salts of same concentration.For interpretation of the references to color in this figure legend, the reader iseferred to the web version of this article.)

.6. Design of hard water sensor

Based on the variation of FRET efficiency or fluorescence inten-ity, depending on the salt concentration we have demonstrated aard water sensor. In the process of hard water sensing first of alllay (laponite) dispersion will be prepared using the sample waterollowed by addition of dyes (Acf and RhB). Then the fluorescencepectra of the solution will be measured. By observing the fluo-escence intensity or FRET efficiency calculated from the observeduorescence spectra it would be possible to sense the hardness ofhe sample water.

Fig. 5 shows the plot of FRET efficiency as a function of saltMgCl2 + CaCl2 mixture) concentration for three different concen-rations viz. 0.05 mg/ml, 0.06 mg/ml and 0.12 mg/ml. The data areaken from spectra shown in Fig. 4.

From Fig. 5 it has been observed that the FRET efficiency for.06 mg/ml and 0.12 mg/ml concentration are 48.2% and 13.5%,espectively. If the FRET efficiency is observed to be higher than8.2%, then the water will be recognized as soft water (salt con-entration <0.06 mg/ml) whereas, if the efficiency lies in between3.5% and 48.2% then the water will be recognized as moder-
tely hard (0.06 mg/ml < salt concentration < 0.12 mg/ml). On thether hand if the observed FRET efficiency is less than 13.5% thenhe water will be recognized as very hard (salt concentration
able 2alues of energy transfer efficiency (E%) for Acf and RhB mixture (1:1 volume ratio)t different salt (MgCl2, CaCl2, and MgCl2 + CaCl2 mixture) concentration in pres-nce of clay. MgCl2 and CaCl2 mixture were prepared by adding 1:1 volume ratiof individual salts of same concentration. Dye concentration was 10−6 M and clayoncentration was 2 ppm.

Salt concentration (mg/ml) FRET efficiency (E%) in presence of

MgCl2 CaCl2 MgCl2 + CaCl2mixture

0.03 78.52 64.27 73.730.05 71.55 55.32 68.340.06 51.59 37.78 48.180.08 32.47 18.75 21.570.12 19.67 10.54 13.480.20 11.34 5.67 07.38

Fig. 5. FRET efficiency of Acf and RhB mixture for the different concentration ofCaCl2 + MgCl2 in presence of clay (values of FRET efficiencies were calculated fromthe spectra of Fig. 4).

>0.12 mg/ml). Therefore with proper calibration it is possible todesign a hard water sensor which can sense hard water veryeasily.

It is interesting to mention that in principle this method canbe used to sense negative ions as well as other cation also. Thepresence of negative ions in between the cationic dye pair candecrease their intermolecular separation and the FRET efficiencywill increase. Whereas, in presence of cation the intermoleculardistance between the dyes will increase, resulting decrease in FRETefficiency. Again for the ions (anion/cation) having same valencythe intermolecular distance between the dyes will be governed onthe basis of their bonding energies as well as their size. Thereforewith proper calibration and selecting suitable FRET pair it is possi-ble to sense both negative as well as positive ions using this method.Detailed investigations are required for this. Work is going on in ourlaboratory in this line.

4. Conclusion

Fluorescence resonance energy transfer (FRET) between twofluorescent dyes Acriflavine and Rhodamine B were investigatedsuccessfully in solution in presence and absence of clay mineralparticle laponite. UV–vis absorption and fluorescence spectroscopystudies reveal that both the dyes present mainly as monomer insolution and there exist sufficient overlap between the fluores-cence spectrum of Acf and absorption spectrum of RhB, which isa prerequisite for the FRET to occur from Acf to RhB. Energy trans-fer occurred from Acf to RhB in solution in presence and absenceof laponite. The energy transfer efficiency increases in presence ofclay laponite in solution. The maximum efficiency was found tobe 78.17% for the mixed dye system (50% RhB + 50% Acf) in claydispersion. In presence of CaCl2 or MgCl2 the FRET efficiency isdecreased to 37.78% and 51.59%, respectively. With suitable cali-bration of these results it is possible to design a hard water sensorthat can sense the water hardness of the range 0.03–0.2 mg/ml.

Acknowledgements

The author SAH is grateful to DST, CSIR and DAE for finan-cial support to carry out this research work through DST
Fast-Track project Ref. No. SE/FTP/PS-54/2007, CSIR project Ref.03(1146)/09/EMR-II and DAE Young Scientist Research Award (No.2009/20/37/8/BRNS/3328). The author SC is grateful to CSIR forfinancial support to carry out this research work through SRF Award(No. 09/714(0014)/2012-EMR-I).

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ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.snb.2013.04.077.

eferences

[1] H. Weingärtner, Water in Ullmann’s Encyclopedia of Industrial Chemistry,Wiley-VCH, Weinheim, 2006 (December).

[2] T.J. Sorg, R.M. Schock, D.A. Lytle, Ion exchange softening: effects on metal con-centrations, Journal of the American Water Works Association 91 (1999) 85–97.

[3] P.B. Sweetser, C.E. Bncker, Spectrophotometric titrations with ethylenedi-aminetetraacetic acid: determination of magnesium, calcium, zinc, cadmium,titanium, and zirconium, Analytical Chemistry 26 (1954) 195–199.

[4] H. Small, T.S. Stevens, W.C. Bauman, Novel ion exchange chromatographicmethod using conductimetric detection, Analytical Chemistry 47 (1975)1801–1809.

[5] P.R. Haddad, P.E. Jackson, Ion Chromatography—Principles and Applications,Elsevier, Amsterdam, 1991.

[6] M.D. Argiiello, J.S. Fntz, Ion-exchange separation and determination of calciumand magnesium, Analytical Chemistry 49 (1977) 1595–1597.

[7] E. Gómez, J.M. Estela, V. Cerdà, Simultaneous spectrophotometric determina-tion of calcium and magnesium in water, Analytica Chimica Acta 249 (1991)513–518.

[8] T.H. Förster, Experimentelle und theoretische Untersuchung des Zwis-chenmolekularen übergangs von Elektrinenanregungsenergie, Zeitschrift furNaturforschung 4A (1949) 321–327.

[9] T.H. Förster, Transfer mechanisms of electronic excitation, Discussions of theFaraday Society 27 (1959) 7–71.

10] G. Haran, Topical review single-molecule fluorescence spectroscopy ofbiomolecular folding, Journal of Physics: Condensed Matter 15 (2003)R1291–R1317.

11] R.B. Best, S.B. Fowler, J.L. Toca Herrera, J. Clarke, A simple method for probing themechanical unfolding pathway of proteins in detail, Proceedings of the NationalAcademy of Sciences of the United States of America 99 (2002) 12143–12148.

12] B. Zagrovic, C.D. Snow, S. Khaliq, M.R. Shirts, V.S. Pande, Native-like mean struc-ture in the unfolded ensemble of small proteins, Journal of Molecular Biology323 (2002) 153–164.

13] R.J.H. Clark, R.E. Hester, Spectroscopy of Inorganic based Materials Advances inSpectroscopy, Wiley, New York, 1996.

14] M.S. Csele, P. Engs, Fundamentals of Light and Lasers, Wiley, New York, 2004.15] P.D. Sahare, V.K. Sharma, D. Mohan, A.A. Rupasov, Energy transfer studies

in binary dye solution mixtures: Acriflavine + Rhodamine 6G and Acri-flavine + Rhodamine B, Spectrochimica Acta, Part A 69 (2008) 1257–1264.

16] D. Dey, D. Bhattacharjee, S. Chakraborty, S.A. Hussain, Effect of nanoclaylaponite and pH on the energy transfer between fluorescent dyes, J. Photochem.Photobiol. A-Chemistry 252 (2013) 174–182.

17] S.A. Hussain, S. Chakraborty, D. Bhattacharjee, R.A. Schoonheydt, Fluorescenceresonance energy transfer between organic dyes adsorbed onto nano-clay andLangmuir–Blodgett (LB) films, Spectrochimica Acta, Part A 75 (2010) 664–670.

18] S.A. Hussain, R.A. Schoonheydt, Langmuir–Blodgett monolayers of cationic dyes
in the presence and absence of clay mineral layers: N,N′-dioctadecyl thiacya-nine, octadecyl rhodamine B and laponite, Langmuir 26 (2010) 11870–11877.
19] T. Szabo, R. Mitea, H. Leeman, G.S. Premachandra, C.T. Johnston, M. Szekeres,I. Dekany, R.A. Schoonheydt, Adsorption of protamine and papine proteins onsaponite, Clays and Clay Minerals 56 (2008) 494–504.

rs B 184 (2013) 268– 273 273

20] S.A. Hussain, S. Banik, S. Chakraborty, D. Bhattacharjee, Adsorption kinetics ofa fluorescent dye in a long chain fatty acid matrix, Spectrochimica Acta, Part A79 (2011) 1642–1647.

21] L.M. Felber, S.M. Cloutier, C. Kündig, T. Kishi, V. Brossard, P. Jichlinski, H.J.Leisinger, D. Deperthes, Evaluation of the CFP-substrate–YFP system for pro-tease studies: advantages and limitations, Biotechniques 36 (2004) 878–885.

22] R.M. Wachter, S.J. Remington, Sensitivity of the yellow variant of green fluo-rescent protein to halides and nitrate, Current Biology 9 (1999) 628–629.

23] S. Jayaraman, P. Haggie, R.M. Wachter, S.J. Remington, A.S. Verkman, Mech-anism and cellular applications of a green fluorescent protein-based halidesensor, Journal of Biological Chemistry 275 (2000) 6047–6050.

24] K. Yoshioka, Y.Y. Yoshioka, F. Fleury, M. Takahashi, pH- and salt-dependentself-assembly of human Rad51 protein analyzed as fluorescence resonanceenergy transfer between labeled proteins, Journal of Biochemistry 133 (2003)593–597.

25] D. Seth, D. Chakrabarty, A. Chakraborty, N. Sarkar, Study of energy transfer from7-amino coumarin donors to rhodamine 6G acceptor in non-aqueous reversemicelles, Chemical Physics Letters 401 (2005) 546–552.

26] R.A. Schoonheydt, Smectite-type clay minerals as nanomaterials, Clays and ClayMinerals 50 (2002) 411–420.

