Controlling the density of nucleic acid oligomers on ®ber opticsensors for enhancement of selectivity and sensitivity

James H. Wattersona, Paul A.E. Piunnoa,b, Christopher C. Wustb, Ulrich J. Krulla,*

aDepartment of Chemistry, Chemical Sensors Group, University of Toronto at Mississauga,

3359 Mississauga Road North, Mississauga, Ont., Canada L5L 1C6bFONA Technologies Inc., 855 Matheson Blvd. East, Unit #14, Mississauga, Ont., Canada L4W 4L6

Abstract

The immobilization of oligonucleotides to solid surfaces is relevant to the development of biosensor and microarray technologies. The

density of oligonucleotide immobilization determines the charge density at the surface by means of ionizable phosphate groups, and may

result in an interfacial dielectric constant, pH and ionic strength that are unlike those of bulk solution. The density of immobilization may

affect the extent of interactions between neighbouring oligomers, as well as interactions between the immobilized oligomers and the

substrate surface. Experiments were done to examine the effects of immobilization density and solution conditions on the sensitivity,

selectivity and dynamic range of hybridization assays done using a ®ber optic nucleic acid biosensor based on total internal re¯ection

¯uorescence (TIRF). Such immobilized nucleic acid ®lms ®rst required activation by thermal denaturation cycling to reach full activity. The

effects of non-selective adsorption of oligonucleotides were dependent on ionic strength, and could not be removed independently of

hybridization. Increased immobilization density resulted in signi®cantly higher sensitivity but reduced dynamic range in all hybridization

assays done. Sensitivity and selectivity were a function of temperature, however, the selectivity of hybridization assays done using these

sensors could not be predicted by consideration of thermal denaturation temperatures alone. # 2001 Elsevier Science B.V. All rights

reserved.

Keywords: Biosensor; Oligonucleotide; Immobilization; Nucleic acid; Fluorescence; Optical ®ber

1. Introduction

The immobilization of biomolecules to solid surfaces is

widely used in the preparation of analytical sensors. Appli-

cations include immunosensor techniques [1±3], which tend

to rely on protein binding as the means of molecular

`̀ recognition'', as well as those which make use of nucleic

acid hybridization [4±9] as the basis for selective recogni-

tion. The use of immobilized nucleic acids to provide for

selective binding interactions is attractive since the selec-

tivity of nucleic acid binding interactions can be quite high

and the advent of polymerase chain reaction and solid phase

nucleic acid synthesis has allowed for relatively simple

nucleic acid preparation and immobilization.

The utility of immobilized selective molecular recogni-

tion elements is dependent upon the retention of selective

binding capacity after the immobilization process is

complete. The binding capacity is dependent upon the

structure of the immobilized molecules in their local

environments, which can be signi®cantly different from

those experienced in bulk solution. The density of immo-

bilization of single-stranded DNA (ssDNA) onto the sur-

face of a solid substrate affects the charge density at the

surface, and the extent to which the immobilized oligomers

interact with the surface of the solid substrate and

with neighbouring nucleic acid oligomers. This has

consequences to issues of selectivity and the extent of

hybridization, as well as the orientation of the immobilized

ssDNA, and therefore affects the kinetics of hybridization

[10]. Clearly, the control of selectivity of binding and the

dynamic range that could be achieved by control of the

concentration of oligonucleotide sequences at an interface

can be complex.

The elucidation of the orientation and packing structure

of nucleic acids immobilized on gold and polystyrene

surfaces has been attempted [11±13]. It was suggested

that the alignment of immobilized oligonucleotides with

respect to the substrate surface may be controlled by

selection of oligonucleotide immobilization density, as

well as through control of the chemical environment at

Sensors and Actuators B 74 (2001) 27±36

* Corresponding author. Tel.: �1-905-828-5437; fax: �1-905-828-5425.

E-mail address: ukrull@credit.erin.utoronto.ca (U.J. Krull).

0925-4005/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved.

PII: S 0 9 2 5 - 4 0 0 5 ( 0 0 ) 0 0 7 0 8 - 5

the surface. Adsorptive interactions of oligonucleotides

immobilized by sulfur±gold interactions on a gold surface

were reduced by blocking unreacted surface sites with

mercaptohexanol [10]. The reduction in oligonucleotide

adsorption to gold resulted in extension of the immobilized

oligonucleotides away from the substrate surface [11]. The

extent of hybridization was found to be affected by the

packing density of immobilized oligonucleotides, with

hybridization being inhibited at higher packing densities

where steric hindrance and electrostatic repulsion were

thought to reduce the stability of hybrids that could

form [11].

