fluorescence studies of coumarin labelled nanocrystalline...
TRANSCRIPT
Fluorescence studies of
coumarin labelled nanocrystalline cellulose
By
Timothy Mack
Submitted in partial fulfilment of the requirements of the degree
Master of Science
Department of Chemistry
McGill University
Montreal, Quebec Canada
April, 2014
©Timothy Mack, 2014
ii
Abstract
Nanocrystalline cellulose is rapidly emerging as a promising material due
to its impressive mechanical and optical properties. The properties of
nanocrystalline cellulose can be tailored towards specific applications through
surface chemical modification. Thus, there is an impetus to further investigate
the surface properties of these nanostructures. One spectroscopic approach is
through the attachment of fluorescent dyes, which can act as probes of the
surface interface. To this effect, a set of coumarin dyes has been synthesized,
which possesses high quantum yields and exhibits a solvatochromic response to
environments of different polarity. This approach employs well established “Click
Chemistry”, between alkyne functionalized nanocrystalline cellulose and azide
functionalized coumarins. Steady-state fluorescence and time-correlated single-
photon counting Stern-Volmer experiments have been conducted. The findings
suggest that the fluorescently tagged nanocrystalline cellulose exhibits different
Stern-Volmer behavior than small molecule analogues. The question of whether
the nanocrystalline cellulose surface can be modelled as a distribution of micro
environments is examined.
iii
Résumé
La nanocellulose cristalline se démontre comme un matériau intéressant
en raison de ses impressionnantes propriétés mécaniques et optiques. Les
propriétés de la cellulose nanocristalline peuvent être adaptées à des
applications particulières par modification chimique de la surface. C’est la raison
pour laquelle il existe une impulsion d'étudier d’avantage les propriétés de
surface de ces nanostructures. Une méthode spectroscopique se fait par la
fixation de fluorophores qui peuvent agir comme sonde du surface. À cet effet,
un ensemble de colorants coumarine ont été synthetisé. Il possède un
rendement quantique élevé et une réponse solvatochrome à des environnements
de polarité différente. Cette approche emploie une réaction bien établie, le “Click
chemistry". Cela se fait entre la nanocellulose cristalline fonctionnalisée avec
des groupes alcynes et de coumarines fontionnalisés par des groupes d'azoture.
Des expériences de Stern-Volmer ont été mené avec les techniques de
fluorescence à l’état stationaire et la méthode de comptage de photon unique
corrélé au temps. Ils suggèrent que les fluorescence de la nanocellulose
crystalline fonctionalisée avec coumarine montre une réponse Stern-Volmer
différent que celle des petites molécules analogues. La question est de savoir si
la surface de la nanocellulose cristalline peut être modélisée comme une
distribution de micro-environnements.
iv
Acknowledgements
First and foremost, I thank my supervisor Dr. Mark Andrews for the
opportunity to work within his research group, for offering extensive feedback
over the past year and for his guidance.
I would like to acknowledge the support of Dr. Rakesh Singh, with whom I
have consulted with daily on this project. It would have been much more difficult
to get through the project without his invaluable advice. I also would like to thank
all the members of the Andrews research group. Tim Morse, Tim Gonzalez and
Dr. Farshid Hajiaboli have provided me with helpful discussions and insight, and
a continuous supply of coffee.
I am grateful for all the help and support I have received within the McGill
chemistry department. In particular, I am in the debt of Dr. George Rizis and Dr.
Jurek Petlicki, for introducing me to the field of polymer science. I specially thank
Dr. Frederic Morin and Nadim Saade, for patiently helping me to troubleshoot
sample characterization. I thank my classmates Yann Desjardins-Langlais,
Alexei Kazarine and Anjelica Gopal for their support and friendship. I am
especially thankful to Jacqueline Riddle for enduring my sometimes compulsive
behavior and overall eccentricity.
Lastly, I thank my entire family for their steadfast support and
encouragement throughout my studies, and for their interest in my work.
v
Table of Contents
Abstract ................................................................................................................................ ii
Résumé .............................................................................................................................. iii
Acknowledgements ............................................................................................................ iv
Table of Contents ................................................................................................................ v
List of Tables ..................................................................................................................... vii
List of Figures ................................................................................................................... viii
List of symbols and abbreviations ....................................................................................... x
Chapter 1: Introduction ........................................................................................................ 1
1.1 Summary ............................................................................................................ 1
1.2 Nanocrystalline cellulose .................................................................................... 2
1.2.1 Historical overview and structural determination of Cellulose ........................... 4
1.2.2 Chemical modification of nanocrystalline cellulose ....................................... 7
1.2.4 Fluorescent labelling of nanocrystalline cellulose .......................................... 8
1.3 Coumarin dyes .......................................................................................................... 9
1.3.1 Solvatochromism of coumarin dyes ............................................................. 10
1.3.2 Solvent rearrangement and the Franck-Condon principle ........................... 11
1.4 Azide-alkyne Huisgen cycloaddition (“Click Chemistry”) ........................................ 15
1.4.1 Triazole linked coumarins ................................................................................ 17
1.5 Fluorescence lifetime .............................................................................................. 17
1.5.1 The relationship between quantum yield and lifetime ...................................... 20
1.5.2 Fluorescence lifetime techniques and application ........................................... 21
1.6 References .............................................................................................................. 22
Chapter 2: Experimental .................................................................................................... 32
2.1 Materials and methods ............................................................................................ 32
2.1.1 Materials and reagents .................................................................................... 32
2.1.2 Coumarin azides .............................................................................................. 32
2.1.3 NCC click chemistry ......................................................................................... 33
2.1.4 Synthesis of 2-propynyl-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside .............. 34
2.1.5 Synthesis of model coumarin gluocoside (DEA-M): ........................................ 34
2.1.5 Instrumentation ................................................................................................ 36
2.1.6 Preparation of Fluorescence and Extinction samples ..................................... 36
2.1.7 Determination of Quantum Yields .................................................................... 37
2.1.7 DLS measurements ......................................................................................... 38
2.1.8 TCSPC data acquisition ................................................................................... 38
2.1.9 TCSPC Data Fitting ......................................................................................... 38
vi
2.1.10 NCC-DEA and NCC-HC PEGDA films .......................................................... 39
2.1.11 2D-Lifetime mapping fits .................................................................................... 40
2.1.12 Stern-Volmer quenching studies .................................................................... 40
2.2 Background Theory ................................................................................................. 41
2.2.1 Time-correlated single-photon counting ............................................................... 41
2.2.2 TCSPC data ..................................................................................................... 43
2.2.3 Non-negative least-squares and indicators of goodness-of-fit ........................ 45
2.2.4 Lifetime distributions ........................................................................................ 46
2.2.5 Fluorescence quenching and Stern-Volmer Plots ........................................... 48
2.3 Lippert-Mataga polarity index (Δf) ........................................................................... 49
2.4 XPS surface characterization .................................................................................. 50
2.5 References .............................................................................................................. 51
Chapter 3: Results and Discussion ................................................................................... 54
3.1 Steady-state characterization.................................................................................. 54
3.1.1 ATR-FTIR analysis ............................................................................................... 54
3.1.2 Extinction of coumarin labelled NCC suspensions .............................................. 55
3.1.3 Aggregation of NCC ............................................................................................. 56
3.1.4 Solvatochromism .................................................................................................. 58
3.1.5 Emission intensity variation of coumarin triazole products .................................. 60
3.1.6 Quantum Yield determination of DEA-M and of NCC-DEA ................................. 63
3.1.7 Emission dependence on excitation wavelength ................................................. 65
3.1.8 Summary of Steady-State characterization ..................................................... 66
3.2 Stern-Volmer Analysis ........................................................................................ 67
3.2.1 Stern-Volmer analysis of DEA-M ..................................................................... 68
3.2.2 NCC-coumarin lifetime fits ............................................................................... 70
3.2.3 Stern-Volmer analysis of NCC-DEA ................................................................ 74
3.3 Lifetime mapping of NCC-coumarin in PEGDA .................................................. 77
3.4 Summary of Stern-Volmer and PEGDA experiments ......................................... 78
3.8 References .......................................................................................................... 79
Chapter 4: Conclusions and Future Work ......................................................................... 83
4.1 Conclusions ............................................................................................................. 83
4.2 Future work ............................................................................................................. 84
4.3 References .............................................................................................................. 86
Appendix A: Characterization data for model compound DEA-M ..................................... 87
Appendix B: Excitation and Emission maxima of NCC-coumarins ................................... 90
Appendix C: N-methyl aniline Extinction spectra .............................................................. 93
Appendix D: PEGDA Emission spectra ............................................................................. 94
vii
List of Tables
Table 2.1: Surface XPS Results .................................................................................................... 50
Table 3.1: NCC-DEA lifetime data ................................................................................................. 72
Table 3.2: NCC-HC lifetime data .................................................................................................. 73
Table 3.3: NCC-AC lifetime data ................................................................................................... 73
Table B1: Data for NCC-DEA ........................................................................................................ 90
Table B2: Data for NCC-HC .......................................................................................................... 91
Table B3: Data for NCC-AC .......................................................................................................... 92
viii
List of Figures
Figure 1.1: Schematic of a Cellulose Fiber. (Copied from Ref2, and reprinted under created
commons license. [CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via InTech
publications. ...................................................................................................................................... 3
Figure 1.2: Transmission electron micrograph of NCC ................................................................... 4
Figure 1.3: Chemical structure of cellulose with carbon numbering system. .................................. 6
Figure 1.4: Examples of chemical modifications at the C6 position. (Copied from Ref.35 and
reprinted with permission from JOHN WILEY AND SONS, Copyright © 2011 Canadian Society for
Chemical Engineering) .................................................................................................................... 8
Figure 1.5: Chemical structure of coumarin (2H-chromen-2-one) ................................................. 10
Figure 1.6: Illustration of the Franck-Condon principle. By Mark M. Somoza (Own work)
(Reprinted under creative commons license.) [GFDL ((http://www.gnu.org/copyleft/fdl.html), CC-
BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/)], ...................................................... 12
via Wikimedia Commons ............................................................................................................... 12
Figure 1.7: Illustration of solvatochromic emission shift. The large black circles represent the
fluorophore while the blue circles represent the solvent. The parabolas represent the potential
energy surfaces of the various states: A, B, C, D (Figure adapted from Ref.68) .......................... 14
Figure 1.8: Azide-alkyne Huisgen cycloaddition reaction scheme ................................................ 16
Figure 1.9: Typical Jablonskii diagram for fluorescence emission. By Jacobkhed (Own work)
(Reprinted under creative commons license.) [GFDL (http://www.gnu.org/copyleft/fdl.html), CC-
BY-SA-1.0 (http://creativecommons.org/licenses/by-sa/2.5-2.0-1.0)], via Wikimedia Commons . 19
Figure 2.1: Stuctures of synthesized azido-coumarins.................................................................. 33
Figure 2.2: Structure of model coumarin glucoside (DEA-M) ........................................................ 35
(2S,3S,4S,5S,6S)-2-(acetoxymethyl)-6-((1-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1H-1,2,3-
triazol-4-yl)methoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate ...................................................... 35
Figure 2.3: Chemical Structures of PEGDA and radical initiator 2-hydroxy-2methylpropiophenone
....................................................................................................................................................... 40
Figure 2.4: Photograph of Mini-Tau fluorescence spectrometer® (Edinburgh photonics) ............ 42
Figure 2.5: Top panel: flow chart for TCSPC electronics. The dotted lines represent electronic
signals, while the solid lines are photon displacements. (Figure adapted from Ref.11) ............... 43
Figure 2.6: Top panel: Typical fluorescence lifetime decay plot, with IRF (blue) and decay data
(red) and fit (black). Bottom panel: Residual plot ......................................................................... 44
Figure 3.1: ATR-FTIR spectra of the clicked NCC. Major peaks for the NCC-DEA (black curve)
are 3334 cm-1 (O-H), 2912 cm-1 (C-H),, 1644 cm-1 (C=O, amide). The major identifiable peaks
for DEA (red curve) are 2972cm-1 (C-H), 2116 cm-1 (N3), 1702 cm-1 (C=O), 1616 cm-1 (C=C).
....................................................................................................................................................... 55
ix
Figure 3.2: Extinction spectra of aqueous NCC-Coumarin suspensions ..................................... 56
Figure 3.3: Size distribution of aqueous NCC-DEA suspension ................................................... 57
Figure 3.4: Confocal microscopy image of NCC-HC suspended in DMF 10x objective. .............. 58
Figure 3.5: Left panel: Solvatochromic shift of emission spectra of NCC-HC, in DMSO (black) and
water (red). Right panel: Visualization of solvatochromism by excitation of various samples with
366nm light: HC in ethanol (left), NCC-HC in water (center) and NCC-HC in dimethyl sulfoxide
(right). The purple glow present on the HC vial is due to the UV-lamp. ....................................... 59
Figure 3.6: Fluorescence intensity emission of NCC-DEA suspensions....................................... 62
Figure 3.7: Comparison of Fluorescence emission intensity of sonicated (red) and unsonicated
(black) aqueous suspensions of NCC-HC ..................................................................................... 62
Figure 3.8: Extinction and emission spectra for quantum yield determination of DEA-M and NCC-
DEA in DMSO ................................................................................................................................ 64
Figure 3.9: Emission spectra of NCC-HC in DMF excited at 351 nm (red) and 405 nm (black) ... 66
Figure 3.10: Stern-Volmer quenching plots for DEA-M in acetonitrile, in terms of integrated
emission intensities (red), and lifetimes (black) ............................................................................. 70
Figure 3.11: Stern-Volmer quenching plots for NCC-DEA in DMSO, in terms of integrated
emission intensities (black), and mean lifetimes (red)................................................................... 76
Figure 3.12: Comparison of lifetime Stern-Volmer quenching plots for NCC-DEA in DMSO,
between a tailfitted stretched exponential fit (black), tail-fitted biexponential (blue) and
triexponential reconvolution fit (red) .............................................................................................. 76
Figure 3.13: Left panel: confocal fluorescence emission image of NCC-DEA embedded in a
photocured PEDGA film. Right panel: Same image, but with 2D lifetime map superimposed ... 78
Figure A1: DEA-M ATR-FTIR ........................................................................................................ 87
Figure A2: DEA-M MS ................................................................................................................... 87
Figure A3: 1H NMR ........................................................................................................................ 88
Figure A4: 13C NMR ....................................................................................................................... 88
Figure D1: Qualitative extinction spectra comparing high concentration N-methyl aniline (black)
and low concentration N-methyl anline (red) in DMSO ................................................................. 93
Figure D1: Emission spectrum of neat PEGDA (MW 575), excited at 400 nm ............................. 94
Figure D2: Emission spectrum of NCC-DEA in crosslinked PEGDA excited at 366nm. The
shoulder at 421 is attributed to the PEGDA fluorescence. ............................................................ 94
x
List of symbols and abbreviations
2D Two-dimensional
ATR Attenuated Total Reflectance
AC Acetyl Coumarin
ACN Acetonitrile
CNCs Cellulose Nanocrystals
DEA Diethyl amino coumarin
DEA-M Model Diethyl amino coumarin
DLS Dynamic Light Scattering
DMF Dimethylformamide
DMSO Dimethyl Sulfoxide
EDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
FLIM Fluorescence lifetime imaging microscopy
FRET Forster resonance energy transfer
FTIR Fourier Transform Infrared Spectrometry
HC Hydroxy coumarin
IRF Instrumental Response Function
MS Mass Spectrometry
NCC Nanocrystalline Cellulose
NMR Nuclear Magnetic Resonance
PEGDA Poly(ethylene glycol) diacrylate
QY Quantum Yield
SV Stern-Volmer
TCSPC Time-Correlated Single-Photon Counting
TEM Transmission Electron Microscopy
TEMPO 2,2,6,6-tetramethylpiperidine-1-oxyl
TMV Tobacco Mosaic Virus
UV Ultraviolet
XPS X-ray photoelectron spectroscopy
1
Chapter 1: Introduction
1.1 Summary
Nanocrystalline cellulose (NCC) consists of rod-like nanoparticles obtained
via the hydrolysis of cellulose.1 These rods have impressive mechanical and
optical properties and are biodegradable.2 NCC dispersed in water can form
stable colloidal mixtures, allowing for functional group modification. Polymer
composites incorporating NCC are being developed for several commercial
applications.3
This thesis aims to study NCC through the fluorescent labelling of various
triazole-substituted coumarin derivatives synthesized through the modular route
of “click” chemistry. This covalent attachment of fluorescent dyes offers
improvement over ionic and hydrogen bonded binding approaches. Coumarin
derivatives are employed as fluorescent probes because their electronic
properties can be systematically modified through functional group
transformation. This allows for a rational approach to probe the local solvent
environment of NCC through solvatochromism and quenching studies.