27] R.H.A. Ras, B. van Duffel, M. Van der Auweraer, F.C. De Schryver, R.A.Schoonheydt, Proceedings of the 12th International Clay Conference, BahiaBlanca, Argentina, 2001.

28] C.E. Marshall, Multifunctional ionization as illustrated by the clay minerals,Clays and Clay Minerals 327 (1954) 364–385.

Biographies

Mr. Dibyendu Dey (M.Sc. 2009, Tripura University, India) is working as a ResearchScholar in Department of Physics, Tripura University. His major fields of interestare fluorescence resonance energy transfer in solution and ultrathin films. He haspublished 3 research papers in international journals and attended several scientificconferences in India.

Prof. D. Bhattacharjee (M.Sc., Kalyani University and Ph.D., IACS, India) is a Professorin the Department of Physics, Tripura University, India. His major fields of interestare preparation and characterization of ultra thin films by Langmuir–Blodgett & self-assembled techniques. He visited Finland and Belgium for postdoctoral research. Hehas undertaken several research projects. He has published more than 60 researchpapers in different national and international journals and attended several scien-tific conferences in India and abroad.

Mr. Sekhar Chakraborty (M.Sc. 2006, Tripura University, India) is working as aSenior Research Fellow in Department of Physics, Tripura University. His majorfields of interest are preparation and characterization of organo-clay hybrid ultrathin films by Langmuir–Blodgett & self-assembled techniques. He has published 8research papers in reputed international journals and attended several national andinternational scientific conferences in India.

Dr. S.A. Hussain (M.Sc. 2001 and Ph.D., 2007, Tripura University, India) is an Assis-tant Professor in the Department of Physics, Tripura University. His major fields ofinterest are thin films and nanoscience. He was a Postdoctoral Fellow of K.U. Leu-
ven, Belgium (2007–2008). He received Jagdish Chandra Bose Award 2008–2009,TSCST, Govt. of Tripura; Young Scientist Research Award by DAE, Govt. of India.He has undertaken several research projects. He has published 50 research papersin international journals and attended several scientific conferences in India andabroad.
http://dx.doi.org/10.1016/j.snb.2013.04.077

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J Biol Phys (2013) 39:387–394

DOI 10.1007/s10867-012-9295-3

ORIGINAL PAPER

Development of a DNA sensor using a molecular

logic gate

D. Bhattacharjee · Dibyendu Dey · S. Chakraborty ·Syed Arshad Hussain · S. Sinha

Received: 11 October 2012 / Accepted: 22 November 2012 / Published online: 9 February 2013

© Springer Science+Business Media Dordrecht 2013

Abstract This communication reports the increase in fluorescence resonance energy

transfer (FRET) efficiency between two laser dyes in the presence of deoxyribonucleic acid

(DNA). Two types of molecular logic gates have been designed where DNA acts as input

signal and fluorescence intensity of different bands are taken as output signal. Use of these

logic gates as a DNA sensor has been demonstrated.

Keywords FRET · DNA · Molecular logic gate · Sensor

1 Introduction

Recent research on molecular logic gate was initiated after the pioneering work of de

Silva [1] using the idea that molecules can be used for processing information just

like the electronic logic gates. Remarkable progress has been made since then in the

development of molecular logic gates [2–14]. The change in fluorescence character-

istics of a dye due to the introduction of some external agent can be considered to

be analogous to the digital responses in electronic logic gates. Molecules can undergo

changes in ground or exited state due to the interference of some external chemical

or biological materials [15]. In most cases, these kinds of changes can be realized

in terms of the basic operations of logic gates using the familiar Boolean logic [16].

The development of molecular systems such as logic gates and circuits is of immense

interest among the researchers that pursue advanced technologies. Basic logic operation

of a molecular logic gate is the same as that of the electronic one, the only differ-

ence is in the input and output signal. Nowadays, these molecular logic gates can

D. Bhattacharjee · D. Dey · S. Chakraborty · S. A. Hussain (B)

Thin Film and Nanoscience Laboratory, Department of Physics, Tripura University,

Suryamaninagar, 799022, Tripura, India

e-mail: [email protected]

S. Sinha

Department of Botany, Tripura University, Suryamaninagar, 799022, Tripura, India

388 D. Bhattacharjee et al.

be used as simple as well as more complex devices [17–19] such as adders and sub-

tractors [2–4], multiplexers/demultiplexers [5–7], encoders/decoders [8, 9], keypad locks

[10–14] etc. Among many applications of molecular logic gates, one of the most interesting

is the investigation of the inside components of a cell, where silicon-based analogues are

not expected to reach [2]. To have some structural and functional ideas about different

biological materials like DNA, RNA, and proteins, etc., the development of techniques for

sensing and monitoring them is in great demand [20]. A homogeneous sensing ensemble,

based on DNA quadruplexes, was reported by Margulies et al. [21] where the sensing

ensemble can provide a direct analysis of the properties of the target proteins.

In designing nanosized devices, the issue of connectivity plays an important role. The

strength of semiconductor devices depends on this connectivity where the output from one

nanosized device is used to control the input of another. Whereas in the case of the molecular

logic gate, it is not easy to pass the output of one gate to serve as an input to the next due

to the difference in nature and properties of output-input characteristics. From this point of

view, it must be stressed that the molecular logic gate or computation need not follow the

conventional semiconductor blue print.

Using some basic logic, molecules can process and manipulate information like elec-

tronic computers and the human brain. There are many molecular logic gates where chemi-

cals are used as inputs and optical signals are the outputs [22]. For the sensing of different

organic [23], inorganic [24], and biological [21, 25] materials, these molecular logic gates

are now being extensively used. To better understand the outputs of the sensors, they are

compared with some well-known digital logic gates and from different outputs of those

logic gates we can have some idea about the different structural features of the test sample.

Fujimoto et al. reported the detection of target DNAs by excimer-monomer switching

of pyrene using the fluorescence resonance energy transfer (FRET) process [26]. A DNA-

based nanomachine was reported by Liu et al. using the FRET phenomenon [20]. Also for

encrypting messages on DNA strands, various methods have been accomplished [27–29].

The present communication reports the effect of DNA on fluorescence resonance energy

transfer (FRET) between two dyes acriflavine (Acf) and rhodamine B (RhB). Acf and

RhB are in principle suitable for energy transfer. Both the dyes are highly fluorescent.

Fluorescence spectrum of Acf overlaps with absorption spectrum of RhB. By using this

FRET process, we are able to construct a photo-regulated fluorescence switch. The output

of the switch is mimicking the electronic NOT and YES/NOT logic gates. This kind

of “ON–OFF” switching of fluorescence intensity can be varied by the introduction of

photochemically active biomolecule DNA. It has been observed that the incorporation of

DNA in the FRET pair modulates the FRET efficiency. This has been used to design the

molecular logic gate, which is capable of sensing the presence of DNA.

2 Experimental section

Both acriflavine (Acf) and rhodamine B (RhB) were purchased from Sigma Chemical Co.,

USA and were used as received. Ultrapure Milli-Q water (resistivity 18.2 M-cm) was

used as solvent. The DNA used is sheared Salmon sperm DNA having a size of nearly

about 2,000 bp with approximate GC content 41.2%, purchased from SRL India and was

used as received. The purity of DNA was checked by UV-Vis absorption and fluorescence

spectroscopy before use. UV-Vis absorption and fluorescence spectra of the solutions were

recorded by a Perkin Elmer Lambda-25 absorption spectrophotometer and Perkin Elmer

Development of a DNA sensor using a molecular logic gate 389

LS-55 fluorescence spectrophotometer, respectively. For fluorescence measurement, the

excitation wavelength was 420 nm. The concentration of the individual dye in aqueous

solution was 10−6

M. In order to create the mixed dye solution, the dye solutions were

mixed with 1:1 volume ratio.

3 Results and discussion

3.1 FRET between Acf and RhB in aqueous solution

Figure 1 shows the fluorescence spectra of pure Acf (curve 1), pure RhB (curve 2), and

their mixture of 1:1 volume ratio (curve 3) in aqueous solution. Spectra shown in Fig. 1

were recorded with excitation wavelength 420 nm (close to the monomer absorption of

Acf). This excitation wavelength was selected in order to avoid the direct excitation of the

RhB molecules. With this excitation wavelength, Acf shows prominent fluorescence with

peak at 500 nm (curve-1 of Fig. 1), whereas RhB fluorescence intensity is almost negligible

with a very weak peak at 578 nm (curve-2 of Fig. 1). From the spectral characteristics, it

has been observed that both the Acf and RhB are mainly present as monomers in aqueous

solution. However, for the fluorescence spectra of Acf-RhB mixed solution (curve 3), the

Fig. 1 Fluorescence spectra of

Acf (1), RhB (2), and Acf + RhB

(3) (1:1 volume ratio) in water

solution. Excitation wavelength

was 420 nm (Acf absorption

maximum) and concentration of

individual dye (pure Acf and

RhB) 10−6

M. Inset (1) shows

the normalized absorption

spectrum of rhodamine B and

fluorescence spectrum of

acriflavine in water solution and

inset (2) shows the excitation

spectra for Acf+RhB mixture

with emission wavelengths at

500 nm (a) and 578 nm (b)


RhB fluorescence intensity increases even with this excitation wavelength (420 nm) as well

as Acf fluorescence decreases compared to their pure counterpart. This may be due to the

transfer of energy from Acf to RhB. This transferred energy excites more RhB molecules

followed by light emission from RhB, which is added to the original RhB fluorescence. As

a result, the RhB fluorescence intensity gets sensitized. In order to confirm this, we measure

the excitation spectra with emission wavelength fixed at Acf (500 nm) and RhB (578 nm)

fluorescence maximum in case of Acf-RhB mixed aqueous solution (inset 2 of Fig. 1).

Interestingly, both of the excitation spectra are almost similar and possess characteristic

absorption bands of Acf monomers. This confirms that the RhB fluorescence in case of

Acf–RhB mixed solution is mainly due to the light absorption by Acf and corresponding

transfer to RhB monomer. Thus, FRET between Acf to RhB has been confirmed.

3.2 FRET between Acf and RhB in the presence of DNA

Figure 2 shows the fluorescence spectra of Acf–RhB mixed aqueous solution (1:1 volume

ratio) in the presence (curve 2) and in absence (curve 1) of DNA. The DNA concentration

was 1 μg/ml. It is interesting to observe that in the presence of DNA, the RhB fluorescence

intensity increases and the Acf fluorescence intensity decreases further compared to that

in the absence of DNA. This indicates that the presence of DNA influences the extent of

energy transfer.