In previous work [14,15], we described an examination of

the thermodynamics of interfacial nucleic acid hybridiza-

tion. The thermal denaturation temperature, Tm (temperature

at which 50% of all duplexes formed are denatured), of the

hybrids formed at a fused silica surface was used to inves-

tigate the thermodynamics of the hybridization process. It

was observed that short immobilized nucleic acid strands

(20mers) that were immobilized on hexaethylene glycol

linkers that were covalently attached to fused silica sub-

strates underwent thermal denaturation transitions that were

signi®cantly broader than those observed for experiments

done in bulk solution. The immobilized nucleic acids exhib-

ited a two±three-fold reduction in the enthalpy change that

accompanied the thermal denaturation transition compared

with experiments done in bulk solution. It was also observed

that immobilized nucleic acids could provide larger devia-

tions in the Tm between fully complementary sequences and

those containing single base-pair mismatches (SBPMs) than

could similar hybridization reactions in bulk solution, with

the effects becoming exacerbated at higher immobilization

densities. These results corroborated the notion that inter-

facial hybridization occurs in a signi®cantly different envir-

onment than that of bulk solution, and that surface-strand

interactions and nearest neighbour interactions may become

signi®cant.

In the present work, we report the effects of oligonucleo-

tide immobilization density on the selectivity and sensitivity

of transduction of hybridization using a fused silica ®ber

optic biosensor based on total internal re¯ection ¯uores-

cence (TIRF). From an analytical standpoint, it is not

possible to simply extrapolate experimental conditions for

a hybridization assay for such biosensors (i.e. temperature,

ionic strength and immobilization density) from data

obtained from experiments done in bulk solution. Conven-

tionally, solution temperature and ionic strength are con-

trolled in order to maximize differences in Tm between fully

complementary and partially complementary sequences,

thereby reducing the effects of non-selective hybridization.

The experiments described herein, were done in an effort to

better characterize the effects of immobilization density on

the sensitivity and selectivity of hybridization assays done in

a variety of conditions using the ®ber optic biosensor.

Particular attention was paid to issues of reproducibility

and non-selective adsorption.

2. Experimental

2.1. Chemicals

Solvents were obtained from BDH (Toronto, Ont.) as

reagent grade and were further puri®ed or dried, when

necessary, by standard distillation methods. Reagent grade

salts were purchased from BDH (Toronto, Ont.). DNA

synthesis reagents were from Dalton Chemical Laboratories

Inc. (Toronto, Ont.). Anhydrous acetonitrile (Dalton) was

dried by distillation from P2O5 prior to receipt, and was

further distilled from calcium hydride under a dry argon

atmosphere prior to use. Tetrahydrofuran (BDH) was ®rst

dried over CaH2, ®ltered and ®nally distilled immediately

prior to use from sodium metal (Aldrich)/benzophenone

(Aldrich). Sterile water for use on its own and with hybri-

dization buffer was produced with the water ®rst double-

distilled in glass, then subsequently treated with diethyl

pyrocarbonate (Aldrich) and sterilized by autoclave. Mole-

cular biology grade polyacrylamide gel electrophoresis

reagents and apparatus were obtained from Bio-Rad (Her-

cules, CA, USA). Silica gel (Toronto Research Chemicals,

Toronto, Ont.) that was used for puri®cation had a particle

size of 30±70 mm.

2.2. Preparation of optical fiber segments

Fused silica optical ®bers of 400 mm core diameter (3M

Powercore Series Optical Fiber, FT-400-URT or FP-400-

UHT) were acquired from Thor Labs Inc., Newton, NJ,

USA. The polymeric outer cladding was removed mechani-

cally by means of a ®ber-stripping tool also obtained through

Thor Labs, Inc. Removal of the outer cladding exposed the

inner cladding layer that coated the core of fused silica.

Individual sensor elements were then made by cutting

optical ®ber pieces of 48 mm length with a ®ber-scoring

device. Each ®ber was cleanly scored by rotating a diamond

pencil about the optical ®ber. Removal of the top portion of

the scored ®ber from the remainder of the optical ®ber

secured in the pin-chuck then yielded cylindrical optical

®ber segments with clean, ¯at termini as evidenced by visual

inspection of the termini at 40� magni®cation.