Furthermore, it enables the use of confocal fluorescence microscopy as a tool to
study the incorporation of NCC into various polymer composites.
Chapter 2 describes the synthesis and preparation of the coumarin
labelled NCC and of a related molecular analogue. A simple synthetic protocol
2
was developed, and the materials were characterized through a number of
commonly employed techniques.
Chapter 3 deals with all the work relating to spectroscopic measurements,
including time-resolved fluorescence lifetime experiments. The fluorescence
decay data could not be fit using a monoexponential decay in a least-squares
procedure. It also explores the use of quenching experiments in order to probe
the accessibility of fluorophores attached to the NCC. Finally, it includes a brief
look at incorporating NCC in films of cross-linked poly(ethylene glycol) diacrylate,
and successfully employing fluorescence lifetime imaging microscopy (FLIM) to
image these films.
The concluding chapter identifies areas in which future work is needed,
and offers an assessment on the relative merits of the research presented.
1.2 Nanocrystalline cellulose
Natural cellulose contains a mixture of crystalline and amorphous (or
paracrystalline) regions, as depicted in Figure 1.1.2,4,5 When cellulose fibers are
heated in strong acid6, such as HCl or H2SO4, the amorphous regions are
selectively hydrolyzed, resulting in a colloidal suspension of the remaining
nanocrystalline sections. These high-aspect ratio nanocrystalline sections are
referred to as NCC or as cellulose nanocrystals (CNCs).
3
Figure 1.1: Schematic of a Cellulose Fiber. (Copied from Ref2, and reprinted
under created commons license. [CC-BY-SA-3.0
(http://creativecommons.org/licenses/by-sa/3.0)], via InTech publications.
NCC can be imaged using transmission electron microscopy7 (Figure
1.2). NCC that is obtained from cotton or black spruce through acid hydrolysis is
rod shaped, with dimensions of approximately 5nm in width and 100-350 nm in
length.8 Although the use of highly concentrated sulphuric acid is currently the
leading method for NCC production9, more environmentally sustainable
procedures10 that avoid the use of acid are being developed. NCC is a material
of broad interest the research and industrial communities. Potential
applications11,12 include polymer nanocomposites13, transparent films14,
catalysis15 as well as optical encryption.16 This interest continues to evolve as
new methods emerge to improve the reproducibility of chemical reactions
involving NCC.17
4
Figure 1.2: Transmission electron micrograph of NCC
1.2.1 Historical overview and structural determination of Cellulose
Cellulose-based materials have been readily employed throughout human
history, yet the extraction of cellulose from natural biomaterials and subsequent
structural identification are relatively new developments. The French chemist,
Anselme Payen, was the first to coin the term “cellulose” in 1838 after
discovering that cellulose was a common structural component of many different
types of vegetation. The term has since been extended to include the structural
material of algae, trees, fungi and even some animal organisms.12 Progress on
5
cellulose structure determination would only continue nearly a century later when
Willstätter and Zechmeister elucidated the cellulose molecular formula in 1921,18
establishing the relationship between cellulose and its monomer glucose.19
X-ray diffraction experiments performed in the twentieth century showed that
cellulose is a polysaccharide, consisting of two repeating D-glucopyranose
units20 (the disaccharide cellobiose).21 Glucopyranose units are linked together
via an equatorial β glycosidic ether bond between the C1’ and C4 positions
(Figure 1.3). These glucopyranose units adopt a 4C1 chair conformation22,23 and
adjacent rings are twisted in relation to each other by 180 , consistent with an
observed 21 screw axis, which reduces steric repulsion24. This geometry is also
stabilized by the inherent hydrogen bonding between the C3 hydroxyl group and
the neighboring ring atom25, stiffening the chain.12 Cellulose consists of an
extended linear chain of thousands of cellobiose units.26 The hydroxyl groups
along the cellulose chain allow for extensive intra and intermolecular hydrogen
bonding and Van der Waals interactions27, driving the formation of highly ordered
crystalline structures.
6
Figure 1.3: Chemical structure of cellulose with carbon numbering system.
A complete understanding of the crystalline nature of cellulose was not
achieved until late in the twentieth century. Initial X-ray diffraction studies on
native cellulose had assigned a monoclinic space group with a unit cell of two
cellulose chains.28 Later, solid-state NMR studies29 pointed to the existence of
two distinct crystalline allomorphs within native cellulose, which are identified as I
α and I β. In 2003, the crystal structures of both allomorphs were solved30,31: I α
adopts a triclinic crystal lattice and Iβ a monoclinic lattice. The ratio of these
allomorphs depends on the biological source of the cellulose. Bacterial
celluloses are mainly I α, whereas I β is dominant in plants and animals.12
Cellulose Iβ is the more thermodynamically stable allomorph. The conversion of
allomorph Iα to Iβ has been demonstrated.32 Other cellulose polymorphs been
discovered and are being investigated, in part due to the increase of accessibility
of hydroxyl groups for chemical modification.33
7
1.2.2 Chemical modification of nanocrystalline cellulose
The ability to functionalize the surface of colloidal particles is important in
order to impart new properties suited for specific function. The hydroxyl groups
along the cellulose chain provide a chemical handle for organic reactions, whose
chemistry is well understood in the mature field of carbohydrate chemistry.34 The
challenge with NCC is the fact that most of the hydroxyl groups remain
inaccessible since they remain buried within the nanocrystal: only the surface
chains of cellulose are chemically accessible. Moreover, due to the chirality of
NCC, only every second D-glucose unit will have an accessible C6 hydroxyl
group.11 Examples of reactions1, 35 are given in Figure 1.4.
The hydroxyl group at the C6 position is more chemically reactive than the
C2 and C3 hydroxyl.12 Thus the C6 position has become the focus of
fundamental group transformations. For example, introduction of carboxylic acid
functionality offers a flexible chemical handle for further derivatization. In
practice, this is accomplished through TEMPO-mediated oxidation36 of primary
alcohols37. This carboxylic acid can then be covalently attached to another
molecule through 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)
coupling.38,39
8
Figure 1.4: Examples of chemical modifications at the C6 position. (Copied from
Ref.35 and reprinted with permission from JOHN WILEY AND SONS, Copyright ©
2011 Canadian Society for Chemical Engineering)
1.2.4 Fluorescent labelling of nanocrystalline cellulose
Several research groups have published studies involving fluorescently
tagged NCC within the past few years.40-44 These studies focus primarily on
synthesis and structural characterization and include little photophysical data.
Zhang et al. published findings focused on limited studies of fluorescence
quenching of pyrene-labelled NCC45. The dearth of work involving time-resolved
9
fluorescence characterization techniques applied to labelled NCC is one of the
justifications for the work performed in this thesis.
One of the main goals of our research was to investigate the solvent-NCC
interface using fluorescent probes. Coumarin fluorophores, which display strong
solvatochromism, are especially well suited for this task. Only two studies
involving coumarins covalently attached to NCC have been published thus far. 43,
47 Neither study has attempted to probe the interface between NCC and its
surroundings through these probes.
1.3 Coumarin dyes
Coumarin46 (here referring solely to the compound as shown in Figure 1.5
and not the class of coumarin molecules) is a naturally occurring compound47
that was first successfully synthesized by William Perkin in the mid-nineteenth
century48 and was henceforth commonly used as an ingredient in perfumes.
Coumarin derivatives offer promising numerous medical applications, as
anticoagulants49, anti-cancer compounds50 and anti-depressants.51 It is also
interesting to note that coumarin glucoside natural products have been
identified52, in addition to synthetic compounds53. Fluorescent coumarins54
have been employed as laser dyes.55,56 They have been successfully used to
image cells through fluorescence lifetime microscopy57 and adsorbed onto native
10
celluloses in order to determine surface polarity.58,59 These fluorophores are
especially attractive when used as fluorescent probes of polymer domains due to
their high sensitivity to their surrounding environment.60-62 This sensitivity
manifests itself in the phenomenon of solvatochromism.63,64
Figure 1.5: Chemical structure of coumarin (2H-chromen-2-one)
1.3.1 Solvatochromism of coumarin dyes
The solvatchromic effect refers to a relative shift of electronic absorption and
or emission of a substance caused by interactions with its host environment. For
example, coumarin-48156 has an emission maximum of 431 nm when dissolved
in hexane. This shifts to 480 nm when the dye is dissolved in ethyl acetate.65 A
relative shift of an emission or absorbance maximum to higher wavelength is
called positive solvatochromism, or a bathochromic shift, whereas a shift to lower
wavelength is called negative solvatochromism, or a hypsochromic shift. The
specific interactions governing solvatochromism are complex, and depend on
specific dye (coumarin) -solvent combinations66. Simply put, dipole-dipole
interactions between the solvent and solute can lead to stabilization or
11
destabilization of ground and/or excited states of the solute dye molecule.
Generally, it is possible to study a solvatochromic trends over a range of solvent
polarity. Although the term “solvent polarity” is imprecise,67 several polarity scale
models have been developed67-70, to yield a polarity index for a given solvent.
Coumarin molecules are featured in photophysical studies that sometimes probe
the effects of solvent polarity.65, 71-72 Viscosity can also play a role in some
coumarin molecules that show solvatochromism.73 This is revealed by a closer
examination of the role of Franck-Condon factors in the phenomenon. We turn to
a brief explanation of these factors next.
1.3.2 Solvent rearrangement and the Franck-Condon principle
In the previous section, a qualitative description of the solvatochromic effect
was presented in terms of spectral shifts due to solute-solvent interactions. This
description can be further expounded by appealing to the Franck-Condon
principle74-76 in relation to solvation dynamics of coumarins. The Franck-Condon
principle states that the likelihood of a particular vibronic transition depends on
the wavefunction overlap of the initial and final vibrational states. Since a
transition occurs on a near-instantaneous timescale, the positions of the nuclei
are unchanged. The resulting excited vibrational state will quickly decay to the
lowest vibrational state through internal conversion. An illustration of this
12
phenomenon is provided in Figure 1.6. It effectively explains why fluorophores
typically exhibit a Stokes’ shift, in other words an absorption wavelength which is
of higher energy than the emitted photon.
Figure 1.6: Illustration of the Franck-Condon principle. By Mark M. Somoza
(Own work) (Reprinted under creative commons license.) [GFDL
((http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0
(http://creativecommons.org/licenses/by-sa/3.0/)],
via Wikimedia Commons
When a solvated coumarin molecule is excited, its excited state dipole differs
from that of its ground state. For many coumarins, the magnitude of the dipole
13
moment is larger in the excited state.77, 78 The change in dipole moment of the
coumarin will induce a reconfiguration of the surrounding solvent molecules, in
order to minimize the repulsive potential energy interactions. This is shown
schematically in Figure 1.7 (state B to state C). The timescale of solvent
rearrangement is on the order of 10-10 seconds68 for low-viscosity solvents, so the
coumarin must have a longer fluorescence lifetime in order to observe the
solvatochromic shift corresponding to an energy minimization. The transition
from the lowest excited state goes through an intermediate Franck-Condon state
(state D in Figure 1.7), in which the coumarin dipole returns to its ground state,
but the solvent is still in its reoriented state. Lastly, the system returns to the
initial ground state upon solvent relaxation.
14
Figure 1.7: Illustration of solvatochromic emission shift. The large black circles
represent the fluorophore while the blue circles represent the solvent. The
parabolas represent the potential energy surfaces of the various states: A, B, C,
D (Figure adapted from Ref.68)
Given the propensity for coumarin dye molecules to respond to changes in
the local polarity of their solvent environment, covalent attachment of different
coumarin moieties to NCC may give insight into the local polarity of the solvent
environment of the dye in the vicinity of the NCC surface. Most likely, there will
be a distribution of sites on the NCC surface. This distribution may manifest itself
in a broadening of the various spectral responses, including perturbations of the
15
fluorescence lifetime. Moroever, non-uniform site distributions might show up in
anomalies in Stern-Volmer quenching, since some dye molecules may be more
accessible to quenching species than others. It remains crucial to such studies
to be able to achieve covalent attachment of the chromophore to the NCC
surface. In the next section we examine some of the chemical methods available
to achieve covalent bonding.
1.4 Azide-alkyne Huisgen cycloaddition (“Click Chemistry”)
Initially developed by Huisgen in the early 1960s79 and popularized by
Sharpless et al. forty years later80, the 1,4-azide-alkyne cycloaddition is an
efficient method of modular organic synthesis.81 The reaction, shown in Figure
1.8, is a cycloaddition between an azide (N3) group and an alkyne to form a
1,2,3-triazole ring. The most frequent variant of the reaction employs a Cu(I)
catalyst, which was first reported in 200282, and whose mechanism was later
elucidated through DFT calculations in 2004.83 The utility of this reaction comes
from the fact that it is a high-yielding reaction which can performed under
aqueous conditions at room temperature. Cu(I) is generated through the
reduction of Cu(II) using an agent such as sodium ascorbate. The procedure is
straightforward. The advantage of using such a reaction in the context of this
thesis stems from the fact that the click reaction can be conducted in water
16
solvent where NCC suspensions can be stabilized.84 Click chemistry in this
fashion has been elaborated by several research groups.39,85 Moreover, the
selectivity to incorporate an alkyne group at the C6 position of NCC through an
EDC coupling reaction provides a chemical handle that can be easily covalently
clicked to an azide functionalized coumarin.86
Figure 1.8: Azide-alkyne Huisgen cycloaddition reaction scheme
We intentionally focused on generating a triazole link between NCC and
the coumarin as a way of enhancing conjugation within the dye molecule, which
process also provides a convenient spectroscopic handle to confirm the
presence of the triazole via x-ray photoelectron spectroscopy (XPS). In some
cases, fluorescence from the clicked coumarin was enhanced by incorporating
the linkage, particularly in the case of the poorly emissive coumarin azides. We
turn to a brief review of the literature on triazole-substituted coumarin molecules.