Fig. 2 Fluorescence spectra of

Acf–RhB (1:1 volume ratio)

mixed aqueous solution in

absence of DNA (1) and in

presence of DNA (2). Excitation

wavelength was 420 nm (Acf

absorption maximum) and

concentration of individual dye

(pure Acf and RhB) 10−6

M.

DNA concentration was 1 μg/ml


Based on the fluorescence spectra of Figs. 1 and 2, the fluorescence energy transfer

efficiency has been calculated using the following equation [30]

E = 1 − FDA

FD

where FDA is the relative fluorescence intensity of the donor in the presence of acceptor and

FD is the fluorescence intensity of the donor in the absence of the acceptor.

It has been observed that the FRET efficiency of the dye pair increases from 11.37%

(absence of DNA) to 79.1% (presence of DNA). These data support the increase in energy

transfer between Acf and RhB in the presence of DNA. It is interesting to mention in

this context that the FRET process is distance-dependent and if the intermolecular distance

between donor and acceptor decreases, then the transfer of energy from donor to acceptor

becomes very efficient. FRET is effective over a distance ranging between 1 and 10 nm

[31]. Also, an increase in spectral overlap integral enhances the energy transfer [32, 33].

In DNA, the nucleotide bases lie horizontally between the two spiraling polymer strands

with negatively charged phosphate backbones attached on either side of the base pair

[34, 35]. The distance between two consecutive base pairs is 0.34 nm [36]. In the present

case, both of the dyes Acf and RhB used are cationic. In the presence of DNA, they

are attached with the DNA strands through the electrostatic attraction with the negatively

charged phosphate backbone of DNA. As a result, both of the dyes come close to each

other, resulting in a favorable condition for energy transfer. Accordingly, the energy transfer

efficiency increases in the presence of DNA. Attachment of the dyes onto the phosphate

backbone of DNA has been shown schematically in Fig. 3. It may be mentioned in this

context that Shu Wang et al. reported that the negatively charged DNA bring a close

electrostatic interaction with the cationic water soluble conjugated polymer backbone

referring to an efficient FRET [37]. DNA strands have also been used in FRET-based

biosensors, where they are used as spacers between FRET dye pairs.

Fig. 3 a Molecular structure of rhodamine B, b molecular structure of acriflavine, c structure of DNA

showing the negatively charged phosphate deoxyribose backbone, d schematic diagram showing the

attachment of Acf & RhB onto phosphate backbone of DNA


3.3 Design of molecular logic gates

Based on the efficiency of FRET between Acf and RhB in the presence and absence of

DNA, two types of molecular logic gates have been proposed, namely NOT and YES/NOT

gates. These molecular logic gates, unlike digital counterparts, sense the presence of a

biological material DNA, which acts as an input signal. The output signal is the fluorescence

intensity of a particular band (500 and 578 nm). Using these logic gates, it is possible to

detect the DNA in aqueous solution up to a very low concentration of 1 μg/ml.

3.4 Design of NOT gate as DNA sensor

Based on the spectral characteristic in Fig. 2, we have designed the logic gates. Here we

consider the fluorescence intensity of the 500-nm band during FRET between Acf and RhB

as the output signal and presence of DNA as input. Fluorescence intensity of 400 units

(shown in Fig. 2) has been chosen as the reference level. Table 1 shows the logic of the

NOT gate. In the absence of DNA (input = 0), fluorescence intensity at the 500-nm band

is greater than the reference level (output = 1). In the presence of DNA (input = 1), the

500-nm fluorescence band intensity is less than the reference level (output = 0). Thus,

an effective NOT gate can be developed that can sense the presence of DNA in aqueous

solution having a concentration as low as 1 μg/ml. Thus, by observing the fluorescence

intensity of the 500-nm band, it is possible to detect the presence of DNA.

3.5 Design of YES-NOT gate as DNA sensor

In this case, the input is similar to that of the NOT gate, where the output signals are the

fluorescence intensities of 500- and 578-nm bands. When the input signal is zero (absence of

DNA), the intensity of the 500-nm band is greater than the reference level (output = 1) and

for the 578-nm band, the fluorescence intensity is less than the reference level (output = 0).

When the input signal is 1 (presence of DNA), the output of the 500-nm band is 0 whereas

the 578-nm band is 1. In this case, the YES-NOT gate strongly confirms the presence and

absence of DNA in the aqueous solution. Table 2 shows the logic of the YES-NOT gate.

Here, by comparing the intensity at 500 nm with the reference level it is possible to detect

the presence or absence of DNA.

It is worthwhile to mention in this context that in the present manuscript the experiments

have been done with sheared salmon sperm DNA having a size of nearly about 2,000 bp

with approximate GC content 41.2%. The actual size of the genomic DNA is approximately

3 × 109 bp, which is sheared to 2,000 bp. Therefore, the sheared DNA in solution contains

a huge number of different kinds of sequences. In order to check the dependence of

experimental results on the specific sequences of DNA, we have also tested all of the

experiments with isolated human DNA (GC content 40%) and found similar results (result

not shown). Therefore, the working principle of the designed logic gate depends on the

interaction of Acf-RhB with phosphate moiety of DNA and is independent of any specific

Table 1 Function table of NOT gate using fluorescence intensity

Input DNA Output (Fluorescence intensity of 500-nm band)

0 (Absence of DNA) 1 (Fluorescence intensity greater than reference level)

1 (Presence of DNA) 0 (Fluorescence intensity less than reference level)


Table 2 Function table of YES-NOT gate using fluorescence intensity

Input DNA Output (Fluorescence intensity Output (Fluorescence intensity

of 500-nm band) of 578-nm band)

0 (Absence of DNA) 1 (Fluorescence intensity greater 0 (Fluorescence intensity less

than reference level) than reference level)

1 (Presence of DNA) 0 (Fluorescence intensity less 1 (Fluorescence intensity greater

than reference level) than reference level)

sequences of DNA. Using this designed logic gate, only the presence or absence of DNA

can be detected.

4 Conclusions

Based on the experimental observation that the presence of DNA increases the fluorescence

resonance energy transfer (FRET) between the two laser dyes, acriflavine (Acf) and

rhodamine B (RhB), two types of molecular logic gates, namely a NOT and a YES-NOT

gate, have been designed. These two molecular logic gates have been found to be efficient in

detecting the presence of DNA in aqueous solution having concentrations as low as 1 μg/ml.

Acknowledgement The author SAH is grateful to DST, CSIR, and DAE for financial support to carry

out this research work through DST Fast-Track project Ref. No. SE/FTP/PS-54/2007, CSIR project Ref.

03(1146)/09/EMR-II, and DAE Young Scientist Research Award (No. 2009/20/37/8/BRNS/3328).

References

1. de Silva, P.A., Gunaratne, N.H.Q., Mccoy, C.P.: A molecular photoionic and gate based on fluorescent

signaling. Nature 364, 42–44 (1993)

2. Margulies, D., Melman, G., Shanzer, A.: A molecular full-adder and full-subtractor, an additional step

toward a moleculator. J. Am. Chem. Soc. 128, 4865–4871 (2006)

3. Pischel, U.: Chemical approaches to molecular logic elements for addition and subtraction. Angew.

Chem. Int. Ed. 46, 4026–4040 (2007)

4. Bozdemir, O.A., Sozmen, F., Büyükcakir, O., Guliyev, R., Cakmak, Y., Akkaya, E.U.: Reaction-based

sensing of fluoride ions using built-in triggers for intramolecular charge transfer (ICT) and photoinduced

electron transfer (PET). Org. Lett. 12, 1400–1403 (2010)

5. Andreasson, J., Straight, S.D., Bandyopadhyay, S., Mitchell, R.H., Moore, T.A., Moore, A.L., Gust, D.:

Molecular 2:1 digital multiplexer. Angew. Chem. Int. Ed. 46, 958–961 (2007)

6. Amelia, M., Baroncini, M., Credi, A.: A simple unimolecular multiplexer/demultiplexer. Angew. Chem.

Int. Ed. 47, 6240–6243 (2008)

7. Perez-Inestrosa, E., Montenegro, J.M., Collado, D., Suau, R.: Molecular 1:2 demultiplexer. Chem.

Commun. A 9, 1085–1087 (2008)

8. Andreasson, J., Straight, S.D., Moore, T.A., Moore, A.L., Gust, D.: Molecular all-photonic encoder-

decoder. J. Am. Chem. Soc. 130, 11122–11128 (2008)

9. Ceroni, P., Bergamini, G., Balzani, V.: Old molecules, new concepts: [Ru(bpy)3]2+

as a molecular

encoder-decoder. Angew. Chem. Int. Ed. 48, 8516–8518 (2009)

10. Margulies, D., Felder, C.E., Melman, G., Shanzer, A.: A molecular keypad lock: a photochemical device

capable of authorizing password entries. J. Am. Chem. Soc. 129, 347–354 (2007)

11. Strack, G., Ornatska, M., Pita, M., Katz, E.: Biocomputing security system: concatenated enzyme-based

logic gates operating as a biomolecular keypad lock. J. Am. Chem. Soc. 130, 4234–4235 (2008)

12. Sun, W., Zhou, C., Xu, C.H., Fang, C.J., Zhang, C., Li, Z.X., Yan, C.H.: A fluorescent-switch-based

computing platform in defending information risk. Chem. Eur. J. 14, 6342–6351 (2008)


13. Suresh, M., Ghosh, A., Das, A.: A simple chemosensor for Hg2+

and Cu2+

that works as a molecular

keypad lock. Chem. Commun. 3906–3908 (2008)

14. Andreasson, J. Straight, S.D., Moore, T.A., Moore, A.L., Gust, D.: An all-photonic molecular keypad

lock. Chem. Eur. J. 15, 3936–3939 (2009)

15. Bozdemir, O.A., Guliyev, R., Büyükcakir, O., Selcuk, S., Kolemen, S., Gulseren, G., Nalbantoglu, T.,

Boyaci, H., Akkaya, E.U.: Selective manipulation of ICT and PET processes in styryl-bodipy derivatives:

applications in molecular logic and fluorescence sensing of metal ions. J. Am. Chem. Soc. 132, 8029–

8036 (2010)