The fused silica ®ber segments and controlled pore glass

(CPG) (CPG Inc., Lincoln Park, NJ, USA) that were used as

solid substrates for automated DNA synthesis were cleaned

prior to modi®cation of the surface according to the two-

stage method of Kern and Puotinen [16]. The ®rst stage

consisted of immersing the solid substrates in a 1:1:5 (v/v)

solution of 30% ammonium hydroxide/30% hydrogen per-

oxide/water and gently agitating at 808C for 5 min. In the

second stage, the substrates were then recovered, thoroughly

washed with sterile water and then gently agitated in a

solution of 1:1:5 (v/v/v) conc. HCl/30% hydrogen perox-

ide/water for 5 min at 808C. The substrates were then

recovered and washed with successive 100 ml portions of

water, methanol, dichloromethane and diethyl ether. The

28 J.H. Watterson et al. / Sensors and Actuators B 74 (2001) 27±36

substrates were then dried under vacuum and stored in vacuo

and over P2O5 until required.

2.3. Surface modification of solid substrates Ð functiona-

lization of substrates with 3-glycidoxypropyltrimethoxy-

silane (GOPS)

The cleaned solid substrates were suspended in an anhy-

drous solution of xylene/3-glycidoxypropyltrimethoxysi-

lane/diisopropylethylamine (100:30:1 v/v/v). The reaction

took place at 808C with stirring over 24 h under an argon

atmosphere. The substrates were then collected and succes-

sively washed with two 50 ml portions of each of methanol,

dichloromethane, diethyl ether, and then were dried and

stored under vacuum and over P2O5 at room temperature

until required.

2.4. Surface modification of solid substrates Ð linkage of

dimethoxytrityl (DMT)±hexaethylene glycol (HEG) onto

GOPS functionalized substrates

DMT±HEG was synthesized as outlined previously [19].

DMT±HEG (700 mg DMT±HEG/100 mg CPG) that had

been dried under vacuum and over P2O5 (>72 h) was dis-

solved in 20 ml of anhydrous pyridine. An excess of NaH

(10 eq.) that had been thoroughly washed with dry hexane

was then introduced to the mixture. The subsequent reaction

was permitted to proceed with stirring for 1 h at room

temperature under an argon atmosphere. The reaction mix-

ture was ®ltered through a sintered glass frit under a positive

pressure of argon into a vessel containing the GOPS func-

tionalized substrates. GOPS functionalized substrates were

separated into three batches, containing both optical ®bers

and CPG. The three batches then underwent the DMT±HEG

coupling reaction, which was permitted to proceed under a

positive pressure of argon at room temperature with gentle

agitation on an oscillating platform stirrer for durations of 4

and 12 h, respectively. Following the coupling reaction, the

substrates were quickly recovered and washed with succes-

sive 150 ml portions of methanol, water, methanol, and

diethyl ether to quench the coupling reaction and remove

any reactants that were non-speci®cally adsorbed. The

DMT-protected HEG-functionalized substrates were dried

under vacuum and over P2O5 and were maintained under

these conditions until further required.

2.5. Surface modification of solid substrates Ð capping of

unreacted silanol and hydroxyl functionalities with

chlorotrimethylsilane (TMS-Cl)

Unreacted silanol and hydroxyl functionalities on the

surface of the solid substrates where undesired oligonucleo-

tide synthesis could occur were capped prior to oligonucleo-

tide synthesis using TMS-Cl according to the method of Pon

[17]. The dried substrates were suspended in a solution

of 1:10 (v/v) TMS-Cl/pyridine for 16 h under an argon

atmosphere at room temperature. The substrates were sub-

sequently recovered and washed with three successive 20 ml

portions of pyridine, methanol and diethyl ether and were

then stored under vacuum and over P2O5 at room tempera-

ture until required.