17
1.4.1 Triazole linked coumarins
Coumarins incorporating a triazole group have been a subject of interest
in the literature in recent years, due to observations that they exhibit increased
fluorescence brightness and red-shifted emissions in comparison with structurally
similar coumarins.87, 88 In the case of coumarin azides, the difference is stark.
Coumarin azides exhibit very low quantum efficiency. This has been attributed to
the presence of the N3 group which is strongly electron withdrawing.86 Post click
reaction, the quantum yield can increase significantly if the triazole ring extends
conjugation.89 It is also possible to cleave the triazole ring, thereby “unclicking”
the product back into alkyne and azide precursor components through high
powered sonication.90 This is unprecedented in the “click” chemistry literature,
where it has been assumed that the cycloaddition product exhibits poor
thermodynamic reversibility. Overall, triazole-substituted coumarins are a
synthetically accessible via dipolar cycloaddtion reactions. This suggests a
simplified entry into coumarin dye molecules that can be covalently bound under
heterogeneous reaction conditions to NCC.
1.5 Fluorescence lifetime
Fluorecence lifetime is a well-established concept that has been
comprehensively described in a number of textbooks.68, 91-92 A fluorescence
18
lifetime is a measure of the kinetic stability of a fluorescence excited state.
Given that the excited state of a fluorophore is thermodynamically unstable, it will
always return to the ground state releasing energy to its surroundings, whether in
the form of an emitted photon or through a non-radiative process, as shown in
Figure 1.9. Observation of single fluorescence lifetime decay will result in one of
these outcomes, which are independent of each other. Consequently, the nature
of fluorescence decay is probabilistic.
19
Figure 1.9: Typical Jablonskii diagram for fluorescence emission. By Jacobkhed
(Own work) (Reprinted under creative commons license.) [GFDL
(http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-1.0
(http://creativecommons.org/licenses/by-sa/2.5-2.0-1.0)], via Wikimedia
Commons
To illustrate this, consider the case where a larger number of identical
fluorophores is observed upon excitation (the theoretical equivalent of a single
fluorophore which is excited and its decay observed a large number of times).
20
Assume that the fluorescence decay is in a monoexponential form provided in
equation 1.1. [I*] is the concentration of fluorophores in the excited state at time
t, [I0*] represents the entirety of the population in the excited state, and is the
fluorescence lifetime. When the time t is equal to , the ratio of [I*]/[I0*] is equal to
1/e. This means that approximately 36.7% of the population will remain in the
excited state after length of time . Put more simply, there is a 63.3% chance
that a given fluorophore will decay within one fluorescence lifetime.
[ ] [ ]
(1.1)
1.5.1 The relationship between quantum yield and lifetime
The relationship between the fluorescence lifetime and quantum yield is
relatively straightforward; however, it is worth to emphasizing because it
underlines the complementarity of fluorescence lifetimes with respect to emission
intensity data. First, the fluorescence lifetime can be defined by two parameters:
a radiative decay constant, and a non-radiative decay constant, as represented
in equation 1.2. The quantum yield (QY) is usually presented in terms of the ratio
of the number of photons absorbed to the number of emitted photons of a
fluorophore in a specific environment. This can also be written in terms of a
radiative and non-radiative decay constant as shown in equation 1.3. Both terms
share a common denominator, and it is trivial to show that the quantum yield is
21
equal to the product of the fluorescence lifetime and the radiative decay constant
(krad). Thus, if the quantum yield of a coumarin dye varies as a function of its
solvent environment, the fluorescence lifetime will also vary accordingly.
(1.2)
(1.3)
1.5.2 Fluorescence lifetime techniques and application
There are two established techniques widely employed in the fluorescence
studies of biological systems68 for acquiring lifetime decay data: pulse and phase
fluorometry.92-93 As their names suggest, the first technique involves time
domain data, whereas the latter collects data in the frequency domain. These
techniques should be considered “equivalent or complementary but by no means
competitive.”.94 These techniques have also been used in conjunction with
confocal microscopy to create a powerful imaging tool: fluorescence lifetime
imaging microscopy95 (FLIM). The use of FLIM is widespread in biophysics, and
while it will be only given rudimentary attention in the context of this thesis, it may
soon become a valuable method to image NCC within biological systems.43 One
advantage of imaging with fluorescence lifetimes is that they are often
22
concentration independent, thus making it possible to distinguish between
different environments where the concentrations of fluorophore are unknown.
1.6 References
1. Dufresne, A., Nanocellulose: a new ageless bionanomaterial. Materials
Today 2013, 16 (6), 220-227.
2. Chengjun, Z.; Qinglin, W., Recent Development in Applications of
Cellulose Nanocrystals for Advanced Polymer-Based Nanocomposites by Novel
Fabrication Strategies. In Nanocrystals - Synthesis, Characterization and
Applications, Neralla, S., Ed. InTech: 2012.
3. Siqueira, G.; Bras, J.; Dufresne, A., Cellulosic Bionanocomposites: A
Review of Preparation, Properties and Applications. Polymers 2010, 2 (4), 728-
765.
4. Astbury, W. T.; van, I.; Preston, R. D.; Cox, E. G.; Preston, J. M., General
discussion. Transactions of the Faraday Society 1933, 29 (140), 71.
5. Frey-Wyssling, A., The Fine Structure of Cellulose Microfibrils. Science
1954, 119 (3081), 80-2.
6. Nickerson, R. F.; Habrle, J. A., Cellulose Intercrystalline Structure - Study
by Hydrolytic Methods. Industrial and Engineering Chemistry 1947, 39 (11),
1507-1512.
7. Dong, X. M.; Kimura, T.; Revol, J.-F.; Gray, D. G., Effects of Ionic Strength
on the Isotropic−Chiral Nematic Phase Transition of Suspensions of Cellulose
Crystallites. Langmuir 1996, 12 (8), 2076-2082.
8. Beck-Candanedo, S.; Roman, M.; Gray, D. G., Effect of reaction
conditions on the properties and behavior of wood cellulose nanocrystal
suspensions. Biomacromolecules 2005, 6 (2), 1048-54.
23
9. Rebouillat, S., State of the Art Manufacturing and Engineering of
Nanocellulose: A Review of Available Data and Industrial Applications. Journal of
Biomaterials and Nanobiotechnology 2013, 04 (02), 165-188.
10. Leung, A. C.; Hrapovic, S.; Lam, E.; Liu, Y.; Male, K. B.; Mahmoud, K. A.;
Luong, J. H., Characteristics and properties of carboxylated cellulose
nanocrystals prepared from a novel one-step procedure. Small 2011, 7 (3), 302-
5.
11. Habibi, Y.; Lucia, L. A.; Rojas, O. J., Cellulose nanocrystals: chemistry,
self-assembly, and applications. Chemical Reviews 2010, 110 (6), 3479-500.
12. Dufresne, A., Nanocellulose, From Nature to High Performance Tailored
Materials. De Gruyter: 2012.
13. Cranston, E. D.; Gray, D. G., Morphological and optical characterization of
polyelectrolyte multilayers incorporating nanocrystalline cellulose.
Biomacromolecules 2006, 7 (9), 2522-30.
14. Yang, H.; Tejado, A.; Alam, N.; Antal, M.; van de Ven, T. G., Films
prepared from electrosterically stabilized nanocrystalline cellulose. Langmuir
2012, 28 (20), 7834-42.
15. Cirtiu, C. M.; Dunlop-Brière, A. F.; Moores, A., Cellulose nanocrystallites
as an efficient support for nanoparticles of palladium: application for catalytic
hydrogenation and Heck coupling under mild conditions. Green Chemistry 2011,
13 (2), 288.
16. Zhang, Y. P.; Chodavarapu, V. P.; Kirk, A. G.; Andrews, M. P.,
Nanocrystalline cellulose for covert optical encryption. Journal of Nanophotonics
2012, 6 (1), 063516-1-063516-9.
17. Labet, M.; Thielemans, W., Improving the reproducibility of chemical
reactions on the surface of cellulose nanocrystals: ROP of epsilon-caprolactone
as a case study. Cellulose 2011, 18 (3), 607-617.
18. Dumitriu, S., Polysaccharides : structural diversity and functional
versatility. Marcel Dekker: New York, 2005.
24
19. Irvine, J. C.; Hirst, E. L., LXIV.?The constitution of polysaccharides. Part
VI. The molecular structure of cotton cellulose. Journal of the Chemical Society,
Transactions 1923, 123 (0), 518.
20. Ferrier, W. G., The crystal and molecular structure of β-D-glucose. Acta
Crystallographica 1963, 16 (10), 1023-1031.
21. Jacobson, R. A.; Wunderlich, J. A.; Lipscomb, W. N., The crystal and
molecular structure of cellobiose. Acta Crystallographica 1961, 14 (6), 598-607.
22. Chu, S. S. C.; Jeffrey, G. A., The refinement of the crystal structures of β-
D-glucose and cellobiose. Acta Crystallographica Section B Structural
Crystallography and Crystal Chemistry 1968, 24 (6), 830-838.
23. Marszalek, P. E.; Oberhauser, A. F.; Pang, Y. P.; Fernandez, J. M.,
Polysaccharide elasticity governed by chair-boat transitions of the glucopyranose
ring. Nature 1998, 396 (6712), 661-4.
24. Hon, D. N. S., Cellulose: a random walk along its historical path. Cellulose
1994, 1 (1), 1-25.
25. Jarvis, M., Chemistry: cellulose stacks up. Nature 2003, 426 (6967), 611-
2.
26. Klemm, D.; Heublein, B.; Fink, H. P.; Bohn, A., Cellulose: fascinating
biopolymer and sustainable raw material. Angewandte Chemie, International
Edition in English 2005, 44 (22), 3358-93.
27. French, A. D.; Miller, D. P.; Aabloo, A., Miniature crystal models of
cellulose polymorphs and other carbohydrates. International Journal of Biological
Macromolecules 1993, 15 (1), 30-6.
28. Gardner, K. H.; Blackwell, J., The structure of native cellulose.
Biopolymers 1974, 13 (10), 1975-2001.
29. Atalla, R. H.; Vanderhart, D. L., Native cellulose: a composite of two
distinct crystalline forms. Science 1984, 223 (4633), 283-5.
30. Nishiyama, Y.; Langan, P.; Chanzy, H., Crystal Structure and Hydrogen-
Bonding System in Cellulose Iβ from Synchrotron X-ray and Neutron Fiber
25
Diffraction. Journal of the American Chemical Society 2002, 124 (31), 9074-
9082.
31. Nishiyama, Y.; Sugiyama, J.; Chanzy, H.; Langan, P., Crystal structure
and hydrogen bonding system in cellulose I(alpha) from synchrotron X-ray and
neutron fiber diffraction. Journal of the American Chemical Society 2003, 125
(47), 14300-6.
32. Debzi, E. M.; Chanzy, H.; Sugiyama, J.; Tekely, P.; Excoffier, G., The Iα
→Iβ transformation of highly crystalline cellulose by annealing in various
mediums. Macromolecules 1991, 24 (26), 6816-6822.
33. Zugenmaier, P., Conformation and packing of various crystalline cellulose
fibers. Progress in Polymer Science 2001, 26 (9), 1341-1417.
34. Capon, B., Mechanism in carbohydrate chemistry. Chemical Reviews
1969, 69 (4), 407-498.
35. Peng, B. L.; Dhar, N.; Liu, H. L.; Tam, K. C., Chemistry and Applications of
Nanocrystalline Cellulose and Its Derivatives: A Nanotechnology Perspective.
Canadian Journal of Chemical Engineering 2011, 89 (5), 1191-1206.
36. Montanari, S.; Rountani, M.; Heux, L.; Vignon, M. R., Topochemistry of
carboxylated cellulose nanocrystals resulting from TEMPO-mediated oxidation.
Macromolecules 2005, 38 (5), 1665-1671.
37. de Nooy, A. E. J.; Besemer, A. C.; van Bekkum, H., Highly selective
nitroxyl radical-mediated oxidation of primary alcohol groups in water-soluble
glucans. Carbohydrate Research 1995, 269 (1), 89-98.
38. Hemraz, U. D.; Boluk, Y.; Sunasee, R., Amine-decorated nanocrystalline
cellulose surfaces: synthesis, characterization, and surface properties. Canadian
Journal of Chemistry-Revue Canadienne De Chimie 2013, 91 (10), 974-981.
39. Filpponen, I.; Argyropoulos, D. S., Regular linking of cellulose
nanocrystals via click chemistry: synthesis and formation of cellulose
nanoplatelet gels. Biomacromolecules 2010, 11 (4), 1060-6.
26
40. Abitbol, T.; Palermo, A.; Moran-Mirabal, J. M.; Cranston, E. D.,
Fluorescent labeling and characterization of cellulose nanocrystals with varying
charge contents. Biomacromolecules 2013, 14 (9), 3278-84.
41. Dong, S.; Roman, M., Fluorescently labeled cellulose nanocrystals for
bioimaging applications. Journal of the American Chemical Society 2007, 129
(45), 13810-1.
42. Ilari, F.; Hasan, S.; Dimitris, S. A., Photoresponsive Cellulose
Nanocrystals. Nanomaterials and Nanotechnology 2011, 1 (1).
43. Mahmoud, K. A.; Mena, J. A.; Male, K. B.; Hrapovic, S.; Kamen, A.;
Luong, J. H., Effect of surface charge on the cellular uptake and cytotoxicity of
fluorescent labeled cellulose nanocrystals. ACS Applied Materials & Interfaces
2010, 2 (10), 2924-32.
44. Nielsen, L. J.; Eyley, S.; Thielemans, W.; Aylott, J. W., Dual fluorescent
labelling of cellulose nanocrystals for pH sensing. Chemical Communications
(Cambridge) 2010, 46 (47), 8929-31.
45. Zhang, L. Z.; Li, Q.; Zhou, J. P.; Zhang, L. N., Synthesis and
Photophysical Behavior of Pyrene-Bearing Cellulose Nanocrystals for Fe3+
Sensing. Macromolecular Chemistry and Physics 2012, 213 (15), 1612-1617.