16. Hayes, J.P.: Introduction to Digital Logic Design. Addison-Wesley Publishing Company, Reading (1993)

17. de Silva, P.A., Uchiyama, S.N.: Molecular logic and computing. Nanotechnology 2, 399–410 (2007)

18. Szacizowski, K.: Digital information processing in molecular systems. Chem. Rev. 108, 3481–3548

(2008)

19. Andreasson, J., Pischel, U.: Smart molecules at work—mimicking advanced logic operations. Rev.

Chem. Soc. 39, 174–188 (2010)

20. Liu, H., Zhou, Y., Yang, Y., Wang, W., Qu, L., Chen, C., Liu, D., Zhang, D., Zhu, D.: Photo-pH dually

modulated fluorescence switch based on DNA spatial nanodevice. J. Phys. Chem. B 112, 6893 (2008)

21. Margulies, D., Hamilton, A.D.: Digital analysis of protein properties by an ensemble of DNA quadru-

plexes. J. Am. Chem. Soc. 131, 9142 (2009)

22. Strack, G., Ornatska, M., Pita, M., Katz, E.: Biocomputing security system: concatenated enzyme-based

logic gates operating as a biomolecular keypad lock. J. Am. Chem. Soc. 130, 4234–4235 (2008)

23. Pais, V.F., Remón, P., Collado, D., Andréasson, J., Inestrosa, E.P., Pischel, U.: OFF-ON-OFF

fluorescence switch with T-Latch function. Org. Lett. 13, 5572–5575 (2011)

24. Kumar, M., Kumar, R., Bhalla, V.: Optical chemosensor for Ag+

, Fe3+

, and cysteine: information

processing at molecular level. Org. Lett. 13, 366–369 (2011)

25. Wang, J., Katz, E.: Digital biosensors with built-in logic for biomedical applications—biosensors based

on a biocomputing concept. Anal. Bioanal. Chem. 398, 1591–1603 (2010)

26. Fujimoto, K., Shimizu, H., Inouye, M.: Unambiguous detection of target DNAs by excimer-monomer

switching molecular beacons. J. Org. Chem. 69, 3271–3275 (2004)

27. Clelland, C.T. , Risca, V., Bancroft, C.: Hiding messages in DNA microdots. Nature 399, 533–534 (1999)

28. Mao, C., LaBean, T.H., Reif, J.H., Seeman, N.C.: Logical computation using self assembly of DNA

triple cross-over molecules. Nature 407, 493–496 (2000)

29. Tanaka, K., Okamoto, A., Saito, I.: Public-key system using DNA as a one-way function for key

distribution. Biosystems 81, 25–29 (2005)

30. Seth, D., Chakrabarty, D., Chakraborty, A., Sarkar, N.S.: Study of energy transfer from 7-amino coumarin

donors to rhodamine 6G acceptor in non-aqueous reverse micelles. Chem. Phys. Lett. 401, 546–552

(2005)

31. Förster, T.H.: Experimentelle und theoretische Untersuchung des Zwischenmolekularen übergangs von

Elektrinenanregungsenergie. Z. Naturforsch. 4A, 321–327 (1949)

32. Hussain, S.A., Chakraborty, S., Bhattacharjee, D., Schoonheydt, R.A.: Fluorescence resonance energy

transfer between organic dyes adsorbed onto nano-clay and Langmuir–Blodgett (LB) films. Spectrochim.

Acta, Part A 75, 664–670 (2010)

33. Förster, T.H.: Modern Quantum Chemistry, Istanbul Lectures, Part III: Action of Light and Organic

Crystals. Academic Press, New York (1965)

34. Malicka, J., Gryczynski, I., Lakowicz, J.R.: DNA hybridization assays using metal-enhanced

fluorescence. Biochem. Biophys. Res. Commun. 306, 213–218 (2003)

35. Mathur, N., Aneja, A., Bhatnagar, P.K., Mathur, P.C.: A new FRET-based sensitive DNA sensor for

medical diagnostics using PNA probe and water-soluble blue light emitting polymer. J. Sens. 2008, 1–6

(2008)

36. Watson, J.D., Crick, F.H.C.: A structure for deoxyribose nucleic acid. Nature 171, 737–738 (1953)

37. Wang, S., Gaylord, B.S., Bazan, G.C.: Fluorescein provides a resonance gate for FRET from conjugated

polymers to DNA intercalated dyes. J. Am. Chem. Soc. 126, 5446–5451 (2004)

De

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Sensors and Actuators B 195 (2014) 382–388

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

jo u r nal homep age: www.elsev ier .com/ locate /snb

evelopment of an ion-sensor using fluorescence resonancenergy transfer

ibyendu Dey, Jaba Saha, Arpan Datta Roy, D. Bhattacharjee, Syed Arshad Hussain ∗

epartment of Physics, Tripura University, Suryamaninagar, 799022, Tripura, India

r t i c l e i n f o

rticle history:eceived 8 November 2013eceived in revised form 15 January 2014ccepted 17 January 2014vailable online 26 January 2014

a b s t r a c t

A method is presented for the sensing of ions by determining the concentration of corresponding salts(KCl, NaCl, MgCl2, CaCl2, FeCl3, FeSO4, AlCl3) in water, based on fluorescence resonance energy transfer(FRET) process. The principle of the proposed sensor is based on the change of FRET efficiency betweentwo laser dyes Acriflavine and Rhodamine B in presence of different ions (K+, Na+, Mg2+, Ca2+, Fe2+, Fe3+,Al3+). Nanodimensional clay platelet laponite was used to enhance the efficiency of the sensor.

eywords:on sensorluorescence resonance energy transferyeslay


aponite

. Introduction

The development of ion sensor technology has emerged as dynamic approach for identifying and quantitating specificnalytes of environment. Now a days the growing need for mul-icomponent analyses and shorter sample preparation methods,ew sensing techniques with decreasing costs are very important.here are several sensing techniques which include ion selective oras sensitive electrodes, thermistors, chemically and biologicallyodified metal or semiconductors [1–3]. Chalcogemide glass sen-

ors are used for the detection of millimole levels of copper, iron,hromium, lead, cadmium and mercury in natural waste waters4]. The method is also used for the detection of heavy metal ions inolutions [4]. But the sensing of ions present in micromole amountould be much interesting and useful. It will be very much interest-ng to sense K+ and Na+ ions in blood samples. In blood the K+ anda+ concentrations are 4.5 and 120 mM respectively [5]. Increase

n concentration of these ions in blood can cause serious healthroblems. Another type of sensor is fluorescent ion sensors. Therere few reports where the detection of heavy metal ions such asg2+, Pb2+ and Cd2+ have been done by fluorescent ion sensors with
igh sensitivity and simplicity [6–9]. But in this method the change
n fluorescent intensity could be perturbed by environmental fac-ors [10,11]. It is interesting to mention in this context that the

∗ Corresponding author. Tel.: +91381 2375317; fax: +913812374802.E-mail addresses: sa [email protected], [email protected] (S.A. Hussain).

ttp://dx.doi.org/10.1016/j.snb.2014.01.065925-4005/© 2014 Elsevier B.V. All rights reserved.

introduction of ratiometric sensors can minimize this environmen-tal perturbation, because it measures the ratio of two emissionsin different environment. The design of ratiometric sensors can bedone by two method (i) ICT (intermolecular charge transfer) and(ii) FRET (fluorescence resonance energy transfer). For many ICTbased ion sensors it is difficult to determine the ratio between tworelatively broad signal emissions. Recently FRET based sensinghas become most effective method for the detection of ions inenvironment. FRET based sensors have been widely used in metalion detection [12,13], sensing of the fluorophores [14–18], Silica[19,20], and polymer particles [21–23]. In one of our recent paperwe have used FRET for the sensing of permanent hard water com-ponents in water for a concentration range of 0.03–0.2 mg/ml [24].

Here in the process of designing ion sensor based on FRET pro-cess, we have used two dyes Acriflavine (Acf) and Rhodamine B(RhB) as energy donor and acceptor respectively. In principle boththe dyes are suitable for fluorescence resonance energy transfer.Both the dyes are highly fluorescent and the fluorescence spectrumof Acf sufficiently overlaps with the absorption spectrum of RhB.P.D. Sahare et al. [25] reported the fluorescence resonance energytransfer in binary solution mixture of these two dyes. Recently wehave demonstrated a pH sensor [26], DNA sensor [35] and hardwater sensor [24] based on the FRET between Acf and RhB.

In the present communication we tried to investigate the
effect of K+, Na+, Mg2+, Ca2+, Fe2+, Fe3+, Al3+ on the FRET efficiencybetween two fluorophores, Acf and RhB in presence and absenceof nanoclay sheet laponite. The energy transfer efficiency has beenaffected if the distance between the donor–acceptor pair has been
dx.doi.org/10.1016/j.snb.2014.01.065


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dx.doi.org/10.1016/j.snb.2014.01.065

ctuators B 195 (2014) 382–388 383

atbo[tebAs

2

2

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Table 1Values of energy transfer efficiency (E%) for Acf and RhB mixture (1:1 volume ratio)with different salts (KCl, MgCl2, FeCl3) in absence and presence of clay. Dye con-centration was 10−6 M, salt concentration was 10 M and clay concentration was2 ppm. The values are calculated from the spectra of Fig.1.

Salt FRET efficiency(E%) without clay

FRET efficiency(E%) with clay

KCl 9.7 56.8


ltered due to the presence of any external agency or change ofhe microenvironment. It has been observed that when distanceetween fluorophores (dyes) is decreased due to adsorptionf the dyes on to nanoclay sheet, the FRET efficiency increases26,27]. Here we have used nanoclay sheet laponite to enhancehe sensitivity of sensing. Our investigation showed that FRETfficiency decreases with increasing salt concentration. It has alsoeen demonstrated that with proper calibration, FRET betweencf and RhB can be used to sense different ions on the basis of theirize and charge up to micromole level.