2.6. Solid phase phosphoramidite synthesis of

oligonucleotides

All solid phase oligonucleotide synthesis was done using

a PE-ABI 392 DNA synthesizer (Perkin-Elmer Applied

Biosystems, Foster City, CA, USA). The pre-programmed

synthesis cycles employed for oligonucleotide assembly

were modi®ed to adjust the reagent delivery times in order

to ensure that the synthesis columns used were completely

®lled. The column used for oligonucleotide synthesis onto

optical ®bers segments was a custom manufactured Te¯on1

synthesis column (6 mm i:d:� 50 mm) capable of holding

eight ®bers. The ®bers were secured by means of insertion

into cylindrical bores (400 mm i:d:� 2 mm deep) machined

into one of the end caps. All end-caps were secured onto the

column bodies with aluminum crimp seals. The columns

used for oligonucleotide synthesis onto DMT±HEG±GOPS

functionalized CPG were custom manufactured Te¯on1

columns (8 mm i:d:� 10 mm). Te¯on1 end ®lters

(0.22 mm pore size, PE-ABI) were used to retain the glass

beads within the column. Synthesis of oligonucleotides for

use as complementary material for immobilized DNA was

carried out on nucleoside functionalized LCAA±CPG sub-

strates pre-packed in polyethylene columns as supplied by

the manufacturer, and has been described previously [14].

Polythymidylic acid icosanucleotides (dT20) were

assembled onto all of the optical ®ber and CPG substrates

functionalized with DMT±HEG linker molecules. Determi-

nation of the density of surface coverage of CPG substrates

with covalently immobilized oligonucleotide±HEG conju-

gates was done by anion-exchange HPLC following meth-

ods developed in our research group that have been reported

elsewhere [18].

Icosanucleotides labeled at the 50-terminus with a ¯uor-

escein moiety were used as complementary material to

hybridize with immobilized dT20 sequences. The 50-¯uor-

escein labeled oligonucleotides were prepared by use of a

¯uorescein phosphoramidite synthon (Dalton) and standard

protocols for oligonucleotide preparation [19]. Additionally,

unlabeled complementary icosanucleotides were prepared

by standard protocols for use in studies of hybridization in

bulk solution.

2.7. Instrumentation for studies of immobilized nucleic

acid films

Fluorescence-based studies of nucleic acid hybridization

at the surface of optical ®bers were carried out via an

automated spectro¯uorimeter instrument developed in our

research group that has been described previously[14]. Laser

J.H. Watterson et al. / Sensors and Actuators B 74 (2001) 27±36 29

radiation (488 nm) from a Coherent Innova 70 CW argon ion

laser (Coherent Laser Products, Palo Alto, CA, USA) was

coupled into a sensing ®ber by ®rst guiding the beam of

source radiation such that it was incident upon the surface of

a dichroic mirror (505 nm cut-off, Omega Optical, Battle-

boro, VT, USA) oriented at 458 to the incident beam. The

sensing ®ber was secured via a Te¯on1 holder with a

waterproof compression-type seal within a stainless steel

hybridization cell with small volume (1 mm i:d:� 50 mm)

that permitted a solution volume of 137 ml to be exposed to

the sensing ®ber. Fluorescence emission from the sensing

®ber with wavelength >505 nm was then directed back

through the dichroic mirror into a Bentham M300 mono-

chromator (f=# � 4:2, 2.5 nm bandwidth, distributed by

Optikon Corporation Limited, Waterloo, Ont.). Fluores-

cence emission exiting the emission monochromator was

detected by a side-on photomultiplier tube (106 A/W respon-

sivity, Model 77348, Oriel Corp.) operated at a potential of

500VDC (PMT Power Supply Model 5502, Products for

Research, Danvers, MA, USA).

The temperature of the solution within the ¯ow cell was

determined by use of a glass-encapsulated bead thermistor

(Fenwal Electronics Inc., distributed by Electrosonic Inc.,

Toronto, Ont.) embedded within the stainless steel block at a

distance not more than 1 mm from the internal wall of the

solution compartment surrounding the sensing ®ber. The

temperature of the ¯ow cell was regulated by use of a Peltier

temperature control accessory (Model 89090A, Hewlett-

Packard Corp., Mississauga, Ont.). Analyte sampling and

delivery was done using an automated sampler and pump

system as previously described [14].