46. Abernethy, J. L., The historical and current interest in coumarin. Journal of
Chemical Education 1969, 46 (9), 561.
47. Bourgaud, F.; Poutaraud, A.; Guckert, A., Extraction of coumarins from
plant material (Leguminosae). Phytochemical Analysis 1994, 5 (3), 127-132.
48. Perkin, W. H., VI.?On the artificial production of coumarin and formation of
its homologues. Journal of the Chemical Society 1868, 21 (0), 53.
49. Lino, C. S.; Taveira, M. L.; Viana, G. S. B.; Matos, F. J. A., Analgesic and
antiinflammatory activities of Justicia pectoralis Jacq and its main constituents:
Coumarin and umbelliferone. Phytotherapy Research 1997, 11 (3), 211-215.
50. Huang, X. Y.; Shan, Z. J.; Zhai, H. L.; Su, L.; Zhang, X. Y., Study on the
anticancer activity of coumarin derivatives by molecular modeling. Chemical
Biology & Drug Design 2011, 78 (4), 651-8.
27
51. Patil, P. O.; Bari, S. B.; Firke, S. D.; Deshmukh, P. K.; Donda, S. T.; Patil,
D. A., A comprehensive review on synthesis and designing aspects of coumarin
derivatives as monoamine oxidase inhibitors for depression and Alzheimer's
disease. Bioorganic and Medicinal Chemistry 2013, 21 (9), 2434-50.
52. Bertrand, C.; Fabre, N.; Moulis, C., A new coumarin glucoside, coumarins
and alkaloids from Ruta corsica roots. Fitoterapia 2004, 75 (2), 242-4.
53. Garazd, Y. L.; Garazd, M. M.; Khilya, V. P., Modified coumarins. 12.
Synthesis of 3,4-cycloannelated coumarin beta-D-glucopyranosides. Chemistry
of Natural Compounds 2004, 40 (1), 6-12.
54. Georgievskii, V. P.; Rybachenko, A. I., Spectral-luminescent properties of
natural coumarin derivatives and their use for group identification. Chemistry of
Natural Compounds 1985, 21 (6), 725-728.
55. Nag, A.; Bhattacharyya, K., Role of Twisted Intramolecular Charge-
Transfer in the Fluorescence Sensitivity of Biological Probes -
Diethylaminocoumarin Laser-Dyes. Chemical Physics Letters 1990, 169 (1-2),
12-16.
56. Jones Ii, G.; Jackson, W. R.; Halpern, A. M., Medium effects on
fluorescence quantum yields and lifetimes for coumarin laser dyes. Chemical
Physics Letters 1980, 72 (2), 391-395.
57. Signore, G.; Nifosi, R.; Albertazzi, L.; Storti, B.; Bizzarri, R., Polarity-
sensitive coumarins tailored to live cell imaging. Journal of the American
Chemical Society 2010, 132 (4), 1276-88.
58. Fischer, K.; Spange, S.; Fischer, S.; Bellmann, C.; Adams, J., Probing the
surface polarity of native celluloses using genuine solvatochromic dyes.
Cellulose 2002, 9 (1), 31-40.
59. Spange, S.; Fischer, K.; Prause, S.; Heinze, T., Empirical polarity
parameters of celluloses and related materials. Cellulose 2003, 10 (3), 201-212.
60. Jones Ii, G.; Jimenez, J. A. C., Azole-linked coumarin dyes as
fluorescence probes of domain-forming polymers. Journal of Photochemistry and
Photobiology B: Biology 2001, 65 (1), 5-12.
28
61. Felorzabihi, N.; Haley, J. C.; Ardajee, G. R.; Winnik, M. A., Systematic
study of the fluorescence decays of amino-coumarin dyes in polymer matrices.
Journal of Polymer Science Part B-Polymer Physics 2007, 45 (17), 2333-2343.
62. Signore, G.; Nifosi, R.; Albertazzi, L.; Bizzarri, R., A novel coumarin
fluorescent sensor to probe polarity around biomolecules. Journal of Biomedical
Nanotechnology 2009, 5 (6), 722-9.
63. Marini, A.; Munoz-Losa, A.; Biancardi, A.; Mennucci, B., What is
solvatochromism? Journal of Physical Chemistry B 2010, 114 (51), 17128-35.
64. Suppan, P., Invited review solvatochromic shifts: The influence of the
medium on the energy of electronic states. Journal of Photochemistry and
Photobiology A: Chemistry 1990, 50 (3), 293-330.
65. Nad, S.; Kumbhakar, M.; Pal, H., Photophysical properties of coumarin-
152 and coumarin-481 dyes: Unusual behavior in nonpolar and in higher polarity
solvents. Journal of Physical Chemistry A 2003, 107 (24), 4808-4816.
66. Wagner, B. D., The Use of Coumarins as Environmentally-Sensitive
Fluorescent Probes of Heterogeneous Inclusion Systems. Molecules 2009, 14
(1), 210-237.
67. Katritzky, A. R.; Fara, D. C.; Yang, H.; Tamm, K.; Tamm, T.; Karelson, M.,
Quantitative measures of solvent polarity. Chemical Reviews 2004, 104 (1), 175-
98.
68. Lakowicz, J. R., Principles of fluorescence spectroscopy. Springer: New
York, 2006.
69. Catalán, J.; López, V.; Pérez, P.; Martin-Villamil, R.; Rodríguez, J.-G.,
Progress towards a generalized solvent polarity scale: The solvatochromism of 2-
(dimethylamino)-7-nitrofluorene and its homomorph 2-fluoro-7-nitrofluorene.
Liebigs Annalen 1995, 1995 (2), 241-252.
70. Kamlet, M. J.; Abboud, J. L.; Taft, R. W., The solvatochromic comparison
method. 6. The .pi.* scale of solvent polarities. Journal of the American Chemical
Society 1977, 99 (18), 6027-6038.
29
71. Hrdlovic, P.; Donovalova, J.; Stankovicova, H.; Gaplovsky, A., Influence of
polarity of solvents on the spectral properties of bichromophoric coumarins.
Molecules 2010, 15 (12), 8915-32.
72. Ravi, M.; Soujanya, T.; Samanta, A.; Radhakrishnan, T. P., Excited-State
Dipole-Moments of Some Coumarin Dyes from a Solvatochromic Method Using
the Solvent Polarity Parameter, E(N)(T). Journal of the Chemical Society-
Faraday Transactions 1995, 91 (17), 2739-2742.
73. Ismail, L. F. M.; Antonious, M. S.; Mohamed, H. A.; Ahmed, H. A. H.,
Fluorescence Properties of Some Coumarin Dyes and Their Analytical
Implication. Proceedings of the Indian Academy of Sciences-Chemical Sciences
1992, 104 (2), 331-338.
74. Franck, J.; Dymond, E. G., Elementary processes of photochemical
reactions. Transactions of the Faraday Society 1926, 21 (February), 536.
75. Condon, E., A Theory of Intensity Distribution in Band Systems. Physical
Review 1926, 28 (6), 1182-1201.
76. Coolidge, A. S.; James, H. M.; Present, R. D., A Study of the Franck-
Condon Principle. The Journal of Chemical Physics 1936, 4 (3), 193.
77. Kumar, S.; Rao, V. C.; Rastogi, R. C., Excited-state dipole moments of
some hydroxycoumarin dyes using an efficient solvatochromic method based on
the solvent polarity parameter, ETN. Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy 2001, 57 (1), 41-47.
78. Raikar, U. S.; Renuka, C. G.; Nadaf, Y. F.; Mulimani, B. G.; Karguppikar,
A. M.; Soudagar, M. K., Solvent effects on the absorption and fluorescence
spectra of coumarins 6 and 7 molecules: determination of ground and excited
state dipole moment. Spectrochimica Acta, Part A: Molecular and Biomolecular
Spectroscopy 2006, 65 (3-4), 673-7.
79. Huisgen, R., Proceedings of the Chemical Society. October 1961.
Proceedings of the Chemical Society 1961, (October), 357.
30
80. Kolb, H. C.; Finn, M. G.; Sharpless, K. B., Click Chemistry: Diverse
Chemical Function from a Few Good Reactions. Angewandte Chemie,
International Edition in English 2001, 40 (11), 2004-2021.
81. Tornøe, C. W.; Christensen, C.; Meldal, M., Peptidotriazoles on Solid
Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar
Cycloadditions of Terminal Alkynes to Azides. The Journal of Organic Chemistry
2002, 67 (9), 3057-3064.
82. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B., A stepwise
huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of
azides and terminal alkynes. Angewandte Chemie, International Edition in
English 2002, 41 (14), 2596-9.
83. Himo, F.; Lovell, T.; Hilgraf, R.; Rostovtsev, V. V.; Noodleman, L.;
Sharpless, K. B.; Fokin, V. V., Copper(I)-Catalyzed Synthesis of Azoles. DFT
Study Predicts Unprecedented Reactivity and Intermediates. Journal of the
American Chemical Society 2004, 127 (1), 210-216.
84. Viet, D.; Beck-Candanedo, S.; Gray, D. G., Dispersion of cellulose
nanocrystals in polar organic solvents. Cellulose 2007, 14 (2), 109-113.
85. Karaaslan, M. A.; Gao, G.; Kadla, J. F., Nanocrystalline cellulose/β-casein
conjugated nanoparticles prepared by click chemistry. Cellulose 2013, 20 (6),
2655-2665.
86. Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill, H. N.; Wang, Q., A
fluorogenic 1,3-dipolar cycloaddition reaction of 3-azidocoumarins and
acetylenes. Organic Letters 2004, 6 (24), 4603-6.
87. Key, J. A.; Koh, S.; Timerghazin, Q. K.; Brown, A.; Cairo, C. W.,
Photophysical characterization of triazole-substituted coumarin fluorophores.
Dyes and Pigments 2009, 82 (2), 196-203.
88. Zhou, Z.; Fahrni, C. J., A fluorogenic probe for the copper(I)-catalyzed
azide-alkyne ligation reaction: modulation of the fluorescence emission via
3(n,pi)-1(pi,pi) inversion. Journal of the American Chemical Society 2004, 126
(29), 8862-3.
31
89. Westlund, R.; Glimsdal, E.; Lindgren, M.; Vestberg, R.; Hawker, C. J.;
Lopes, C.; Malmstrom, E., Click chemistry for photonic applications: triazole-
functionalized platinum(II) acetylides for optical power limiting. Journal of
Materials Chemistry 2008, 18 (2), 166-175.
90. Brantley, J. N.; Wiggins, K. M.; Bielawski, C. W., Unclicking the click:
mechanically facilitated 1,3-dipolar cycloreversions. Science 2011, 333 (6049),
1606-9.
91. Birch, D. S.; Imhof, R., Time-Domain Fluorescence Spectroscopy Using
Time-Correlated Single-Photon Counting. In Topics in Fluorescence
Spectroscopy, Lakowicz, J., Ed. Springer US: 2002; Vol. 1, pp 1-95.
92. Valeur, B., Principles of Steady-State and Time-Resolved Fluorometric
Techniques. In Molecular Fluorescence, Wiley-VCH Verlag GmbH: 2001; pp 155-
199.
93. O'Connor, D. V. P. D., Time-correlated single photon counting. Academic
Press: London; Orlando, 1984.
94. Valeur, B., Pulse and Phase Fluorometries: An Objective Comparison. In
Fluorescence Spectroscopy in Biology, Hof, M.; Hutterer, R.; Fidler, V., Eds.
Springer Berlin Heidelberg: 2005; Vol. 3, pp 30-48.
95. Becker, W., Fluorescence lifetime imaging--techniques and applications.
Journal of Microscopy 2012, 247 (2), 119-36.
32
Chapter 2: Experimental
2.1 Materials and methods
2.1.1 Materials and reagents
All chemicals and reagents were purchased from Sigma-Aldrich, unless
otherwise specified. Carboxylated NCC was obtained from Bio Vision
Technology Inc. An aqueous colloidal suspension of C6 alkyne-functionalized
NCC (NCC-alkyne, 8 % by weight) was provided by Dr. Singh, McGill Unversity.
It was prepared through EDC coupling of propargyl amine with carboxylated
NCC. Quartz fluorescence cells (10 x 10 mm, 3500 l) were purchased from
Hellma Analytics Inc. ACS grade solvents were used for all reactions, as well as
fluorescence and absorbance characterisation studies.
2.1.2 Coumarin azides
3-Azido-7-hydroxy-2H-chromen-2-one (HC), 3-Azido-2-oxo-2H-chromen-
7-yl acetate (AC), and 3-Azido-7-(diethylamino)-2H-chromen-2-one (DEA
coumarin) were synthesized following the procedures developed by Sivakumar et
al.1 HC and AC were selected because their non-azide analogues exhibit strong
solvatochromism, have high quantum efficiencies and have long lifetimes in
water.2 DEA was selected for its red-shifted absorption and emission
33
wavelengths.1 Related diethylamino coumarins exhibit low quantum yields and
fluorescence lifetimes in polar solvents.3
HC AC DEA
Figure 2.1: Stuctures of synthesized azido-coumarins
2.1.3 NCC click chemistry
NCC-alkyne suspended in distilled water (10 mL, 8% by weight aq.) was
added to a large vial under stirring. HC dissolved in THF (0.1 mmol, 10 mL) was
added slowly and the mixture was left to stir for several minutes. Aqueous
copper sulphate pentahydrate solution (0.15 mL, 0.15 eq) was added dropwise to
the mixture, followed by the addition of aqueous sodium ascorbate solution (0.45
mL, 0.45 eq). The reaction was left stirring at room temperature for 24 h. Next,
the reaction mixture was transferred in equal portions in two centrifuge tubes.
Ethanol (30 mL, 95%) was added to each tube, and the mixtures were
centrifuged (10min, 4500 rpm). The supernatant was decanted off, and the
precipitate was suspended again in ethanol (40 ml, 95%) and centrifuged. This
process was repeated once more, and this time the precipitate was suspended in
34
water (5 mL), collected and stored cold. The concentration of the NCC-HC
suspension was determined to be 0.4% by weight through gravimetric analysis.
NCC-AC and NCC-DEA suspensions were prepared in the same manner
as above. The concentrations obtained were 0.4% by weight for NCC-AC and
0.1% by weight for NCC- DEA.
2.1.4 Synthesis of 2-propynyl-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside
2-propynyl-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside was synthesized by
the method of Mereyala et al.4
2.1.5 Synthesis of model coumarin gluocoside (DEA-M):
The coumarin-glucoside model compound was synthesized from the
reaction of 2-propynyl-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside and DEA
coumarin. To a large vial, and 2-propynyl-2,3,4,6-tetra-O-acetyl-β-D-
glucopyranoside (60 mg, 1.55x10-4 mol) and DEA coumarin (40mg, 1.55x10-4
mol) were added. Tert-butyl alcohol (10 mL) was added, and the mixture was
heated to 40 °C and vigorously stirred until the compounds had dissolved.