. Materials and methods

.1. Material

Both the dyes Acf and RhB were purchased from Sigma Chemicalo., USA and used as received. Ultrapure Milli-Q water (resistivity8.2 M cm) was used as solvent. The dyes used in our studiesre cationic in nature. The clay mineral used in the present workas Laponite, obtained from Laponite Inorganic, UK and used as

eceived. The size of the clay platelet is less than 0.05 m and CECs 0.739 meq/g determined with CsCl [28]. All the salts KCl, NaCl,

gCl2, CaCl2, FeCl3, FeSO4, and AlCl3 were purchased from Thermoisher Scientific India Pvt. Ltd. and used as received. Dye and saltolutions were prepared in Milli-Q water. For spectroscopic mea-urement the dye solution concentration was optimized at 10−6 M.he clay dispersion was prepared by using Milli-Q water and stirredor 24 h with a magnetic stirrer followed by 30 min ultrasonica-ion before use. The concentration of clay was kept fixed at 2 ppmhroughout the experiment. To check the effect of clay on the spec-ral characteristics, the dye solutions (Acf and RhB) were preparedn the clay suspensions (2 ppm). In order to check the effect of salt onpectral characteristics in presence of clay, first of all the salts weredded in the clay dispersion individually at different concentration.hen the dyes were added in the salt mixed clay dispersions. In allases the clay concentration was 2 ppm and the dye concentrationor both Acf and RhB was 10−6 M. The pH of the (Acf + RhB) mixedqueous solution was measured as 5.8 in this present experiment.

.2. UV–vis absorption and fluorescence spectra measurement

UV–vis absorption and fluorescence spectra of the solutionsere recorded by a Perkin Elmer Lambda-25 Spectrophotometer

nd Perkin Elmer LS-55 Fluorescence Spectrophotometer respec-ively. For fluorescence measurement the excitation wavelengthas 420 nm (close to the absorption maxima of Acf).

. Results and discussions

.1. FRET between Acf and RhB in aqueous solution and clayispersion

The absorption and emission maxima of Acf are centered at49 and 502 nm respectively which is assigned due to the Acfonomers [25]. On the other hand RhB absorption spectrum

ossess prominent intense 0–0 band at 553 nm along with a weakump at 520 nm which is assigned due to the 0–1 vibronic transi-ion [29]. The RhB fluorescence spectrum shows prominent band at71 nm which is assigned due to the RhB monomeric emission [29].he corresponding absorption and emission spectra of the aboveesults are shown in Fig. 1 of the supporting information.

Fig. 1a shows the fluorescence spectra of pure Acf, RhB andheir mixture in water solution in presence and absence of saltKCl, MgCl2, and FeCl3). All the spectra were measured with exci-ation wavelength 420 nm (close to absorption maximum of Acf).

MgCl2 7.4 47.4FeCl3 5.2 38.5

This excitation wavelength was chosen in order to excite theAcf molecule directly and to avoid the direct excitation of RhBmolecule. From the figure it has been observed that the fluores-cence intensity of pure Acf (curve 1 of Fig. 1a) is much higher, onthe other hand the fluorescence intensity of pure RhB (curve 2 ofFig. 1a) is almost negligible. However, the Acf–RhB mixture fluo-rescence spectrum is (curve 3 of Fig. 1a) very interesting. Here theAcf emission decreases with respect to pure Acf and on the otherhand RhB emission increases with respect to pure RhB (curve 3of Fig. 1a). This is mainly due to the transfer of energy from Acfmolecule to RhB molecule via fluorescence resonance energy trans-fer. In order to confirm this, excitation spectra was recorded withmonitoring emission wavelength 500 nm (Acf emission maximum)and 571 nm (RhB emission maximum) and observed that both theexcitation spectra are very similar to the absorption spectrum of Acfmonomer (Fig. 2 of supporting information). This confirms that theRhB fluorescence is mainly due to the light absorption by Acf andcorresponding transfer to RhB monomer. Thus FRET between Acfand RhB has been confirmed. FRET efficiencies have been calculatedusing the following equation [30]

E = 1 − FDA

FD

where FDA is the fluorescence intensity of the donor in the presenceof acceptor and FD is the fluorescence intensity of the donor in theabsence of the acceptor.

In order to sense different ions we have introduced differentsalts in the Acf–RhB mixed aqueous solution and the FRET betweenAcf and RhB has been measured. The change in FRET efficiency dueto the presence of ions/salts has been examined in order to sense thepresence of corresponding ions. Fluorescence spectra of Acf–RhBmixture in presence of KCl (curve 4), MgCl2 (curve 5), FeCl3 (curve 6)have also been shown in Fig. 1a. It has been observed that in all thecases the FRET efficiency decreased. However, the change in FRETefficiency is very small. The FRET efficiency changes from 11.37% (inabsence of salt) to 9.2% (in presence of KCl) or 7.4% (in presence ofMgCl2) or 5.2% (in presence of FeCl3). The corresponding efficienciesare listed in Table 1.

In order to increase the FRET efficiency between Acf and RhBwe have introduced nanoclay platelet laponite in the Acf–RhB mix-ture. Fluorescence spectra of pure Acf (curve 1), RhB (curve 2) andAcf–RhB mixture (curve 3) in presence of laponite are shown inFig. 1b. The corresponding FRET efficiencies are also listed in Table 1.It has been observed that FRET efficiency increases to 78.17% inpresence of clay for Acf–RhB mixture which was 11.37% in absenceof clay platelet. Now salts are introduced in the Acf–RhB mixture inpresence of clay laponite. Corresponding fluorescence spectra (KCl(curve 4), MgCl2 (curve 5), FeCl3 (curve 6), are also shown in Fig. 1b.The FRET efficiencies are also listed in Table 1.

It is worthwhile to mention in this context that clay particlesare negatively charged and have layered structure with a cation
exchange capacity [31,32]. Both the dyes Acf and RhB under inves-tigation are positively charged. Accordingly they are adsorbed ontothe clay layers. On the other hand FRET process is very sensi-tive to distances between the energy donor and acceptor and

384 D. Dey et al. / Sensors and Actuators B 195 (2014) 382–388

F me rato clay suc

oottbs

elt(cot

iaopcd

3

ealFt

rAfeTitSt

and smaller energy transfer between the dye molecules. The simi-lar kind of study is also done for the divalent and trivalent ions andis shown in Fig. 2b and c respectively. The corresponding FRET effi-ciencies are also tabulated in Table 2. The trends of energy transfer

Fig. 2. (a) Fluorescence spectra of pure Acf with clay (1), pure RhB with clay (2) andAcf + RhB (1:1 volume ratio) in clay suspension (3), with KCl (4), NaCl (5) and (b)

ig. 1. (a) Fluorescence spectra of pure Acf (1), pure RhB (2) and Acf + RhB (1:1 voluf pure Acf with clay (1), pure RhB with clay (2) and Acf + RhB (1:1 volume ratio) in

lay concentration was 2 ppm and salt concentration was 10 M.

ccurs only when the distance between the D–A pair is of therder of 1–10 nm [33,34]. Therefore in the present case, clay par-icles play an important role in determining the concentration ofhe dyes on their surfaces or to make possible close interactionetween energy donor and acceptor in contrast to the pure aqueousolution.

It is important to mention in this context that in one of ourarlier works it has been observed that the presence of nanoclayaponite increased the FRET efficiency between N,N′-dioctadecylhiacyanine perchlorate (NK) and octadecyl rhodamine B chlorideRhB) [27]. Effect of nanoclay laponite on the energy transfer effi-iency between Acf and RhB has also been studied [26]. It has beenbserved that the presence of clay platelet increase the energyransfer efficiency.

From Table 1 it has been observed that FRET efficiency increasesn presence of nanoclay platelets. Again the presence of salts causes

decrease in FRET efficiencies. Here in presence of clay the changer variation of FRET efficiency due to presence of salts is more com-ared to that in absence of clay platelet. Therefore incorporation oflay laponite in the present system lower the error level in sensingifferent ions or increases the ion sensing efficiency/sensitivity.

.2. Ions with variable size

In order to have an idea about the effect of ion size on FRETfficiency, we have selected three different sets of salts – (i) NaClnd KCl (both are monovalent), (ii) MgCl2 and CaCl2 (both are diva-ent), (iii) FeCl3 and AlCl3 (both are trivalent), and measured theRET efficiency between Acf and RhB in presence of these salts. Allhe experiments were performed in presence of clay.

Fig. 2a shows the fluorescence spectra of Acf + RhB (1:1 volumeatio) in presence and absence of NaCl and KCl along with purecf and RhB fluorescence spectra. The corresponding energy trans-

er efficiencies are listed in Table 2. It has been observed that thenergy transfer efficiencies decrease in presence of different salts.he interesting thing is that the transfer of energy from Acf to RhB
s larger in case of NaCl than KCl. It is very much possible due tohe fact that the molecular size of Na+ ion is smaller than K+ ion.o the space occupied by the K+ ions on the clay templates is largerhan the Na+ ions resulting in a larger intermolecular separation
io) in water solution (3), with KCl (4), MgCl2 (5), FeCl3 (6). (b) Fluorescence spectraspension (3), with KCl (4), MgCl2 (5), FeCl3 (6). Dye concentration was 10−6 M and

fluorescence spectra of pure Acf with clay (1), pure RhB with clay (2) and Acf + RhB(1:1 volume ratio) in clay suspension (3), with CaCl2 (4), MgCl2 (5). (c) Fluorescencespectra of pure Acf with clay (1), pure RhB with clay (2) and Acf + RhB (1:1 volumeratio) in clay suspension (3), with FeCl3 (4), AlCl3 (5). Dye concentration was 10−6 Mand clay concentration was 2 ppm and salt concentration was 10 M.

D. Dey et al. / Sensors and Actuators B 195 (2014) 382–388 385

Table 2Values of energy transfer efficiency (E%) for Acf and RhB mixture (1:1 volume ratio)with different salts (KCl, NaCl, MgCl2, CaCl2, FeCl3, AlCl3) in presence of clay. Dyeconcentration was 10−6 M, salt concentration was 10 M and clay concentrationwas 2 ppm. The values are calculated from the spectra of Fig.2.

Salt FRET efficiency (E%) with clay

KCl 56.8NaCl 62.15MgCl2 47.4CaCl2 41.1FeCl3 38.5AlCl3 45.6

Fig. 3. Fluorescence spectra of pure Acf with clay (1), pure RhB with clay (2) andAcw

etlm

3

tAaFc

TVwsc

Table 4Values of energy transfer efficiency (E%) for Acf and RhB mixture (1:1 volume ratio)with different salt concentration in presence of clay. Dye concentration was 10−6 Mand clay concentration was 2 ppm. The values are calculated from the spectra of Fig.3 given in the supporting information.