2.8. Acquisition of thermal denaturation profiles from

nucleic acid films immobilized onto fused silica optical

fibers

All thermal denaturation pro®les for hybridization occur-

ring at the surface of the optical ®ber sensors were acquired

by monitoring the intensity of ¯uorescence emission at

542 nm over the temperature range of ca. 20±808C using

a temperature ramp rate of 0.38C minÿ1 to ensure that

equilibrium conditions were satis®ed, as described pre-

viously [15]. All sensors were cleaned by sonication in

ethanol in a 40 W bath sonicator for 90 min to remove

adsorbed impurities from the sensor surface prior to ana-

lysis. Thermal denaturation pro®les were obtained for

optical sensors that were exposed to ¯uorescein labelled

cDNA in various dilutions of a stock phosphate buffered

saline (PBS) hybridization buffer (1.0 M NaCl, 50 mM

PO4ÿn, pH 7.0). Removal of complementary oligonucleo-

tide associated with the sensor surface from previous ana-

lyses was done prior to each subsequent experiment by

¯ushing 15 ml of 808C water through the ¯ow-cell

(3 ml minÿ1, 5 min) and by ¯ushing 1 ml of 90% forma-

mide in TE buffer (10 mM Tris±HCl, 5 mM EDTA, pH 8.3)

through the ¯ow cell.

2.9. Hybridization assays of nucleic acid films immobilized

onto fused silica optical fibers

All sensors were cleaned by sonication in ethanol in a

40 W bath sonicator for 90 min to remove adsorbed impu-

rities from the sensor surface prior to analysis. Hybridization

assays were done for optical sensors that were exposed to

solution phase oligonucleotides labelled on the 50-terminus

with ¯uorescein. In all cases, oligonucleotides were dis-

solved in various dilutions of a stock PBS hybridization

buffer (1.0 M NaCl, 50 mM PO4ÿn, pH 7.0), with oligonu-

cleotide concentrations in the range of 0.001±0.1 mM. Dilu-

tions of the stock buffer by factors of 1.0, 0.5 and 0.1 were

used for ionic strength studies. Each sample solution had a

total volume of 1 ml, and was washed past the sensor at a

¯ow rate of 3 ml minÿ1. Following the addition of each

sample, a 1 ml solution of PBS buffer identical to that used

to prepare the oligonucleotide solution was washed past the

sensor to remove non-selectively adsorbed material. Assays

were done using solution temperatures of 28, 36, and 448C.

All hybridization assays were done in triplicate. Removal of

complementary oligonucleotide that had associated with a

sensor surface from previous analyses were done prior to

each subsequent experiment by ¯ushing 15 ml of 808C water

through the ¯ow-cell (3 ml minÿ1, 5 min) and by ¯ushing

1 ml of 90% formamide in TE buffer (10 mM Tris±HCl,

5 mM EDTA, pH 8.3) through the ¯ow cell.

3. Results and discussion

3.1. Control and characterization of oligonucleotide

immobilization density

The immobilization of dT20 onto the surface of fused

silica optical ®ber substrates was achieved by means of a

modi®cation to the method of Maskos and Southern [20].

The fused silica optical ®ber substrates were ®rst functio-

nalized with glycidoxypropyltrimethoxysilane (GOPS).

Linker molecules were then covalently attached to the

GOPS layer. The linker species used was HEG, protected

on one terminus with DMT in order to ensure single-site

reactivity and to minimize the risk of formation of closed-

ring structures with unreacted sites on the epoxysilane ®lm.

The modi®ed optical ®ber substrates were then subjected to

standard b-cyanoethyl-phosphoramidite oligonucleotide

synthesis protocols to prepare by stepwise synthesis the

dT20 oligonucleotides on the surface of the substrates.

The packing density of immobilized oligonucleotides was

altered by means of controlling the duration of the reaction

of DMT±HEG conjugates with the GOPS-functionalized

substrates. Two different immobilized densities were

obtained by permitting the DMT±HEG coupling reaction

to proceed for 4 and 8 h, respectively. In order to character-

ize the density of immobilization, oligonucleotide synthesis

was carried out as described above on the ®bers and

30 J.H. Watterson et al. / Sensors and Actuators B 74 (2001) 27±36

concurrently on GOPS-functionalized controlled-pore glass

(CPG), which has a well-de®ned surface area. The oligo-

nucleotide±HEG conjugates were then cleaved from the

surface of the CPG by means of exposure to concentrated

ammonium hydroxide for approximately 3 h, lyophilized

and re-dissolved in water. The quantity of immobilized

ssDNA as well as the quality of the products prepared by

automated synthesis were subsequently determined by

anion-exchange HPLC. Quantitation of the cleaved HEG±

dT20 conjugates was achieved by co-injection with a known

quantity of dT20. The results of the HPLC analysis are shown

in Table 1. The peaks corresponding to HEG±dT20 were

determined to have retention times of ca. 33 min. Distribu-

tions of peaks in the region of 33 min were due to the

formation of a HEG-linker ®lm in which some of the HEG

molecules had coupled onto one another to provide a dis-

tribution of linker lengths, as was described earlier [15,18].