Distilled water was added in slowly (10 mL), followed by the addition of solutions
copper sulphate pentahydrate (0.31 mL, 0.2 eq) and sodium ascorbate (0.62 mL,
0.4 eq). The reaction was left for 24 h, and was followed by extraction with
35
dichloromethane (3 x 20 mL). The organic layer was dried over anhydrous
sodium sulphate and concentrated under reduced pressure to afford a brown
solid. The product was dissolved in a minimal amount of dichloromethane and
filtered through an alumina pad to remove any remaining copper, and evaporated
to dryness. Finally, the product was recrystallized from methanol to give a bright
yellow product; 48.5 mg, 50% yield. 1H, 13C, ATR-FTIR, ESI-MS and melting
point data are provided in appendix A. The structure of the model analogue is
given in Figure 2.2.
Figure 2.2: Structure of model coumarin glucoside (DEA-M)
(2S,3S,4S,5S,6S)-2-(acetoxymethyl)-6-((1-(7-(diethylamino)-2-oxo-2H-chromen-
3-yl)-1H-1,2,3-triazol-4-yl)methoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate
36
2.1.5 Instrumentation
Fluorescence emission spectra were taken on a Cary Eclipse
Fluorescence Spectrometer. Extinction spectra were obtained using a Cary 300
Series UV-VIS Spectrometer. Dynamic light scattering data was acquired using
a BI-200SM goniometer and BI-2030 correlator from Brookhaven Instruments.
Confocal fluorescence images were imaged with a Horiba DynaMyc
Fluorescence lifetime mapping microscope, and a DeltaDiode-375L light source.
ATR-FTIR data were acquired on a Spectrum TWO IR Spectrometer from Perkin
Elmer. All measurements were done under ambient conditions. XPS data were
acquired with a VG Escalab 3 MKII.
2.1.6 Preparation of Fluorescence and Extinction samples
Clicked NCC-coumarin solutions were prepared in the following manner.
First, the stock suspensions were shaken vigorously using a Vortex mixer. This
was followed by pipetting (25 uL for NCC-AC and NCC-HC, 50uL for NCC-DEA)
of the aqueous stock suspension into a series of vials. Each vial was then
diluted to 3 mL with solvent, before being transferred to the quartz cuvettes. As a
consequence, there was always an amount of water present in all our samples.
This was necessary because early attempts to dry out NCC-coumarin and
resuspend them through sonication proved unsucessful. The quartz cuvettes
37
were washed in Piranha solution (3:1 H2SO4: 30% H2O2) and heavily rinsed with
distilled water before each series of measurements.
2.1.7 Determination of Quantum Yields
Quantum yields of DEA-M and NCC-DEA were determined by the relative
method5 with the fluorescent standard quinine bisulphate (0.1 M H2SO4,
λexc=366nm, QY=0.53).6 Quinine sulphate is an appropriate standard because its
absorption and emission profiles overlap with that of DEA-M and NCC-DEA. The
formula for obtaining quantum yields as given in equation 2.1 is only valid for
dilute samples (optical density < 0.05). QY is the quantum yield, OD is the
optical density, I is the integrated intensity of the fluorescence emission curve,
and n is the index of refraction of the solvent. The integrated intensities values
were obtained by using the integration function built in to the Cary Eclipse
operating software. Extinction measurements were baseline corrected with
respect to a solvent blank before optical densities were measured, and inner filter
corrections were not applied.
(2.1)
38
2.1.7 DLS measurements
Dynamic light scattering (DLS) measurements were taken using 532 nm
solid-state laser, with the photomultiplier tube positioned at a 90 angle with
respect to the laser. Size distributions were obtained using a non-negative least-
squares fitting procedure, using commercial Brookhaven software. The
hydrodynamic diameter reported for DLS assume a spherical geometry for the
particles.
2.1.8 TCSPC data acquisition
Time-correlated single-photon counting decays were obtained with an
Edinburgh photonics Mini-Tau fluorescence spectrometer. The excitation
wavelength was 405 nm, and the laser start rate was set to 2 MHz. Stop rates
were set at 1%, and measurements were acquired until 10000 counts had been
registered in one channel. Measurement of the IRF was achieved by adding a
few drops of LUDOX® (HS-40) to distilled water.
2.1.9 TCSPC Data Fitting
Fluorescence lifetime measurements of DEA-M and of NCC-coumarins in
various solvents were obtained. The lifetimes for DEA-M in could always be fit
satisfactorily to a monoexponential. A fit consisting of several exponentials or a
39
single stretched exponential were found to be adequate for NCC-coumarin.
Biexponential Tail fits and three-exponential reconvolution fits were performed on
commerical Edinburgh photonics software. Stretched exponential fits were
performed in Matlab® (Mathworks), using the Matlab function fminsearch.
2.1.10 NCC-DEA and NCC-HC PEGDA films
Poly(ethylene glycol) diacrylate (PEGDA, Mn 575, 1 mL) was added to a
large vial along with aqueous NCC-DEA (200 uL, 0.1% by weight) and radical
initiator 2-hydroxy-2methylpropiophenone (0.03 mL). Structures of PEGDA and
2-hydroxy-2methylpropiophenone are provided in Figure 2.3. The viscous
mixture was quickly stirred with a glass pipet and then coated on a clean glass
slide. The glass slide was left to dry for 24 h, under a sheet of aluminum foil.
The following day, the glass slide was placed in a plastic petri dish, and purged
with nitrogen gas. The petri dish was then placed inside a UV curing chamber
and irradiated for one minute. A 2D lifetime map of the film was obtained with a
Horiba DynaMyc Fluorescence lifetime mapping microscope.
40
Figure 2.3: Chemical Structures of PEGDA and radical initiator 2-hydroxy-
2methylpropiophenone
2.1.11 2D-Lifetime mapping fits
2D lifetime mapping fits were performed using the DAS6 software
(Horiba). A series of point time-resolved fluorescence decay measurements
were taken systematically over the focal plane of the image. This was
accomplished by dividing the area of the image into a 2D grid, and acquiring a
fluorescence decay measurement at every cell within this grid. These decay
measurements were fitted using a monoexponential.
2.1.12 Stern-Volmer quenching studies
Quenching studies involving N-methyl aniline (98%) as a quencher were
performed on DEA-M in acetonitrile, NCC-DEA in dimethylsulfoxide and NCC-HC
in dimethylsulfoxide. The solutions were not degassed prior to measurements.
After recording initial lifetime and integrated intensity values, N-methyl aniline
was added (1-10 uL) to the fluorescent solutions (3 mL) in quartz cuvettes using
41
a volumetric Hamilton syringe (10 uL). The cuvettes were shaken to ensure
proper mixing and left to equilibrate for a few minutes before lifetime or
fluorescence emission intensity measurements were taken.
2.2 Background Theory
2.2.1 Time-correlated single-photon counting
Time-correlated single-photon counting (TCSPC) (also referred to as
pulse fluorometry) is the lifetime measurement technique featured in this thesis.
Entire textbooks7,8 have been devoted to the subject, and systems are now
commercially available. One example is shown in Figure 2.4. In this section the
instrumentation will be briefly summarized. It is by no means an exhaustive
description, and is intended only to provide readers with a basic sense of how the
technique is performed.
A TCSPC setup is similar to many steady-state emission fluorescence
spectrometers in which the excitation source and detector are offset by 90 .
The laser is pulsed and a neutral density filter is placed in front of the laser in
order to control the amount of photons incident on the sample. Only one photon
on average per excitation pulse should be detected. If the intensity of the light
reaching the sample is too high, then a “pileup effect”9-10 is observed. This pileup
effect will artificially skew the lifetime decay data towards shorter lifetimes. An
42
emission filter is placed in front of the detector in order to mitigate the amount of
scattered light, including the Raman contribution.
Figure 2.4: Photograph of Mini-Tau fluorescence spectrometer® (Edinburgh
photonics)
The timing mechanism of the instrument, depicted in figure 2.5, is based
on two signals: the start and stop pulse. The start pulse is sent from the laser at
the moment the excitation pulse is emitted, and the stop pulse is sent by the
detector once it detects an emitted photon from the sample. These pulses are
sent to a resistor-capacitor charging circuit (time-to-amplitude converter).
Capacitor charging begins from the moment the start signal is sent and
subsequent discharge occurs when the stop signal arrives. This analog output is
then converted to a digital signal, and binned into a time channel (multichannel
43
analyzer). The process is repeated several thousands of times at which point a
fluorescence decay curve is collected.
Figure 2.5: Top panel: flow chart for TCSPC electronics. The dotted lines
represent electronic signals, while the solid lines are photon displacements.
(Figure adapted from Ref.11)
2.2.2 TCSPC data
TCSPC data usually consists of two separate measurements: a
fluorescence decay curve associated with the experimental sample and a curve
44
known as the instrumental response function (IRF) that is collected by measuring
the fluorescence decay profile of a non-fluorescent scattering solution. The IRF
is a measure of the lamp width and detection electronics. Examples of these
curves are displayed in Figure 2.6. The measure of the IRF can be omitted when
the sample lifetime is longer than the full width half maximum of the IRF.
Once the decay data and IRF have been collected, the data must be
processed. Several methods exist for processing these data, however the most
popular and accessible method remains the least-squares method.12-13
Figure 2.6: Top panel: Typical fluorescence lifetime decay plot, with IRF (blue)
and decay data (red) and fit (black). Bottom panel: Residual plot
45
2.2.3 Non-negative least-squares and indicators of goodness-of-fit
The least-squares method is a popular method for data analysis in TCSPC
due to its relative ease of implementation and speed. The method involves fitting
a model decay function to the fluorescence decay data by iterating a number of
parameters through an efficient minimization algorithm, such as the Nead-Melder
simplex.14-15 The previously mentioned IRF is usually incorporated into the
model decay function through convolution (a product of the functions respective
Fourier transforms). The main drawback with this method is that an a priori
assumption of the fluorescence decay model must be made. In the case of most
simple organic fluorophores, this is a seemingly trivial point since the majority of
these molecules exhibit monoexponential decay. Nevertheless, experimenters
can mistakenly assume a wrong fluorescence decay model, in which case the
lifetime obtained is erroneous. Researchers must scrutinize least-squares fits
carefully and assess whether or not the result is physically plausible.
In order to assess the goodness-of-fit of a particular model, three
measures are commonly used: 5, 16 the reduced chi-squared of the function, the
residual plot and the autocorrelation of residuals. The reduced chi-squared is a
statistical parameter defined in equation 2. Fk and Sk are the number of photon
counts of the model decay function and actual decay function respectively, in the
kth channel. Wk is a weighing factor corresponding to the statistical form of the
46
data, and n is the number of degrees of freedom. The reduced chi-squared is
also the statistical parameter which is minimized in the least-squares iteration.
An ideal fit would have a reduced chi-squared of unity. The residual is defined in
equation 2.3. It is very similar to the reduced chi-squared, and is often included
below a plot of the fluorescence decay data as in the bottom panel in Figure 2.6.
An ideal residual plot would appear randomly distributed, centered at zero and
would not have any discernable skews. Finally the residual data can be further
analyzed through autocorrelation to see if it truly is random. The autocorrelation
plot should also appear randomly distributed around zero.
∑
[ ]
(2.2)
[ ] (2.3)
2.2.4 Lifetime distributions
Fluorescence lifetime decays cannot always be fit with a
monoexponential. Some fluorescence systems exhibit nonexponetial decay, or
consist of multiple fluorescence lifetimes. In the case of the latter, a series of
several monoexponential terms may be sufficient to fit the decay data. However,
a statistically plausible fit does not guarantee an accurate physical description.
Extreme caution is required in such cases, and while biexponential fits are
common17, venturing to fit three or four exponentials should only be done if the
47
lifetimes can be justified with prior knowledge of the system. The number of
discrete lifetimes obtained from a fit is equal to the number of exponentials used.
Ware et al. first showed that any fit consisting of two or more exponential
terms could always be fit by a distribution, with a mean lifetime and standard
deviation.18 If a biexponential is used, then the weighted average <τ> is
approximately equal to the mean lifetime of the distribution. The expression for
the weighted average is given in equation 2.4, where Bk simply represents the
weighted prefactor of the exponential term. Distributions are typically in the form
of a Gaussian19, or stretched exponential.20
The stretched exponential function, shown in equation 2.5, has been
employed previously in fitting the decays of coumarins in certain polymer
systems, where it is assumed a continuum of micro-environments exist.21 It has
also been used in regards to NCC, albeit in terms of fitting solid-state 13C free
induction decays.22 The stretched exponential also only adds one extra
parameter, β, in the fitting procedure. The parameter β is a constant that ranges
between 0 and 1. In the limit that β approaches 1, the monoexponential form is
recovered. A low value of β is a wide distribution, whereas a high value of β is a
narrow distribution. The mean lifetime of the distribution is calculated from
equation 2.6, where Г(x) is the gamma function.20 The mean lifetime obtained
using the stretched exponential function will be compared to the weighted
48
average of the multiple exponentials in regards to the lifetime analysis of the
NCC-coumarins.
⟨ ⟩ ∑
(2.4)
(
)
(2.5)
⟨ ⟩
(
) (2.6)
2.2.5 Fluorescence quenching and Stern-Volmer Plots
Fluorescence quenching studies are an efficient way to probe the
accessibility of fluorophores.23 Common solute quenchers of biological systems
include oxygen, acrylamide and iodide.24 The quenching efficiency a particular
quencher varies depending on the nature of the fluorophore and the
environment.25 The simplest form of the data analysis to perform is given by the
Stern-Volmer equation, as presented in equation 2.7. Here kq is the quenching
rate constant, I0 and τ0 are the initial fluorescence intensity and initial lifetime
respectively, and [Q] is the concentration of quencher. This equation is only true
if τ0 is a monoexponential lifetime.24 A typical experiment would consist of
49
recording the initial fluorescence lifetime and integrated intensity for a given
fluorophore, adding quencher and repeating the measurements.
[ ]
(2.7)
There are two typical quenching mechanisms can be determined from a
Stern-Volmer analysis in the form of equation 2.7. A purely dynamic or collisional
quenching mechanism would result in the plots of I0/I vs [Q] and τ0 / τ vs [Q] to be
linear plots with identical slopes. A purely static quenching mechanism would
still result in a linear plot of I0/I vs [Q], but would result in a flat line for the plot of
τ0 / τ vs [Q]. The overall fluorescence intensity would decrease if fewer
fluorophores reach the excited state, but lifetimes are not affected because they
are concentration independent. Statically quenched fluorophores simply do not
appear in TCSPC data.
2.3 Lippert-Mataga polarity index (Δf)
The Lippert-Mataga polarity index is a commonly within solvatochromatic
studies of coumarins.3, 26 The Lippert-Mataga polarity index of a solvent is
determined by the difference of two functions: the total polarization function and
the polarization induction function.5 These functions are denoted f(ε) and f(n2)
50
and are functions of the solvent dielectric constant ε, and the square of the
solvent index of refraction, n2. They are shown below in equation 2.8.