Salts FRET efficiency (E%) with clay

Salt con = 10 M Salt con = 100 M Salt con = 1000 M

KCl 56.8 48.7 41.2NaCl 62.15 55.7 51.2MgCl2 47.4 42.5 36.8CaCl2 41.1 36.5 31.2FeSO4 46.8 41.7 35.8

cf + RhB (1:1 volume ratio) in clay suspension (3), with FeCl3 (4), FeSO4 (5). Dyeoncentration was 10−6 M and clay concentration was 2 ppm and salt concentrationas 10 M.

fficiencies are similar to that for monovalent salts. Here both thesewo cases it have been observed that energy transfer efficiency isarger for the salts with smaller ion sizes. In later section of this

anuscript this has been explained with schematic diagram.

.3. Ions with variable valency and same size

In order to check the effect of valency of ions on the energyransfer efficiency we have measured the fluorescence spectra of
cf + RhB mixture (1:1 volume ratio) in aqueous clay dispersion inbsence and presence of salt FeSO4 (divalent) and FeCl3 (trivalent).ig. 3 shows the corresponding spectra. Energy transfer efficien-ies calculated from the spectra of Fig. 3 are listed in Table 3. From
able 3alues of energy transfer efficiency (E%) for Acf and RhB mixture (1:1 volume ratio)ith different salts (FeSO4, FeCl3) in presence of clay. Dye concentration was 10−6 M,

alt concentration was 10 M and clay concentration was 2 ppm. The values arealculated from the spectra of Fig.3.

Salt FRET efficiency (E%) with clay

FeSO4 46.8FeCl3 38.5

FeCl3 38.5 32.7 27.8AlCl3 45.6 39.8 33.5

Fig. 3 and calculated values of efficiencies it has been observedthat the energy transfer efficiency decreases in presence of boththe salts. However, the extent of decrease in energy transfer effi-ciency is more in presence of FeCl3 compared to that in presence ofFeSO4. In this study we have selected the salts in such a way thattheir molecular size remains same but their charge changes. HereFeSO4 provides a Fe2+ ion and FeCl3 provides a Fe3+ ion in aqueousmedium. So the larger electrostatic repulsion of the Fe3+ ion pro-vides a larger intermolecular separation between the Acf and RhBin compared to Fe2+ ion on to the clay templates when the ionsand dyes were adsorbed on to clay surface. As a result the FRETefficiency decreases more in case of FeCl3. In later section of thismanuscript this has also been explained with schematic diagram.

3.4. Effect of variation of salt concentration on FRET efficiency

In the previous sections we have seen that presence of laponiteparticle increases the FRET efficiency between Acf and RhB,whereas, presence of salt/ions decreases the FRET efficiency. Againthe decrease in energy transfer efficiency is proportional to the ionsize and valency. Now in order to check the effect of variation ofsalt concentration on the FRET efficiency, we have measured thefluorescence spectra of Acf and RhB mixture with different salt con-centration (10,100 and 1000 M) in clay dispersion and the energytransfer efficiency has been calculated. The spectra are availablein Fig. 3 of the supporting information and the corresponding effi-ciencies are listed in Table 4. It has been observed that the FRETefficiency decreases with increasing salt concentration for all thesalts. The increase in salt concentration basically increases theamount of cations in the solvent and as a result a larger area ofthe clay layers is occupied by the salt cations. Accordingly a com-paratively smaller amount of dye molecules are attached to theclay templates resulting in a less probability of occurrence of FRETbetween Acf and RhB.

As a whole our investigations suggest that it is possible to sensethe ions by observing the change in FRET efficiency with ion size,valency and varying salt concentration.

3.5. Schematic diagram

A schematic diagram showing the organization of Acf and RhBin absence and presence of clay laponite and salt is shown in Fig. 4.Normally in absence of both clay and salt the distance between Acfand RhB molecules in aqueous solution is larger resulting lowerenergy transfer efficiency but in presence of clay only the dyes areadsorbed by cation exchange reaction on to the clay surface andaccordingly the distance between Acf and RhB decreases as shown
in Fig. 4b. These results can increase the energy transfer efficiency.In presence of both clay and salt, the probability of adsorption of saltcations are larger in compared to the cationic dyes. This is becausein the process of dye-clay-salt solution preparation initially the

386 D. Dey et al. / Sensors and Actuators B 195 (2014) 382–388

F epresel nt ion

stacdRctarOvFbal

3

iid

ig. 4. Schematic representation of (a) donor, acceptor and clay sheet. Schematic rarger size ions, (d) clay and salt with smaller size ions, (e) clay and salt with trivale

alt was added to the clay dispersion followed by the dye addi-ion. Accordingly, most of the negative charges on the clay surfacere neutralized by the salt cations and there exist very few unoc-upied negative charges on the clay surface to adsorb the cationicyes (Fig. 4c–f). Accordingly the average distance between Acf andhB become larger. This results a decrease in energy transfer effi-iency. Now as the size of the salt cations increase the space onhe clay template decreases farther for the dye molecules to bettached even if the number of salt molecules are same and as aesult the FRET efficiency also decreases farther (Fig. 4c and d).n the other hand if the size of the ions are kept constant but thealency is increased then also there will be a farther decrease of theRET efficiency for the larger valency of the salt ions (Fig. 4e and f)ecause the number of negative ions present on the clay templatere neutralized more by the high valency salt molecule than theow valency salt molecule.

.6. Design of ion sensor

Based on the variation of FRET efficiency or fluorescencentensity, depending on the type of salts we have demonstrated anon sensor. In the process of ion sensing first of all clay (laponite)ispersion will be prepared using the sample water (in presence

ntation of FRET between Acf and RhB in presence of (b) clay, (c) clay and salt withs, (f) clay and salt with divalent ions.

of ions) followed by addition of dyes (Acf and RhB). Then thefluorescence spectra of the solution will be measured. By observ-ing the fluorescence intensity or FRET efficiency calculated fromthe observed fluorescence spectra it would be possible to sensedifferent ions of the sample water.

Fig. 5 shows the plot of FRET efficiency as a function of differentsalts (KCl, NaCl, MgCl2, CaCl2, FeSO4, FeCl3, AlCl3) of concentration10 M. The data are taken from spectra shown in Figs. 1b, 2a–c and3. From Fig. 3 it has been observed that the FRET efficiency for KCland NaCl are 56.8% and 62.15% respectively. If the FRET efficiencyis observed to be higher than 56.8% but lower than 62.15% then itshows the presence of Na+ ion whereas, if the efficiency is lower56.8% then it shows the presence of K+ ion. Therefore with propercalibration it is possible to design an ion sensor which can senseions on the basis of their size (Fig. 5a–c) and similar observationcan be done for the sensing of ions of different valency but samesize (as shown in Fig. 5b).

It is interesting to mention that in principle this method canbe used to sense negative ions also. The presence of negative ions
in between the cationic dye pair can decrease their intermolecularseparation and the FRET efficiency will increase. Therefore withproper calibration and selecting suitable FRET pair it is possibleto sense both negative as well as positive ions using this method.

D. Dey et al. / Sensors and Actuators B 195 (2014) 382–388 387

F alt (3)( fficie

DW

4

flspsscitieRCttiom

A

tN

[

ig. 5. FRET efficiency of Acf and RhB mixture for (a) KCl (1), NaCl (2) and without s3) (d) FeSO4 (1), FeCl3 (2) and without salt (3) in presence of clay. (Values of FRET e

etailed investigations are required for the sensing of negative ions.ork is going on in our laboratory in this line.

. Conclusion

Fluorescence resonance energy transfer (FRET) between twouorescent dyes Acriflavine and Rhodamine B were investigateduccessfully in solution in presence and absence of clay mineralarticle laponite. UV–vis absorption and fluorescence spectroscopytudies reveal that both the dyes present mainly as monomer inolution and there exist sufficient overlap between the fluores-ence spectrum of Acf and absorption spectrum of RhB, whichs a prerequisite for the FRET to occur from Acf to RhB. Energyransfer occurred from Acf to RhB. The energy transfer efficiencyncreases in presence of clay laponite in solution. The maximumfficiency was found to be 78.17% for the mixed dye system (50%hB + 50% Acf) in clay dispersion. In presence of KCl, NaCl, MgCl2,aCl2, FeSO4, FeCl3, and AlCl3 the FRET efficiency is decreasedo 56.8%, 62.15%, 47.4%, 41.1%, 46.8%, 38.5% and 45.6% respec-ively from 78.17%. With suitable calibration of these results its possible to design an ion sensor that can sense the presencef different ions in water up to a concentration of 10 M orore.

cknowledgement

The author SAH is grateful to DST and CSIR for financial supporto carry out this research work through DST Fast-Track project Ref.o. SE/FTP/PS-54/2007, CSIR project Ref. 03(1146)/09/EMR-II.

[

(b) CaCl2 (1), MgCl2 (2) and without salt (3) (c) FeCl3 (1), AlCl3 (2) and without saltncies were calculated from the spectra of Figs. 2 and 3.)

Appendix A. Supplementary data

Supplementary material related to this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.snb.2014.01.065.

References

[1] C.R. Lowe, Chemoselective biosensors, Curr. Opin. Chem. Biol. 3 (1999)106–111.

[2] K.R. Rogers, M. Mascini, Biosensors for field analytical monitoring, Field Anal.Chem. Technol. 2 (1998) 317–331.

[3] J. Janata, M. Josowicz, P. Vanysek, V. De, Chemical sensors, Anal. Chem. 70 (1998)179–208.

[4] Y.G. Vlasov, E.A. Bychkov, A.V. Lizgin, Chalcogenide, glass chemical sensors:research and analytical applications, Talanta 41 (1994) 1059–1063.

[5] H. He, H. Li, G. Mohr, B. Kovacs, T. Werner, O.S. Wolfbeis, Novel type of ion-selective fluorosensor based on the inner filter effect: an optrode for potassium,Anal. Chem. 65 (1993) 123–127.

[6] J. Han, K. Burgess, Fluorescent indicators for intracellular pH, Chem. Rev. 110(2010) 2709–2728.

[7] H. Wang, D. Wang, Q. Wang, X. Li, C.A. Schalley, Nickel(II) and iron(III) selectiveoff-on-type fluorescence probes based on perylene tetracarboxylic diimide,Org. Biomol. Chem. 8 (2010) 1017–1026.

[8] J. Wang, X. Qian, Two regioisomeric and exclusively selective Hg(II)sensor molecules composed of a naphthalimide fluorophore and an o-phenylenediamine derived triamide receptor, Chem. Commun. (2006)109–111.