These data show that these two densities provide two

different physical environments for the immobilized oligo-

nucleotides. The low-density sample consisted of immobi-

lized dT20±HEG conjugates separated by approximately

263 AÊ between adjacent strands, assuming uniform oligo-

nucleotide distribution. Since the length of the dT20±HEG

conjugate is ca. 100 AÊ in length, the low-density sample then

represents the system wherein there may be the onset of

some interaction between neighbouring strands. The high-

density sample consisted of immobilized dT20±HEG con-

jugates separated by approximately 100 AÊ between adjacent

strands. This close packing is much more likely to facilitate

interactions between neighbouring strands than at the lower

packing density.

3.2. Activation of nucleic acid films for hybridization

Regardless of the density of immobilization of ssDNA,

initial hybridization assays done using freshly prepared

®bers resulted in no signi®cant ¯uorescence increase fol-

lowing introduction of ¯uorescently labelled complemen-

tary DNA (cDNA). In attempts to achieve activation of

hybridization, ®bers were washed sequentially with water,

a solution of 90% formamide in TE buffer, and again with

sterile water, all at 808C. Sensors showed no response to

complementary material even after 10 such hybridization

assay cycles were attempted. The results indicate that heat-

ing of the immobilized nucleic acid ®lm alone is not

suf®cient to provide activation for response to cDNA. It

was observed that the process of selective hybridization

could be activated by subjecting freshly prepared ®bers to a

series of thermal denaturation experiments. The sensor

surfaces were exposed to a phosphate buffered solution

containing cDNA, and subsequently underwent a tempera-

ture ramp of 0.38C minÿ1, over a range from 20 to 808C.

Fig. 1 illustrates the response of a sensor coated with dT20 at

low packing density to a solution containing 10ÿ7 M dA20-

50-¯uorescein, before and after the sensor was subjected to

two thermal denaturation cycles using a solution containing

10ÿ7 M dA20-50-¯uorescein as the complementary material.

It may be that the presence of cDNA throughout the thermal

denaturation experiments provided a driving force for the

removal of some impurities adsorbed during the synthesis of

the nucleic acid ®lm. Evidence for this was that the back-

ground ¯uorescence was observed to decrease as a result of

the activating thermal denaturation experiments, and spec-

tral data suggested that trityl cation might have been the

species that was desorbed [21].

The response of a biosensor to successive hybridization

assays with 10ÿ7 M solutions of cDNA did not remain

constant after the initial activation cycles, but rather

decreased to a steady value after approximately three cycles,

as may be observed in Fig. 1. This suggests that the binding

activity of the immobilized oligonucleotides is dynamic

even after the process of activation. It was originally pos-

tulated that this reduction in signal was due to occlusion of

the surface by impurities present in the buffer solutions, but

this may only partially account for the loss of sensitivity

Table 1

Density of immobilization of dT20±HEG conjugate onto GOPS-functionalized substrates as determined by anion-exchange high performance liquid

chromatography [18]

Sample Reaction duration

(DMT±HEG-substrate) (h)

Total surface area

of CPG used (AÊ 2)

Molecules dT20±HEG

immobilized

Mean separation

distance (AÊ )

Low density 4 3.04 � 1019 5.59 � 1014 263

High density 8 4.12 � 1019 5.25 � 1015 100

Fig. 1. Response of sensor with low dT20 immobilization density to

10ÿ7 M solutions of dA20-50-fluorescein in 1� PBS buffer, before and

after activation of the film. Activation of the sensor was achieved by

exposure of the sensor to two thermal denaturation cycles.

J.H. Watterson et al. / Sensors and Actuators B 74 (2001) 27±36 31

since full signal (relative to maximum sensitivity shown)

was not achieved even after sonication of the sensor and

reintroduction of identical analyte solutions. It may be that

loss of the trimethylsilane capping species was occurring,

since the susceptibility of this functionality to cleavage from

the substrate at elevated temperatures was established by

wettability experiments (data not shown). In this case, the

variation in signal may be the result of the changing nature

of the interactions between the immobilized oligonucleo-

tides and the substrate surface.