(2.8)
2.4 XPS surface characterization
XPS survey scans were taken on a VG Escalab 3 MKII with a power of
216 W using Mg Kα radiation. The atomic % of carbon, oxygen and nitrogen are
reported in table 2.1 (XPS cannot detect Hydrogen). The NCC-coumarin
samples have an elevated carbon:oxygen ratio compared with the starting NCC-
alkyne. This is consistent with what has been previously observed in the
literature,27 due to the high carbon content of the coumarin dyes. The nitrogen
content is lower in the case of the NCC-coumarins than in the case of NCC-
alkyne. It was expected to increase due the three nitrogen atoms per clicked
coumarin. We attribute this inconsistency to residual ammonium persulfate
present in the starting NCC-alkyne, which is also present as an impurity in the
NCC obtained from Bio Vision Technology Inc.
Table 2.1: Surface XPS Results
sample C (at%) O (at%) N (at%)
NCC-alkyne 63.0 33.3 1.0
NCC-HC 74.5 24.1 0.6
NCC-AC 69.5 29.1 0.9
NCC-DEA 79.5 18.5 0.6
51
2.5 References
1. Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill, H. N.; Wang, Q., A
fluorogenic 1,3-dipolar cycloaddition reaction of 3-azidocoumarins and
acetylenes. Organic Letters 2004, 6 (24), 4603-6.
2. Nizomov, N.; Kholov, A. U.; Ishchenko, A. A.; Ishchenko, V. V.; Khilya, V.
P., Electronic structure and spectral fluorescence properties of umbelliferone and
herniarin. Journal of Applied Spectroscopy 2007, 74 (5), 626-634.
3. Nad, S.; Kumbhakar, M.; Pal, H., Photophysical properties of coumarin-
152 and coumarin-481 dyes: Unusual behavior in nonpolar and in higher polarity
solvents. Journal of Physical Chemistry A 2003, 107 (24), 4808-4816.
4. Mereyala, H. B.; Gurrala, S. R., A highly diastereoselective, practical
synthesis of allyl, propargyl 2,3,4,6-tetra-O-acetyl-β-d-gluco, β-d-
galactopyranosides and allyl, propargyl heptaacetyl-β-d-lactosides. Carbohydrate
Research 1998, 307 (3-4), 351-354.
5. Lakowicz, J. R., Principles of fluorescence spectroscopy. Springer: New
York, 2006.
6. Adams, M. J.; Highfield, J. G.; Kirkbright, G. F., Determination of absolute
fluorescence quantum efficiency of quinine bisulfate in aqueous medium by
optoacoustic spectrometry. Analytical Chemistry 1977, 49 (12), 1850-1852.
7. O'Connor, D. V. P. D., Time-correlated single photon counting. Academic
Press: London; Orlando, 1984.
8. Becker, W., Advanced time-correlated single photon counting techniques.
Springer: Berlin; New York, 2005.
9. Coates, P. B., The correction for photon `pile-up' in the measurement of
radiative lifetimes. Journal of Physics E: Scientific Instruments 1968, 1 (8), 878.
10. Davis, C. C.; King, T. A., Correction methods for photon pile-up in lifetime
determination by single-photon counting. Journal of Physics A: General Physics
1970, 3 (1), 101-109.
52
11. Valeur, B., Principles of Steady-State and Time-Resolved Fluorometric
Techniques. In Molecular Fluorescence, Wiley-VCH Verlag GmbH: 2001; pp 155-
199.
12. Ware, W. R.; Doemeny, L. J.; Nemzek, T. L., Deconvolution of
Fluorescence and Phosphorescence Decay Curves - Least-Squares Method.
Journal of Physical Chemistry 1973, 77 (17), 2038-2048.
13. O'Connor, D. V.; Ware, W. R.; Andre, J. C., Deconvolution of fluorescence
decay curves. A critical comparison of techniques. The Journal of Physical
Chemistry 1979, 83 (10), 1333-1343.
14. Lagarias, J. C.; Reeds, J. A.; Wright, M. H.; Wright, P. E., Convergence
properties of the Nelder-Mead simplex method in low dimensions. Siam Journal
on Optimization 1998, 9 (1), 112-147.
15. Nelder, J. A.; Mead, R., A Simplex-Method for Function Minimization.
Computer Journal 1965, 7 (4), 308-313.
16. Straume, M.; Frasier-Cadoret, S.; Johnson, M., Least-Squares Analysis of
Fluorescence Data. In Topics in Fluorescence Spectroscopy, Lakowicz, J., Ed.
Springer US: 2002; Vol. 2, pp 177-240.
17. Birch, D. S.; Imhof, R., Time-Domain Fluorescence Spectroscopy Using
Time-Correlated Single-Photon Counting. In Topics in Fluorescence
Spectroscopy, Lakowicz, J., Ed. Springer US: 2002; Vol. 1, pp 1-95.
18. James, D. R.; Ware, W. R., A Fallacy in the Interpretation of Fluorescence
Decay Parameters. Chemical Physics Letters 1985, 120 (4-5), 455-459.
19. James, D. R.; Liu, Y. S.; Demayo, P.; Ware, W. R., Distributions of
Fluorescence Lifetimes - Consequences for the Photophysics of Molecules
Adsorbed on Surfaces. Chemical Physics Letters 1985, 120 (4-5), 460-465.
20. Berberan-Santos, M. N.; Bodunov, E. N.; Valeur, B., Mathematical
functions for the analysis of luminescence decays with underlying distributions 1.
Kohlrausch decay function (stretched exponential). Chemical Physics 2005, 315
(1-2), 171-182.
53
21. Felorzabihi, N.; Haley, J. C.; Ardajee, G. R.; Winnik, M. A., Systematic
study of the fluorescence decays of amino-coumarin dyes in polymer matrices.
Journal of Polymer Science Part B-Polymer Physics 2007, 45 (17), 2333-2343.
22. Lemke, C. H.; Dong, R. Y.; Michal, C. A.; Hamad, W. Y., New insights into
nano-crystalline cellulose structure and morphology based on solid-state NMR.
Cellulose 2012, 19 (5), 1619-1629.
23. Johnson, D. A.; Yguerabide, J., Solute accessibility to N epsilon-
fluorescein isothiocyanate-lysine-23 cobra alpha-toxin bound to the acetylcholine
receptor. A consideration of the effect of rotational diffusion and orientation
constraints on fluorescence quenching. Biophysical Journal 1985, 48 (6), 949-55.
24. Eftink, M., Fluorescence Quenching: Theory and Applications. In Topics in
Fluorescence Spectroscopy, Lakowicz, J., Ed. Springer US: 2002; Vol. 2, pp 53-
126.
25. Eftink, M. R.; Selva, T. J.; Wasylewski, Z., Studies of the Efficiency and
Mechanism of Fluorescence Quenching Reactions Using Acrylamide and
Succinimide as Quenchers. Photochemistry and Photobiology 1987, 46 (1), 23-
30.
26. Signore, G.; Nifosi, R.; Albertazzi, L.; Storti, B.; Bizzarri, R., Polarity-
sensitive coumarins tailored to live cell imaging. Journal of the American
Chemical Society 2010, 132 (4), 1276-88.
27. Huang, J. L.; Li, C. J.; Gray, D. G., Cellulose Nanocrystals Incorporating
Fluorescent Methylcoumarin Groups. Acs Sustainable Chemistry & Engineering
2013, 1 (9), 1160-1164.
54
Chapter 3: Results and Discussion
3.1 Steady-state characterization
3.1.1 ATR-FTIR analysis
ATR-FTIR spectra of the azido-coumarins were compared to the
corresponding spectra of the clicked products. Azide groups display a strong
asymmetric stretching vibration band, typically between 2167 and 2080 cm-1.1
The FTIR spectrum of DEA coumarin presented in Figure 3.1 displays a strong
peak at 2116 cm-1, attributed to the azide group. The DEA clicked product shows
no evidence of the azide group indicating that the compound is not physisorbed
onto the NCC, and suggesting that the DEA azido has cycloadded to form the
triazole. FTIR bands in this region were not observed for the 1,2,4 triazoles, so
this technique cannot provide definitive evidence for covalent binding of DEA
through the triazole.2 The more surface-sensitive technique of X-ray
photoelectron spectroscopy (XPS) is required to detect the presence of the
triazole.3-4 Soxhlet extraction of the NCC-DEA produced no evidence of free
DEA.5
55
Figure 3.1: ATR-FTIR spectra of the clicked NCC. Major peaks for the NCC-DEA
(black curve) are 3334 cm-1 (O-H), 2912 cm-1 (C-H),, 1644 cm-1 (C=O, amide).
The major identifiable peaks for DEA (red curve) are 2972cm-1 (C-H), 2116 cm-1
(N3), 1702 cm-1 (C=O), 1616 cm-1 (C=C).
3.1.2 Extinction of coumarin labelled NCC suspensions
The term extinction is used to describe the UV-VIS spectra in our
experiments because there is a scattering contribution to the spectra that arises
from suspended NCC. Extinction spectra of the raw aqueous coumarin triazole
NCC products were compared to the extinction spectrum of the NCC-alkyne, and
these are shown in Figure 3.2. The gradual increase in extinction from 800 nm to
200 nm in all three curves is consistent with what has been reported in the
literature for similar coumarin molecules attached to NCC.5 The absorption peak
maxima at 339 nm for NCC-HC, 341nm for NCC-AC and 422nm for NCC-DEA
56
arise from coumarin absorption. Since the absorbers are not distributed
homogenously in solution, we did not attempt to quantify the amount of coumarin
bound to NCC through the Beer-Lambert relation.
Figure 3.2: Extinction spectra of aqueous NCC-Coumarin suspensions
3.1.3 Aggregation of NCC
Dynamic light scattering (DLS) measurements were performed on the
NCC-coumarin samples in order to assess their uniformity. Ideally, a monomodal
distribution should be observed for DLS measurements of a non-aggregated
population of cylindrical particles. This result is has been observed in the case of
DLS studies carried out on the tobacco mosaic virus6 (TMV), which is a rod-like
particle with comparable dimensions to that of NCC. However, the data in Figure
57
3.3 is bimodal, consisting of two distinct populations: one with a mean value of 98
nm, and the second with a mean value of 611 nm. Thus, a portion of the NCC-
coumarin population in solvent media is aggregated.
Evidence for NCC-Coumarin particle aggregation is further supported by
our results from confocal fluorescence imaging. NCC-coumarin was
mechanically dispersed in several different solvents. In every case, partial
aggregation was observed. Most of the non-aqueous suspensions contained
aggregates that could be imaged by confocal fluorescence microscopy at low
magnification (10x), as shown in Figure 3.4.
Figure 3.3: Size distribution of aqueous NCC-DEA suspension
58
Figure 3.4: Confocal microscopy image of NCC-HC suspended in DMF 10x
objective.
3.1.4 Solvatochromism
As expected, the NCC-coumarin extinction and emission spectra exhibited
strong solvatochromism. An illustration of the effect of solvatochromism on
emission for NCC-HC is presented in Figure 3.5. Here the emission in dimethyl
sulfoxide (DMSO) and water are contrasted. The emission maximum for water is
red-shifted by 43 nm. Also, note that a small shoulder can be seen at 421 nm in
the emission spectrum of NCC-HC in water. Control experiments of emission
spectra of NCC-alkyne suggest that this feature is related to the NCC. The
background fluorescence intensity of NCC-alkyne was weak when suspended in
all of the solvents employed in this thesis. Nevertheless, noticeable peaks,
59
including one at 421 nm, can be seen upon examination of the baseline. The
panel on the right is a photograph showing the solvatochromic effect in the two
solvents.
The solvatochromic shifts are collected in the Tables presented in
Appendix B. The solvents are listed in decreasing order of polarity of the Lippert-
Mataga polarity index.7 The Lipper-Mataga is just one of the frequently
employed in the coumarin photophysical literature.8-9 A detailed analysis of the
electronic origins of solvatochromism in our NCC-Coumarin materials is beyond
the scope of this thesis. We use the Lippert-Mataga index here as a reasonable
starting point for discussion.
Figure 3.5: Left panel: Solvatochromic shift of emission spectra of NCC-HC, in
DMSO (black) and water (red). Right panel: Visualization of solvatochromism by
excitation of various samples with 366nm light: HC in ethanol (left), NCC-HC in
water (center) and NCC-HC in dimethyl sulfoxide (right). The purple glow
present on the HC vial is due to the UV-lamp.
60
3.1.5 Emission intensity variation of coumarin triazole products
Figure 3.6 shows the emission spectra of NCC-DEA suspended in several
different solvents. The samples were excited at their respective excitation
maxima, as given in Appendix B. The fluorescence intensity is greatest in
DMSO, and weakest in water. Similar behavior was exhibited in emission from
NCC-HC and NCC-AC in the various solvents. This result is surprising when
taking into consideration the trends observed in the literature. In the case of
DEA, structurally similar coumarins are well known to exhibit very low quantum
yields in the presence of high polarity solvents. Low quantum yields have been
argued to be caused by preferential decay through a non-radiative pathway
opened by the solvent.10 In non polar solvents quantum yields of near-unity are
observed.11-12 Moreover, higher emission intensities from DEA-M were observed
when it was dissolved in solvents of lower polarity than DMSO and DMF. Thus, it
seems probable that the observed order of emission intensity is not localized to
the photophysics of the coumarin molecule alone when it is covalently bound to
NCC. The NCC platform also plays a role.
Our data suggest that NCC aggregation diminishes fluorescence intensity.
We observed that sonication and vortexing influenced increased fluorescence
intensity. An example of this effect is shown in Figure 3.7 which compares
unsonicated and sonicated samples of NCC-HC. The sonicated sample has
61
28% greater emission intensity than the unsonicated sample. We attribute this
increase to a break up of large aggregates, and not to sonication induced
breaking of the covalent bond between the dye and NCC. Break up of
aggregates was confirmed by dynamic light scattering which showed an
increased number of smaller particels, and decrease of larger ones.
When dye-labeled NCC is aggregated, it is possible that the exciting light
cannot reach some population of coumarin molecules because of light scattering
or blocking of the optical path. Studies involving colloidal systems sometimes
attempt to match the solvent and colloid indices of refraction to reduce the
influence of colloidal scatter.
62
Figure 3.6: Fluorescence intensity emission of NCC-DEA suspensions.
Figure 3.7: Comparison of Fluorescence emission intensity of sonicated (red)
and unsonicated (black) aqueous suspensions of NCC-HC
63
3.1.6 Quantum Yield determination of DEA-M and of NCC-DEA
The quantum yield of DEA-M and NCC-DEA were determined relative to a
quinine sulphate standard. A relative quantum yield of 37% was determined for
DEA-M. This decreased to 29% for NCC-DEA. The error difference is
statistically significant within ±1, based on the propagated relative error of the
integrated fluorescence intensities and optical densities at a 95% confidence
interval.