[9] Q. He, E.W. Miller, A.P. Wong, Christopher J. Chang, A selective fluores-cent sensor for detecting lead in living cells, J. Am. Chem. Soc. 128 (2006)9316–9317.

10] S.C. Dodani, Q. He, C.J. Chang, A turn-on fluorescent sensor for detecting nickel
in living cells, J. Am. Chem. Soc. 131 (2009) 18020–18021.
11] H. Takakusa, K. Kikuchi, Y. Urano, S. Sakamoto, K. Yamaguchi, T. Nagano, Designand synthesis of an enzyme-cleavable sensor molecule for phosphodiesteraseactivity based on fluorescence resonance energy transfer, J. Am. Chem. Soc. 124(2002) 1653–1657.

http://dx.doi.org/10.1016/j.snb.2014.01.065
















































































































































































































































































3 Actuat

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

88 D. Dey et al. / Sensors and

12] G. Chen, Y. Jin, L. Wang, J. Deng, C. Zhang, Gold nanorods-based FRET assay forultrasensitive detection of Hg2+ C, Chem. Commun. 47 (2011) 12500–12502.

13] Z. Zhou, M. Yu, H. Yang, K. Huang, F. Li, T. Yi, C. Huang, FRET-based sen-sor for imaging chromium(III) in living cells C, Chem. Commun. (2008)3387–3389.

14] K. Wang, Z. Tang, C.J. Yang, Y. Kim, X. Fang, W. Li, Y. Wu, C.D. Medley, Z. Cao, J.Li, P. Colon, H. Lin, W. Tan, Molecular engineering of DNA: molecular beacons,Angew. Chem. Int. Ed. 48 (2009) 856–870.

15] U.H.F. Bunz, V.M. Rotello, Gold nanoparticle – fluorophore complexes: sensi-tive and discerning “noses” for biosystems sensing, Chem. Int. Ed. 49 (2010)3268–3279.

16] B. Tang, N. Zhang, Z. Chen, K. Xu, L. Zhuo, L. An, G. Yang, Probing hydroxylradicals and their imaging in living cells by use of FAM-DNA-Au nanoparticles,Chemistry 14 (2008) 522–528.

17] B.Y. Wu, H.F. Wang, J.T. Chen, X.P. Yan, Fluorescence resonance energy transferinhibition assay for fetoprotein excreted during cancer cell growth using func-tionalized persistent luminescence nanoparticles, J. Am. Chem. Soc. 133 (2011)686–688.

18] Y. Chen, M.B. O’Donoghue, Y.F. Huang, H. Kang, J.A. Phillips, X. Chen, M.C.Estevez, C.J. Yang, W. Tan, A surface energy transfer nanoruler for measur-ing binding site distances on live cell surfaces, J. Am. Chem. Soc. 132 (2010)16559–16570.

19] S.H. Kim, M. Jeyakumar, J.A. Katzenellenbogen, Dual-mode fluorophore-dopednickel nitrilotriacetic acid-modified silica nanoparticles combine histidine-tagged protein purification with site-specific fluorophore labeling, J. Am. Chem.Soc. 129 (2007) 13254–13264.

20] J. Lei, L. Wang, J. Zhang, Ratiometric pH sensor based on mesoporous silicananoparticles and Förster resonance energy transfer, J. Chem. Commun. 46(2010) 8445–8447.

21] J. Chen, F. Zeng, S. Wu, J. Zhao, Q. Chen, Z. Tong, Reversible fluorescence modula-tion through energy transfer with ABC triblock copolymer micelles as scaffolds,Chem. Commun. (2008) 5580–5582.

22] M.Q. Zhu, L. Zhu, J.J. Han, W. Wu, J.K. Hurst, A.D.Q. Li, Spiropyran-based pho-tochromic polymer nanoparticles with optically switchable luminescence, J.Am. Chem. Soc. 128 (2006) 4303–4309.

23] E.S. Childress, C.A. Roberts, D.Y. Sherwood, C.L.M. LeGuyader, E.J. Harbron,Ratiometric fluorescence detection of mercury ions in water by conjugatedpolymer nanoparticles, Anal. Chem. 84 (2012) 1235–1239.

24] D. Dey, D. Bhattacharjee, S. Chakraborty, S.A. Hussain, Development of hardwater sensor using fluorescence resonance energy transfer, Sens. Acutators B:Chem. 184 (2013) 268–273.

25] P.D. Sahare, V.K. Sharma, D. Mohan, A.A. Rupasov, Energy transferstudies in binary dye solution mixtures: Acriflavine + Rhodamine 6Gand Acriflavine + Rhodamine B, Spectrochim. Acta Part A 69 (2008)1257–1264.

26] D. Dey, D. Bhattacharjee, S. Chakraborty, S.A. Hussain, Effect of nanoclaylaponite and pH on the energy transfer between fluorescent dyes, J. Photochem.Photobiol. A: Chem. 252 (2013) 174–182.

27] S.A. Hussain, S. Chakraborty, D. Bhattacharjee, R.A. Schoonheydt, Fluorescenceresonance energy transfer between organic dyes adsorbed onto nano-clay
and Langmuir–Blodgett (LB) films, Spectrochim. Acta Part A 75 (2010)664–670.
28] T. Szabo, R. Mitea, H. Leeman, G.S. Premachandra, C.T. Johnston, M. Szekeres,I. Dekany, R.A. Schoonheydt, Adsorption of protamine and papine proteins onsaponite, Clays Clay Miner. 56 (2008) 494–504.

ors B 195 (2014) 382–388

29] S.A. Hussain, S. Banik, S. Chakraborty, D. Bhattacharjee, Adsorption kinetics ofa fluorescent dye in a long chain fatty acid matrix, Spectrochim. Acta Part A 79(2011) 1642–1647.

30] D. Seth, D. Chakrabarty, A. Chakraborty, N. Sarkar, Study of energy transfer from7-amino coumarin donors to rhodamine 6G acceptor in non-aqueous reversemicelles, Chem. Phys. Lett. 401 (2005) 546–552.

31] R.A. Schoonheydt, Smectite-Type clay minerals as nanomaterials, Clay ClayMiner. 50 (2002) 411–420.

32] R.H.A. Ras, B. van Duffel, M. van der Auweraer, F.C. De Schryver, R.A.Schoonheydt, Proceedings of the 12th International Clay Conference, BahiaBlanca, Argentina, 2001.

33] T.H. Förster, Experimentelle und theoretische untersuchung des zwischen-molekularen übergangs von elektrinenanregungsenergie, Z. Naturforsch. 4A(1949) 321–327.

34] T.H. Förster, Transfer mechanisms of electronic excitation, Diss. Faraday Soc.27 (1959) 7–71.

35] D. Bhattacharjee, D. Dey, S. Chakraborty, S.A. Hussain, S. Sinha, Develop-ment of a DNA sensor using a molecular logic gate, J. Bio. Phys. 39 (2013)387–394.

Biographies

Mr. Dibyendu Dey (M.Sc. 2009, Tripura University, India) is working as a researchscholar in Department of Physics, Tripura University. His major fields of interestare Fluorescence Resonance Energy Transfer in solution and ultrathin films. He haspublished 3 research papers in international journals and attended several scientificconferences in India.

Ms. Jaba Saha (M.Sc. 2012, Tripura University, India) is working as a research scholarin Department of Physics, Tripura University. Her major fields of interest are fluo-rescence resonance energy transfer in solution and ultrathin films and their sensingapplications.

Mr. Arpan Datta Roy (M.Sc. 2013, Tripura University, India) is working as a researchscholar in Department of Physics, Tripura University. His major fields of interest arestudy of biomolecules using fluorescence resonance energy transfer.

Prof. D. Bhattacharjee (M.Sc., Kalyani University & Ph.D., IACS, India) is aProfessor in the Department of Physics, Tripura University, India. His majorfields of interest are preparation and characterization of ultra thin films byLangmuir–Blodgett and self-assembled techniques. He visited Finland and Belgiumfor postdoctoral research. He has undertaken several research projects. Hehas published more than 66 research papers in different national and inter-national journals and attended several scientific conferences in India andabroad.

Dr. S.A. Hussain (M.Sc. 2001 and Ph.D., 2007, Tripura University, India) is an Assis-tant Professor in the Department of Physics, Tripura University. His major fields ofinterest are Thin Films and Nanoscience. He was a Postdoctoral Fellow of K.U. Leu-
ven, Belgium (2007–2008). He received Jagdish Chandra Bose Award 2008–2009,TSCST, Govt. of Tripura; Young Scientist Research Award by DAE, Govt of India.He has undertaken several research projects. He has published 56 research papersin international journals and attended several scientific conferences in India andabroad.



































































































































































































































































































































































































































































































































































































































































































































Delhi – 110089, India Email: [email protected] www.ripublication.com

Proceedings of the Conference on Recent Trends of Research in Physics (CRTRP-2012)

Editors: B. K. De, D. Bhattacharjee, S. Chattopadhyaya, S. A. Hussain, A. Guha

ISBN: 978-81-904362-9-8 (2013)

Molecular Logic Gates using FRET Phenomenon.

Dibyendu Dey, D. Bhattacharjee, S. Chakraborty and Syed Arshad Hussain*

Department of Physics Tripura University, Suryamaninagar-799022, India [email protected], [email protected],[email protected]

Abstract: Fluorescence resonance energy transfer (FRET) between two dyes acriflavine (Acf) and rhodamine B (RhB) were investigated. It was observed that the energy transfer efficiency from Acf to RhB is sensitive to the presence of dsDNA. In presence of dsDNA the FRET efficiency of the dye pair increases up to 79.1%. Using this property we are able to construct two photoregulated molecular logic gates viz NOT and YES-NOT gate. These molecular logic gates can be used for sensing the presence dsDNA. Keywords: molecular logic gate, FRET. 1. Introduction: The research on molecular logic is initialized by the recognition of a pioneer work by de Silva et al [1]. The change in fluorescence property of a dye due to the introduction of some external agent could be considered to be analogous to the digital responses in electronic logic gates. Molecules can undergo changes in ground or exited state due to the interference of some external chemical or biological material [2]. This kind of change can be realized by the change in fluorescence intensity, and the output can be related to the operation of some well known digital logic gates. Using some basic logic, the molecules can process and manipulate information as like electronic computers and human brain. Using this concept some simple logic gates [3] as well as few complex circuitry [4-6] can be constructed, such as adder/subtractors [7-9], encoders/decoders [10, 11], multiplexers/ demultiplexers [12-14], and keypad locks [15-19]. There are many molecular logic gates where chemicals are used as inputs and optical signals are the outputs [20, 21]. Now a day for the sensing of different organic [22], inorganic [23] and biological [24-26] materials these molecular logic gates are being used. For the easier understanding of the outputs of the sensors they are compared with some well known digital logic gates and from different outputs of those logic gates we can have some idea about the different structural features and properties of the test sample.