3.3. Non-selective adsorption of oligonucleotides

The effects of non-selective adsorption of oligonucleo-

tides in bulk solution were examined by exposing an acti-

vated sensor surface to ¯uorescein labelled cDNA and

¯uorescein labelled non-complementary oligonucleotides

(ncDNA), and observing the resultant ¯uorescence intensity

before and after washing the sensor surface with 1 ml PBS

buffer under identical temperature conditions. Fig. 2a and b

show the results of such experiments for a sensor with low

dT20 packing density. Fig. 2a shows the results for experi-

ments done when the sensor was exposed to both fully

complementary (dA20-50-¯uorescein) and fully non-comple-

mentary oligonucleotides (dT20-50-¯uorescein) (10ÿ7 M). In

each case, cDNA or ncDNA was added and no further

washing of the sensor surface with PBS buffer was done.

The results show that for all ionic strengths examined, the

relative ¯uorescence intensity increase for the cDNA was

indistinguishable from that of ncDNA, within experimental

error. It was also observed that the magnitude of the increase

was dependent upon the ionic strength of the buffer used,

with larger signals obtained when buffers with higher ionic

strength were used. This increased signal was likely due to

the screening of electrostatic repulsion between immobi-

lized oligonucleotides and ssDNA in solution, and by

screening repulsive effects between the phosphate backbone

of ssDNA and any ionized hydroxyl moieties on the sensor

surface.

Fig. 2b shows the results for experiments done when the

sensor was exposed to both cDNA and ncDNA (10ÿ7 M),

after washing the sensor surface with 1 ml PBS buffer. For

all ionic strengths examined, the relative ¯uorescence inten-

sity increase observed after washing with PBS buffer was

signi®cantly larger when the sensor had been exposed to

complementary oligonucleotides than that observed when

non-complementary oligonucleotides were introduced,

implying that the difference of signal was largely represen-

tative of hybridization. Again, it was observed that the

magnitude of the ¯uorescence intensity increase was depen-

dent upon the ionic strength of the buffer used, with larger

signals observed when the complementary oligonucleotides

were dissolved in buffers of higher ionic strength.

Of importance to analytical experiments were the differ-

ences in sensitivity of response to both cDNA and ncDNA

for the sensor in buffers of different ionic strengths. Fig. 2c

shows the difference in response of a sensor with coated with

ssDNA at a low packing density to ¯uorescein labelled

cDNA, relative to the response of ¯uorescein labelled

ncDNA at in PBS buffers of various ionic strengths (1,

0.5 and 0.1 M NaCl). Examination of Fig. 2b and c shows

Fig. 2. (a) Response of sensor with low dT20 immobilization density to

10ÿ7 M solutions of dA20-50-fluorescein and 10ÿ7 M solutions of dT20-50-fluorescein as the non-complementary material. The results include

experiments that used 1� PBS buffer, 0:5� PBS buffer, and 0:1� PBS

buffer, with no washing step performed after introduction of solution phase

ssDNA. (b) Response of sensor with low dT20 immobilization density to

10ÿ7 M solutions of dA20-50-fluorescein and 10ÿ7 M solutions of dT20-50-fluorescein as the non-complementary material. The results include

experiments that used 1� PBS buffer, 0:5� PBS buffer, and 0:1� PBS

buffer, with a washing step performed after introduction of solution phase

ssDNA (1 ml PBS buffer). (c) Difference in response of sensor with low

dT20 immobilization density to 10ÿ7 M solutions of dA20-50-fluorescein

and 10ÿ7 M solutions of dT20-50-fluorescein for solutions containing

1� PBS, buffer, 0:5� PBS buffer, and 0:1� PBS buffer, with a washing

step performed after introduction of solution phase ssDNA (1 ml PBS

buffer).

32 J.H. Watterson et al. / Sensors and Actuators B 74 (2001) 27±36

that the largest response to both cDNA and ncDNA occurred

when the PBS buffer containing 1 M NaCl was used.