We can only speculate on causes of the reduction in relative quantum
yield for NCC-DEA: aggregation effects, some sort of quenching effect caused by
substituents local to the dye on the NCC platform, or quenching by adventitious
Cu(II) that was not removed from the NCC samples during work-up (DEA-M was
purified of Cu(II), which is known to quench coumarin fluorescence). Thus, the
difference in quantum efficiencies between the model compound and the clicked
species might reflect difference in radiationless pathways stemming from multiple
sources.
The extinction and emission spectrum of DEA-M and NCC-DEA in DMSO
are compared in Figure 3.8. The fact that both molecules have similar emission
profiles suggests that the local intrinsic glucose environment of NCC does not
contribute to the observed solvatochromic shift, meaning that the DMSO solvent
64
on average has access to the clicked compound in a way that is similar to the
free model compound.
Figure 3.8: Extinction and emission spectra for quantum yield determination of
DEA-M and NCC-DEA in DMSO
65
3.1.7 Emission dependence on excitation wavelength
In a few cases, emission spectra showed significant dependence on
excitation wavelength. The emission spectra of NCC-HC in DMF at two different
excitation wavelengths are displayed in Figure 3.9. The emission spectrum
obtained by exciting at 405 nm has an emission maximum of 488 nm, which is
red-shifted with compared to the 462 nm peak emission obtained by exciting at
351 nm. Two distinct peaks are obtained when excitation measurements are
performed. Signore et al.13 have previously reported similar data for another
coumarin derivative, and attribute it to the presence of a second emissive state
tied to the medium polarity. This secondary emissive state could be detected as
an additional fluorescence lifetime when the lower excitation maximum
wavelength was used. Lifetime measurements conducted at various
wavelengths would be an ideal way to verify whether our system behaves
similarly. However, only a 405 nm excitation source was available for our study.
A HC molecular analogue would also be desirable to study the impact of the
NCC on the emission dependence.
66
Figure 3.9: Emission spectra of NCC-HC in DMF excited at 351 nm (red) and 405
nm (black)
3.1.8 Summary of Steady-State characterization
Alkyne functionalized NCC was covalently attached to three coumarin
azide derivatives by the Huisgen cycloaddition reaction. The procedure was also
successfully adapted to the synthesis of a coumarin glucoside molecular
analogue (DEA-M). The clicked NCC products were characterized in several
different solvents by dynamic light scattering (DLS), confocal fluorescence
67
microscopy, ATR-FTIR spectroscopy, UV-VIS spectroscopy and fluorescence
emission spectroscopy. DLS and confocal microscopy gave evidence of NCC
aggregates when the centrifuged product was suspended in water, post-reaction.
Aggregation of the clicked NCC may complicate quantitative fluorescence
intensity measurements, but this effect can be slightly mitigated in aqueous
suspensions through sonication. The labelled NCC exhibits typical
solvatochromic behavior across several solvents. Suspensions of NCC-
coumarin in dimethyl sulfoxide showed the highest fluorescence intensity, which
was contrary to the trends observed in molecular coumarin analogues. Further
work is required to investigate fluorescence emission dependence on excitation
wavelength in certain NCC-HC suspensions.
3.2 Stern-Volmer Analysis
N-methylaniline will quench coumarin fluorescence. We used this
quencher to determine if there might be differences in the way the small molecule
analog DEA-M might undergo quenching, as compared with NCC-DEA.
Differences might reflect, for example, the different steric environment of DEA on
the NCC platform.
Fluorescence intensity data were obtained by integration of the
corresponding fluorescence emission spectra. Inner filter excitation corrections14-
68
15 for the emission intensities were not applied because the dataset was
restricted to low concentrations of quencher (<10-2 M). This is justified by
analyzing the UV-VIS spectra of N-methyl aniline in DMSO, in Appendix C. At
low concentrations of N-methyl aniline, the extinction intensity is not significant at
405 nm, whereas at high concentrations of N-methyl aniline this assumption is no
longer valid. If a simple inner filter correction is employed14, then emission
intensities would increase by approximately 1-7%, depending on the
concentration of the quencher for the given data point.
All fluorescence measurements were performed in aerated solutions.
Oxygen predominantly quenches coumarin triplet state emission
(phosphorescence)16, though it has been shown to quench fluorescence in
certain coumarins.17 Quenching studies were carried out on samples that were
not deoxygenated.
3.2.1 Stern-Volmer analysis of DEA-M
A 10-5 M solution of DEA-M in acetonitrile was prepared and a quenching
analysis was performed using N-methyl aniline. Lifetimes were obtained from
monoexponential fits to the time-resolved decay data. The lifetime value of DEA-
M in acetonitrile was 2.86 ns (2.22 ns in DMSO). The Stern-Volmer (SV) plots
are shown in Figure 3.10. The data for both the case of integrated intensity and
69
lifetime values are linear with respect to N-methylaniline. This is a typical SV plot
result for a fluorophore quenched predominantly through dynamic interactions
(collisional quenching). Dynamic quenching manifests itself both in the
fluorescence intensity and lifetime data, whereas static quenching will only affect
the fluorescence intensity plot. This result is fully consistent with studies involving
similar DEA compounds quenched by N-methyl aniline.18
The slope for the intensity measurement was determined to be 0.27 ± 0.01
M-1, and the slope for the lifetime measurement was determined to be 0.23 ±
0.01 M-1. The errors were determined for a 95% confidence interval, using the
student’s t-test. The two slopes do not agree within a 95% confidence interval.
The slope for intensity is roughly 17% greater than that of the lifetime. This
discrepancy is typically observed, and is often attributed to the emergence of a
static quenching component at higher quencher concentrations.19 This is easily
explained by the fact that at higher quencher concentrations, there is an
increased probability that a particular quencher will be adjacent to a fluorophore
at the moment of excitation. This results in an instantaneous static quenching.
Afterwards, this quencher diffuses away and may quench a nearby excited
fluorophore. Thus, the observed fluorescence intensity will be affected by the
quenching of two fluorophores, whereas the lifetime decay data is not affected at
all by the first event.
70
Figure 3.10: Stern-Volmer quenching plots for DEA-M in acetonitrile, in terms of
integrated emission intensities (red), and lifetimes (black)
3.2.2 NCC-coumarin lifetime fits
Fluorescence lifetimes were obtained for the NCC-coumarins suspended
in varying solvents. The solvents that were investigated were constrained by the
suspensions which could be excited using the 405nm laser on the TCSPC
instrument. The fluorescence lifetimes varied between solvents. This is
consistent with the steady-state emission intensities, which also showed solvent
dependence. This is expected, since the two observables are related.
71
Fluorescence decay data of the NCC-coumarin suspensions could not be
satisfactorily fit to a monoexponential. Reasonable fits were obtained either by
tail-fitting the decay using a stretched exponential, or as a sum of two
exponential terms. A three-exponential reconvolution fit also gave an acceptable
fit, when a third exponential term was added to compensate for colloidal scatter.
In all cases, a single weighted average lifetime was determined. These data are
tabulated in tables 3.1, 3.2 and 3.3. A systematic comparison of the DEA-M and
NCC-DEA lifetime values was beyond the scope of this thesis. However, the
lifetimes of NCC-DEA were lower than those of DEA-M in both acetonitrile and
DMSO.
72
Table 3.1: NCC-DEA lifetime data
Tail average Streched Exponential Reconvolution
average
Solvent <τ>
(ns)
χ2 <τ> (ns) β χ2 <τ> (ns) χ2
dioxane 2.75 1.04 2.57 0.94 0.98 2.38 1.07
ethanol 2.5 1.06 2.28 0.91 1.02 2.16 1.08
water 2.67 1.02 2.23 0.84 0.94 1.44 1.14
DMSO 2.39 1.00 2.26 0.95 1 2.13 1
ACN 2.32 1.03 2.05 0.89 1 1.89 1.08
73
Table 3.2: NCC-HC lifetime data
Tail average Streched Exponential Reconvolution
average
Solvent <τ>
(ns)
χ2 <τ> (ns) β χ2 <τ> (ns) χ2
DMF 3.59 1.03 3.36 0.94 0.95 3.15 1.05
DMSO 3.32 1.06 2.83 0.88 1.04 2.56 1.34
water 4.16 1.03 4.03 0.97 0.96 3.92 1.03
Table 3.3: NCC-AC lifetime data
Tail average Streched Exponential Reconvolution
average
Solvent <τ>
(ns)
χ2 <τ> (ns) β χ2 <τ> (ns) χ2
DMF 3.62 1 3.38 0.94 0.95 3.19 1.04
DMSO 3.41 1.12 2.92 0.88 1.08 2.72 1.11
water 4.15 1.02 4.03 0.97 0.96 3.94 1.05
74
A stretched exponential has been used in NMR experiments to fit free
induction decays of solid-state 13C T1 relaxations in NCC.20 In this study, the
authors argued that NCC particles manifest a distribution of environments, made
up of crystalline and amorphous regions. Similarly, it is reasonable to argue that
NCC-coumarin might exhibit some sort distribution of lifetimes reflecting
heterogeity in the local-environment. We believe that this distribution in
heterogeneity is rather narrow based on the fitted values of β, which are
approximately 0.9. This means that covalently bound coumarin has an emissive
lifetime that is only slightly perturbed by the surrounding NCC micro-environment.
3.2.3 Stern-Volmer analysis of NCC-DEA
Stern-Volmer quenching of NCC-DEA was determined in a manner to that
of DEA-M. For the NCC samples, DMSO was used as the solvent instead of
acetonitrile because the NCC particles were more stable against aggregation.
The data are plotted in Figure 3.12. The NCC-DEA SV intensity plot has a slope
of 0.07±0.01. This is only 27% of the slope found for the small molecule DEA-M
case. The difference between the two slopes is not surprising. The effect is well-
known in studies of fluorescence quenching of chromaphores attached to
macromolecules.21 Yguerabide et al.21 developed theoretical models for the
accessibility of fluorophores immobilized on the surface of large proteins. Their
75
work suggests that quenching rates can be decreased by a factor of 2-5
compared to the free fluorophore, due to the reduced translational diffusion rate
of the protein, the reduced rotational mobility as well as orientational factors. The
extent of quenching rate reduction depends on the molecular weight of the
protein, and reaches an asymptotic value when the molecular weight is
approximately 50 kDa. Considering that a single NCC particle has a molecular
weight22 which exceeds this value, then it can be argued that NCC should
behave in a similar manner to that of a massive protein.
The SV lifetime plot has a slope of 0.03±0.01. This slope is significantly
lower than that of the intensity plot, which suggests that quenching appears to
have a dominant static component.23 In purely static quenching, the SV slope of
the lifetime plot is 0. This is because the lifetime of a fluorophore is not affected
by static quenching. The intensity SV plot would still have a slope, because the
overall intensity of the sample would still diminish. Thus, the greater the static
quenching component, the lower the slope of the lifetime SV plot will be in
comparison with the slope for the intensity data.
The slope of the SV lifetime plot did not change appreciably depending on
what method was used to calculate the average lifetime, as can be seen in
Figure 3.13. The mean lifetime of narrow lifetime distributions can be
approximated by the weighted average of two monoexponential lifetimes.24
76
Figure 3.11: Stern-Volmer quenching plots for NCC-DEA in DMSO, in terms of
integrated emission intensities (black), and mean lifetimes (red)
Figure 3.12: Comparison of lifetime Stern-Volmer quenching plots for NCC-DEA
in DMSO, between a tailfitted stretched exponential fit (black), tail-fitted
biexponential (blue) and triexponential reconvolution fit (red)
77
3.3 Lifetime mapping of NCC-coumarin in PEGDA
FLIM was employed to assess how well the NCC-coumarin could be
dispersed into a polymer matrix. The use of FLIM has several advantages over
intensity-based confocal fluorescence imaging. It is a concentration independent
technique, and thus resistant to photobleaching.
NCC-DEA was dispersed in a matrix of photo-cured poly(ethylene glycol)
diacrylate (PEGDA). The NCC-DEA was not uniformly dispersed in the film, and
aggregated regions could be clearly identified as can be seen in Figure 3.14.
The aggregated region was imaged using two-dimensional (2D) fluorescence
lifetime imaging, with a monoexponential fit. The contour of the aggregate was
successfully picked out with respect to the PEGDA background. PEGDA
displays weak background fluorescence,25 with a maximum at 421 nm, as shown
in Appendix E. No large lifetime deviations were viewed across the aggregate.
This observation agrees with the assertion that the NCC-coumarin has a single,
narrow distribution of lifetime values. This mapping method could potentially be
used to estimate the extent to which aggregation effects the fluorescence
lifetime.
78
Figure 3.13: Left panel: confocal fluorescence emission image of NCC-DEA
embedded in a photocured PEDGA film. Right panel: Same image, but with 2D
lifetime map superimposed
3.4 Summary of Stern-Volmer and PEGDA experiments
Lifetime measurements were performed on a solution of DEA-M in
acetonitrile and suspensions of NCC-coumarins in several solvents. The DEA-M
lifetime was obtained from a single exponential fit, and displayed typical dynamic
quenching Stern-Volmer plots in which the slopes of emission intensity and
lifetime were nearly identical. The lifetimes of the NCC coumarin hybrids could
not be fit to a single exponential. They were fitted instead to a stretched
exponential distribution with β values of ~0.9. It is hypothesized that the inability
to fit a single discrete lifetime is due to variations of environments inherent to the
NCC, which in turn is responsible for variations in the lifetime of the NCC-
79
coumarin combination. The result is a narrow continuum of lifetimes,
symmetrically centered on a mean value. The Stern-Volmer plots for NCC-
coumarin show that the quenching rate constant is reduced by over 50% in
comparison with the single molecule analogue, DEA-M. Moreover, evidence of
static quenching can be seen in the intensity and lifetime slopes. Lastly, NCC-
DEA was incorporated into a film of photo-cured PEGDA. NCC-DEA aggregates
were visible throughout the film via confocal fluorescence microscopy, and could
be successfully picked out through a 2D lifetime mapping procedure.
3.8 References
1. Lieber, E.; Rao, C. N. R.; Chao, T. S.; Hoffman, C. W. W., Infrared
Spectra of Organic Azides. Analytical Chemistry 1957, 29 (6), 916-918.
2. Krishnakumar, V.; Xavier, R. J., FT Raman and FT-IR spectral studies of
3-mercapto-1,2,4-triazole. Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy 2004, 60 (3), 709-14.
3. Ding-Bo, W.; Bao-Hua, C.; Bing, Z.; Yong-Xiang, M., XPS study of
aroylhydrazones containing triazole and their chelates. Polyhedron 1997, 16 (15),
2625-2629.
4. Devadoss, A.; Chidsey, C. E., Azide-modified graphitic surfaces for
covalent attachment of alkyne-terminated molecules by "click" chemistry. Journal
of the American Chemical Society 2007, 129 (17), 5370-1.