In the present communication we have investigated FRET between two laser dyes Acf and RhB. It was observed the FRET efficiency changes in presence of dsDNA. Based on this

effect we have demonstrated two photoregulated molecular logic gates viz NOT and YES-NOT gate. Use of these logic gates as dsDAN sensor has also been demonstrated. 2. Experimental: Two dyes Acf and RhB as well as dsDNA were purchased from Sigma-Aldrich Chemical Co. and used as received. Ultrapure Milli-Q water was used for solution preparation. Fluorescence spectra were recorded using a spectrophotometer (LS-55, Perkin Elmer). The striking wavelength was 420 nm. 3. Logic gates based on the Energy Transfer between laser dyes: We investigated the fluorescence resonance energy transfer between two laser dyes acriflavine (Acf) and rhodamine B (RhB). Details have been communicated for publication [27]. These two dyes are in principle suitable for energy transfer. It was observed that the energy transfer efficiency increases enormously in presence of dsDNA. Based on this fact we developed a chemical logic gate, which can be used as dsDNA sensor.

450 500 550 600 650

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Figure 1: (a) The spectroscopic characteristics show the FRET between Acf-RhB in absence of dsDNA (1) and in presence of dsDNA (2). Inset showing the fluorescence spectra of pure Acf and RhB exited at 420 nm wavelength. Figure 1 shows the fluorescence spectra of Acf and RhB aqueous solution in presence (curve 2) and in absence (curve 1) of dsDNA. Inset shows the fluorescence spectra of pure Acf and RhB. All spectra of figure 1 were recorded with excitation wavelength 420 nm. This excitation wavelength was selected in order to avoid the direct excitation of the RhB molecules. With this excitation wavelength RhB fluorescence intensity is almost negligible for pure RhB spectrum (inset II of Fig.1). However for Acf-RhB mixed solution fluorescence spectrum (curve 1 of figure 1) the RhB fluorescence intensity increases even with this excitation wavelength (420 nm), as well as

Acf fluorescence decreases compare to their pure counterpart. This may be due to transfer of energy from Acf to RhB. This transferred energy excites more RhB molecules followed by light emission from RhB, which is added to the original RhB fluorescence. As a result the RhB fluorescence intensity gets sensitized. Thus FRET between Acf to RhB has been confirmed. This study is reported in one of our communications relating FRET [27]. It is very interesting to observe that in presence of dsDNA the transfer of energy is much more efficient for the same concentration and volume ratio of Acf and RhB in water solution. The FRET efficiency of the dye pair increases from 11.37% (absence of dsDNA) to 79.1% (presence of dsDNA). Based on the energy transfer between Acf and RhB we propose a molecular logic gate. Using this gate it is possible to detect dsDNA in solution up to a concentration of 1 µg/ml. It is a kind of biological sensor and to understand the output of this sensor in an easier manner we are trying to mimic its output with some well-known digital logic gates. In this system particular fluorescence intensity is taken as reference. Here the chosen reference level is 400 au (fluorescence intensity).

Here we consider the output is 1 when fluorescence intensity at 500 nm is greater than the reference level and the output is zero when the intensity is below the reference level. The input is considered as ‘1’ when dsDNA is present in the system and as ‘0’ when dsDNA is absent. From fig. 1a it is observed that the fluorescence intensity at 500 nm is less than the reference level (400 au) when dsDNA is present and greater than the reference level when dsDNA is absent. The corresponding functional table is given in table 1 which is similar to the operation of a digital NOT gate.

Table 1: Function table of NOT logic

Input (dsDNA) Output (fluorescence intensity at 500 nm) 0

(absence of dsDNA) 1

(intensity is greater than reference level) 1

(presence of dsDNA) 0

(intensity is less than reference level)

Table 2: Function table of YES-NOT logic

Input (dsDNA) Output (fluorescence intensity at 500 nm)

Output (fluorescence intensity at 578 nm)

0 (absence of dsDNA)

1 (intensity is greater than

reference level)

0 (intensity is less than reference

level) 1

(presence of dsDNA) 0

(intensity is less than reference level)

1 (intensity is greater than

reference level) By considering the fluorescence intensity of both Acf and RhB at 500 nm and 578 nm respectively we can construct a logical YES/NOT gate. In this case the output of RhB at 578 nm is directly proportional to the input (that is the presence or absence of dsDNA). At 578 nm the fluorescence intensity is less than 400 au in absence of dsDNA and in presence of dsDNA the

intensity is greater than 400 au, which are denoted as 0 and 1 respectively. The functional table is given in table 2. Both the gates can be used to sense the presence of dsDNA. In our devices the input is either the presence (logic 1) or absence (logic 0) of dsDNA. The output is the fluorescence intensity at particular wavelengths. We assumed specific fluorescence intensity (400) as the reference. If the fluorescence intensity is greater than the reference than the output is logic 1 else it is 0. So just by observing the output of the YES-NOT gate i.e., whether the fluorescence intensity is greater than the reference level it is possible to detect the presence of dsDNA in the system. In the present case the reference level is taken at 33% higher than the fluorescence intensity at 578 nm in absence of dsDNA. This has been done in order to confirm that the increase in intensity is due to presence of dsDNA. It was observed that even if the concentration of dsDNA is 0.1µg/ml the fluorescence intensity at 578 nm crosses the reference level. So our device can sense the presence of dsDNA up to a concentration of 0.1µg/ml. 4. Conclusion: Fluorescence resonance energy transfer (FRET) between two dyes acriflavine (Acf) and rhodamine B (RhB) were successfully investigated in presence and absence of dsDNA for the realization of NOT and YES/NOT gate. It was observed that the FRET efficiency increases from 11.37% to 79.1% in presence of dsDNA. Using this effect we have demonstrated two photoregulated fluorescence switches. The functionality of the switches mimic with the electronic NOT and YES-NOT logic gate. Use of these two molecular logic gates as dsDNA sensor has also been demonstrated. Acknowledgement: The author SAH is grateful to DST for financial assistance through DST Fast-Track project to carry out this research work. References: [1] de Silva, A. P. Gunaratne, H. Q. N.;and McCoyNature 364 42 (1993) [2] O. Altan Bozdemir, Ruslan Guliyev, Onur Buyukcakir, Sencer Selcuk, Safacan Kolemen, Gulcihan Gulseren, Tugrul Nalbantoglu, Hande Boyaci and Engin U. Akkaya JACS 132 8029 (2010) [3] Sauvage, J.-P. Acc. Chem. Res. 31 611 (1998) [4] de Silva, A. P.; Uchiyama and S. Nat. Nanotechnol. 2 399 (2007) [5] Szacizowski, K. Chem. Rev.108 3481 (2008). [6] Andreasson, J Pischel, U. Chem. Soc. Rev. 39 174 (2010) [7] Margulies, D Melman and G Shanzer JACS 128 4865 (2006) [8] Pischel, U. Angew. Chem. Int. Ed. 46 4026 (2006) [9] Bozdemir, O. A Guliyev, R. Buyukcakir, O. Selcuk, S. Kolemen, S.Gulseren, G Nalbantoglu, T Boyaci, H.Akkaya, E. U. J. Am. Chem. Soc. 132 8029 (2010) [10]. Andreasson, J. Straight, S. D.; Moore, T. A.; Moore, A. L.; Gust, D. J. Am. Chem. Soc. 130 11122 (2008) [11] Ceroni, P. Bergamini, G. Balzani, V. Angew. Chem. Int. Ed. 48 8516 (2009). [12] Perez Inestrosa, E. Montenegro, J.M. Collado, D. Suau, R. Chem. Commun. 11 1085 (2008)

[13] Andreasson, J. Straight, S. D. Bandyopadhyay, S. Mitchell, R. H. Moore, T. A. Moore, A. L. Gust, D. Angew. Chem. Int. Ed. 46 958 (2007). [14] Amelia, M. Baroncini, M. Credi, A. Angew. Chem. Int. Ed. 47 6240 (2008) [15] Margulies, D. Felder, C. E. Melman, G. Shanzer A. J. Am. Chem. Soc. 129 347 (2007) [16] Strack, G. Ornatska, M. Pita, M. Katz, E. J. Am. Chem. Soc. 130 4234 (2007) [17] Sun, W. Zhou, C. Xu, C.-H. Fang, C.-J. Zhang, C. Li, Z.-X. Yan, C.-H. Chem. Eur. J. 14 6342 (2008) [18] Suresh, M. Ghosh, A. Das, A. Chem. Commun. 48 3906 (2008) [19] Andreasson, J. Straight, S. D. Moore, T. A. Moore, A. L. Gust, D. Chem. Eur. J. 15 3936 (2009) [20] S.A. Hussain, P.K. Paul, D. Dey , D. Bhattacharjee , S. Sinha Chemical Physics Letters 450 49 (2007) [21] Kazuhisa Fujimoto, Hisao Shimizu and Masahiko Inouye J. Org. Chem. 69 3271 (2004) [22] H. Liu, Y. Zhou, Y. Yang, W. Wang, L. Qu, C. Chen, D. Liu, D. Zhang and D. Zhu J. Phys. Chem. B 112 6893 (2008) [23] Clelland, C. T. Risca, V. Bancroft C. Nature 399 533 (1999) [24] Mao, C. LaBean, T. H. Reif, J. H. Seeman, N. C. Nature 407 493 (2000) [25] Tanaka, K. Okamotoa, A. Saito I. BioSystems, 81 25 (2005) [26] Sahare, P. D. Sharma, V. K. Mohan, D. Rupasov Spectrochimica Acta Part A 69 1257 (2008) [27] D. Dey, D. Bhattacharjee, S. Chakraborty, S.A Hussain, J. Photochem. Photobiol. A-Chem 252 (2013) 174– 182

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