5. Huang, J. L.; Li, C. J.; Gray, D. G., Cellulose Nanocrystals Incorporating
Fluorescent Methylcoumarin Groups. Acs Sustainable Chemistry & Engineering
2013, 1 (9), 1160-1164.
80
6. Santos, N. C.; Castanho, M. A., Teaching light scattering spectroscopy:
the dimension and shape of tobacco mosaic virus. Biophysical Journal 1996, 71
(3), 1641-50.
7. Lakowicz, J. R., Principles of fluorescence spectroscopy. Springer: New
York, 2006.
8. Kamlet, M. J.; Abboud, J. L.; Taft, R. W., The solvatochromic comparison
method. 6. The .pi.* scale of solvent polarities. Journal of the American Chemical
Society 1977, 99 (18), 6027-6038.
9. Catalán, J.; López, V.; Pérez, P.; Martin-Villamil, R.; Rodríguez, J.-G.,
Progress towards a generalized solvent polarity scale: The solvatochromism of 2-
(dimethylamino)-7-nitrofluorene and its homomorph 2-fluoro-7-nitrofluorene.
Liebigs Annalen 1995, 1995 (2), 241-252.
10. Cigan, M.; Donovalova, J.; Szocs, V.; Gaspar, J.; Jakusova, K.;
Gaplovsky, A., 7-(Dimethylamino)coumarin-3-carbaldehyde and its
phenylsemicarbazone: TICT excited state modulation, fluorescent H-aggregates,
and preferential solvation. Journal of Physical Chemistry A 2013, 117 (23), 4870-
83.
11. Nad, S.; Kumbhakar, M.; Pal, H., Photophysical properties of coumarin-
152 and coumarin-481 dyes: Unusual behavior in nonpolar and in higher polarity
solvents. Journal of Physical Chemistry A 2003, 107 (24), 4808-4816.
12. Jones Ii, G.; Jackson, W. R.; Halpern, A. M., Medium effects on
fluorescence quantum yields and lifetimes for coumarin laser dyes. Chemical
Physics Letters 1980, 72 (2), 391-395.
13. Signore, G.; Nifosi, R.; Albertazzi, L.; Storti, B.; Bizzarri, R., Polarity-
sensitive coumarins tailored to live cell imaging. Journal of the American
Chemical Society 2010, 132 (4), 1276-88.
14. Kubista, M.; Sjoback, R.; Eriksson, S.; Albinsson, B., Experimental
Correction for the Inner-Filter Effect in Fluorescence-Spectra. Analyst 1994, 119
(3), 417-419.
81
15. Borissevitch, I. E., More about the inner filter effect: corrections of Stern–
Volmer fluorescence quenching constants are necessary at very low optical
absorption of the quencher. Journal of Luminescence 1999, 81 (3), 219-224.
16. Aspée, A.; Alarcon, E.; Pino, E.; Gorelsky, S. I.; Scaiano, J. C., Coumarin
314 Free Radical Cation: Formation, Properties, and Reactivity toward Phenolic
Antioxidants. The Journal of Physical Chemistry A 2011, 116 (1), 199-206.
17. Kubin, R. F.; Fletcher, A. N., The Effect of Oxygen on the Fluorescence
Quantum Yields of Some Coumarin Dyes in Ethanol. Chemical Physics Letters
1983, 99 (1), 49-52.
18. Nad, S.; Pal, H., Electron Transfer from Aromatic Amines to Excited
Coumarin Dyes: Fluorescence Quenching and Picosecond Transient Absorption
Studies. The Journal of Physical Chemistry A 1999, 104 (3), 673-680.
19. Eftink, M., Fluorescence Quenching: Theory and Applications. In Topics in
Fluorescence Spectroscopy, Lakowicz, J., Ed. Springer US: 2002; Vol. 2, pp 53-
126.
20. Lemke, C. H.; Dong, R. Y.; Michal, C. A.; Hamad, W. Y., New insights into
nano-crystalline cellulose structure and morphology based on solid-state NMR.
Cellulose 2012, 19 (5), 1619-1629.
21. Johnson, D. A.; Yguerabide, J., Solute accessibility to N epsilon-
fluorescein isothiocyanate-lysine-23 cobra alpha-toxin bound to the acetylcholine
receptor. A consideration of the effect of rotational diffusion and orientation
constraints on fluorescence quenching. Biophysical Journal 1985, 48 (6), 949-55.
22. Filpponen, I.; Argyropoulos, D. S., Regular linking of cellulose
nanocrystals via click chemistry: synthesis and formation of cellulose
nanoplatelet gels. Biomacromolecules 2010, 11 (4), 1060-6.
23. Johansson, J. S., Binding of the volatile anesthetic chloroform to albumin
demonstrated using tryptophan fluorescence quenching. Journal of Biological
Chemistry 1997, 272 (29), 17961-17965.
24. James, D. R.; Ware, W. R., A Fallacy in the Interpretation of Fluorescence
Decay Parameters. Chemical Physics Letters 1985, 120 (4-5), 455-459.
82
25. Chiu, Y. C.; Brey, E. M.; Perez-Luna, V. H., A study of the intrinsic
autofluorescence of poly (ethylene glycol)-co-(L-lactic acid) diacrylate. Journal of
Fluorescence 2012, 22 (3), 907-13.
83
Chapter 4: Conclusions and Future Work
4.1 Conclusions
Three coumarin azides were covalently attached to the surface of alkyne
functionalized NCC through a Huisgen cycloaddition. The procedure is efficient
and can be adapted to a wide arrange of fluorophores. Resulting triazole linked
NCC-coumarins could be suspended in a number of polar organic solvents. The
emission maxima wavelengths of the NCC-DEA and model compound DEA-M in
DMSO were identical, which suggests that the coumarins had the same access
to solvent molecules on average. The derivatives were strongly fluorescent in
DMSO. NCC-coumarins retained the inherent solvatochromic response of
coumarin dyes, and might be used as a polarity probe of the NCC surroundings.
Aggregation was shown to reduce fluorescence intensities; however its effect on
lifetimes is still unknown.
To our knowledge, this is the first report of fluorescently labelled NCC
lifetime measurements. Our findings suggest that the NCC-coumarins exhibit a
lifetime distribution, which points to an underlying micro-environmental
heterogeneity of NCC. Stern-Volmer analyses demonstrated that quenching
rates are significantly reduced for NCC-coumarins in comparison with free
molecular analogues. This result suggests that fluorescently labelled NCC could
84
be an effective biocompatible lifetime probe of environments which would
normally quench free fluorophores.
NCC-coumarin was incorporated into a photo-curable PEGDA matrix and
could be easily identified through fluorescence lifetime mappings. This approach
should be amenable as a general method to assess the distribution of NCC
within a rigid matrix.
4.2 Future work
Reproducibility of the click reaction remains a concern which needs to be
addressed if this method is to be employed as a platform for future studies. It
remains to be seen whether modifications to the procedure can be employed to
reduce the extent of aggregation. Efforts to further purify NCC-coumarin through
the removal of trace amounts of Cu (II) should also be considered.
While the coumarins selected for this study displayed strong
solvatochromism, it may be worthwhile to select fluorophores with
correspondingly red-shifted absorption and emission. This would reduce the
extinction overlap between the NCC and coumarin dye, as well as other
fluorescence peaks attributed to the NCC.
85
Quantum yields should be determined through the use of an integrating
sphere1 instead of the relative method in order to assess the relative amount of
scatter compared to emission.
Extending the study to include NCC-coumarin suspended in highly viscous
environments2-3 may also be of interest. This, in conjunction with varying
temperature4 and polarization anisotropy studies, should also be considered to
provide insight into the fluorescence dynamics of NCC-coumarin. The ability to
perform lifetime measurements with several excitation wavelengths would be
helpful to establish the existence of multiple emissive states within NCC-
coumarin.
While the use of Non-negative least-squares5 fitting provides a simple
means to obtain lifetime values, researchers must make an a priori assumption
about the form of the fluorescence decay model. Switching to fitting techniques
which do not require such an assumption, such as the Maximum Entropy
Method6, is preferable in order to avoid biasing and ultimately misinterpreting the
lifetime results. This should be easily achievable since the iterative simplex can
be easily modified for more complex techniques.
Finally, fluorescence lifetime data should be collected for a large range of
fluorescently tagged NCC systems. The conjectures drawn in this thesis rely on
a small data set, and require further studies to generalize the findings.
86
4.3 References
1. Suzuki, K.; Kobayashi, A.; Kaneko, S.; Takehira, K.; Yoshihara, T.; Ishida,
H.; Shiina, Y.; Oishi, S.; Tobita, S., Reevaluation of absolute luminescence
quantum yields of standard solutions using a spectrometer with an integrating
sphere and a back-thinned CCD detector. Physical Chemistry Chemical Physics
2009, 11 (42), 9850-60.
2. Demchenko, A. P., The red-edge effects: 30 years of exploration.
Luminescence 2002, 17 (1), 19-42.
3. Demchenko, A. P., Red-edge-excitation fluorescence spectroscopy of
single-tryptophan proteins. European Biophysics Journal 1988, 16 (2), 121-9.
4. Galley, W. C.; Purkey, R. M., Role of heterogeneity of the solvation site in
electronic spectra in solution. Proceedings of the National Academy of Sciences
of the United States of America 1970, 67 (3), 1116-21.
5. Ware, W. R.; Doemeny, L. J.; Nemzek, T. L., Deconvolution of
Fluorescence and Phosphorescence Decay Curves - Least-Squares Method.
Journal of Physical Chemistry 1973, 77 (17), 2038-2048.
6. Livesey, A. K.; Brochon, J. C., Analyzing the Distribution of Decay
Constants in Pulse-Fluorimetry Using the Maximum Entropy Method. Biophysical
Journal 1987, 52 (5), 693-706.
87
Appendix A: Characterization data for model compound DEA-M
Figure A1: DEA-M ATR-FTIR
Figure A2: DEA-M MS
88
dea_purifm_cmpd_jun16.esp
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5
Chemical Shift (ppm)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Norm
alized Inte
nsity
6.073.003.012.983.014.101.000.011.961.011.021.031.020.011.920.990.990.990.940.88
water
8.5
48.4
0
7.4
37.4
17.2
6
6.7
06.6
9
6.5
66.5
5
5.2
1
5.1
95.1
65.1
1 4.9
94.9
3
4.9
04.6
94.6
7
4.2
84.2
74.1
94.1
94.1
64.1
6
3.7
3
3.5
03.4
93.4
73.4
53.4
3
2.1
2 2.0
42.0
21.9
91.5
7 1.5
7
1.2
61.2
51.2
3
Figure A3: 1H NMR
C13_20000deam.esp
170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10
Chemical Shift (ppm)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Norm
alized Inte
nsity
170.7
7170.2
4169.5
4169.4
2
156.8
6155.8
3
151.6
4
143.6
4
134.8
1
130.0
5
123.9
3
116.6
6
110.0
9106.9
7
99.2
597.0
0
77.4
3
76.5
872.8
471.8
871.1
068.2
962.3
161.8
5
45.0
1
20.7
8
20.6
0
12.4
1
Figure A4: 13C NMR
89
1H NMR (CDCl3, 400 MHz, 22 °C): 1.25 (t, J = 8 Hz, 6H), 1.99 (s, 3H),
2.02 (s, 3H), 2.04 (s, 3H), 2.12 (s, 3H), 3.46 (q, J = 8Hz, 4H), 3.74 (dq, J = 8Hz, 4
Hz, 1H), 4.23 (ddd, J= 32 Hz, 12Hz, 4 Hz, 2H), 4.68 (d, J = 8 Hz, 1H),
4.96 (dd, J = 24Hz, 12Hz, 2H), 5.04 (t, J = 8Hz, 1H), 5.11 (t, J = 8Hz, 1H),
5.19 (t, J = 12Hz, 1H), 6.55 (d, J = 4 Hz, 1H), 6.68 (dd, J = 8Hz, 4Hz, 1H),
7.42 (d, J = 8Hz, 1H), 8.40 (s, 1H), 8.54 (s, 1H).
13C NMR (CDCl3, 75 MHz, 22 °C): 170.77, 170.24, 169.54, 169.42, 156.86,
155.83, 151.64, 143.64, 134.81, 130.05, 123.93, 116.66, 110.09, 106.97, 99.25,
97.00, 72.84, 71.88, 71.10, 68.29, 62.31, 61.85, 45.01, 20.78, 20.66, 20.60,
12.41.
ATR-FTIR (powder, cm-1) 1745 (s), 1733 (s), 1704 (s), 1623(s), 1599(s),
1525 (m), 1434(m), 1369 (m), 1354 (m), 1247 (s), 1226 (s), 1180 (m), 1134 (m),
1090 (m), 1050 (s), 1039 (s), 1007 (m), 821 (m)
m.p 154-156 °C
ESI-MS: (M + Na) for C30H36O12N4Na calcd 677.62, found 667.16
1H NMR was acquired on a Varian-Mercury 400 MHz spectrometer, and
13C NMR was collected on a Varian-Mercury 300 MHz spectrometer. ESI-MS
was performed on a Thermo-Finnigan LCQ-Duo with 4.5kV spray voltage.
90
Appendix B: Excitation and Emission maxima of NCC-coumarins
Table B1: Data for NCC-DEA
Solvent Lippert-Mataga
Polarity Index
Excitation
maximum (nm)
Emission
Maximum (nm)
Water 0.32 425 498
DMSO 0.26 413 489
DMF 0.27 410 486
Acetonitrile 0.30 412 483
Ethanol 0.30 417 481
Dioxane 0.03 417 473
Isopropanol 0.28 412 480
91
Table B2: Data for NCC-HC
Solvent Lippert-Mataga
Polarity Index
Excitation
maximum (nm)
Emission
Maximum (nm)
Water 0.32 360 472
DMSO 0.26 346 434
DMF 0.27 351 459
Acetonitrile 0.30 344 418
Ethanol 0.30 344 418
Dioxane 0.03 345 418
Isopropanol 0.28 354 423
92
Table B3: Data for NCC-AC
Solvent Lippert-Mataga
Polarity Index
Excitation
maximum (nm)
Emission
Maximum (nm)
Water 0.32 360 476
DMSO 0.26 345 431
DMF 0.27 352 458
Acetonitrile 0.30 339 413
Ethanol 0.30 345 417
Dioxane 0.03 345 414
Isopropanol 0.03 345 417
93
Appendix C: N-methyl aniline Extinction spectra
Figure D1: Qualitative extinction spectra comparing high concentration N-methyl
aniline (black) and low concentration N-methyl anline (red) in DMSO
94
Appendix D: PEGDA Emission spectra
Figure D1: Emission spectrum of neat PEGDA (MW 575), excited at 400 nm
Figure D2: Emission spectrum of NCC-DEA in crosslinked PEGDA excited at
366nm. The shoulder at 421 is attributed to the PEGDA fluorescence.