Although the non-selective response of the sensor to non-

complementary ssDNA was essentially removed when the

low ionic strength buffer was used, the difference in

response to cDNA relative to that of ncDNA was largest

when the high ionic strength buffer was used. For the

experiments using the PBS buffer containing 1 M NaCl,

the difference in response to complementary and non-com-

plementary oligonucleotides was 10-fold larger than that

observed in experiments done using PBS buffer with low

ionic strength (0.1 M NaCl). Thus, greater sensitivity is

obtained when using buffers of higher ionic strength, despite

the increased contribution from non-selective adsorption of

oligonucleotides in solution. Consequently, PBS buffer con-

taining 1 M NaCl was used for the remaining experiments to

examine other experimental parameters (sensitivity and

selectivity).

3.4. Comparison of sensitivity of biosensors with different

oligonucleotide packing densities

Experiments were done to examine the effect of oligo-

nucleotide packing density on the response calibration

curves of the ®ber optic sensors with respect to the con-

centration of complementary oligonucleotide concentration.

Fig. 3 shows calibration curves generated using activated

sensors with low and high oligonucleotide packing density,

obtained using solution temperatures of 35 and 448C, in PBS

buffer containing 1 M NaCl to maximize sensitivity. After

the addition of complementary oligonucleotides and incu-

bation for 15 min, the sensors were rinsed with 1 ml PBS

buffer at the stated temperature, in order to remove non-

selectively adsorbed oligonucleotides. It is apparent from

the results shown in Fig. 3 that at each temperature exam-

ined, the sensor with high oligonucleotide immobilization

density exhibited a signi®cantly greater response to the

presence of complementary oligonucleotides than did the

sensor with low immobilization density. Interestingly, it was

also observed that the dynamic range had an upper limit of

about 1� 10ÿ8 M cDNA for the sensor with high oligonu-

cleotide packing density, whereas deviations from linearity

did not begin until cDNA concentrations reached about

3� 10ÿ8 M for the sensor with low packing density. This

result can be explained in terms of the variation in thermal

stability of hybrids formed at surfaces with different oligo-

nucleotide packing densities. In a previous paper [15], we

reported the thermal denaturation temperatures of dA20-50-¯uorescein hybridized to immobilized dT20 with different

packing densities. We found that the Tm values for hybrids

formed at interfaces with high oligonucleotide packing

density were consistently lower (�68C) than those observed

using sensors with low and medium packing densities. It was

postulated that at higher packing densities, interactions

between neighbouring oligonucleotides become signi®cant,

and these interactions reduce thermodynamic stability of

hybrids formed in the interfacial environment. Conse-

quently, at comparable temperatures, the hybrids formed

at the surface with high oligonucleotide packing density are

less stable than those formed in an environment with lower

packing density where nearest-neighbour interactions are

not as likely to occur. The larger absolute number of

immobilized oligonucleotides may account for greater sen-

sitivity for sensors coated with higher oligomer packing

densities. However, the onset of deviations from linearity

occurs at a lower cDNA concentration for surfaces with high

immobilization density in comparison to those with low

packing density, suggesting that the optimal oligonucleotide

immobilization density will require a compromise between

sensitivity and dynamic range. High oligonucleotide pack-

ing density is better suited for low cDNA concentrations,

while higher cDNA concentrations may be more easily

quanti®ed with a sensor coated with a low oligonucleotide

packing density.

3.5. Biosensor selectivity

A comparison of the selectivity of hybridization was done

by exposing an optical ®ber coated with a high density of

dT20 to solutions containing ¯uorescein labelled ssDNA

with varying degrees of complementarity. In each experi-

ment, the ®ber was exposed sequentially to cDNA solutions

containing dA20-50-¯uorescein, d(A19G)-50-¯uorescein,

d(A9GA10)-50-¯uorescein, d(A9G2A9)-50-¯uorescein and a

mixture of equimolar amounts of all of these oligonucleo-

tides. All samples were prepared in PBS buffer containing

1 M NaCl to maximize sensitivity. The sensor was incubated

in the cDNA sample for 10 min, rinsed with 1 ml PBS buffer

and the resulting ¯uorescence was then measured. The

sensor was subsequently cleaned by washing sequentially

with 15 ml sterile water, 1 ml 90% formamide in TE buffer

and again with 15 ml sterile water, all at 808C. Two different

cDNA concentrations were chosen, and they represented

two different regions of the calibration curves in Fig. 3. The

2:5� 10ÿ9 M cDNA concentration was suf®ciently low that

Fig. 3. Response of sensors with low and high dT20 immobilization

densities to solutions of various concentrations of dA20-50-fluorescein in

1� PBS buffer at 28 and 408C. Wash steps were performed after each

addition of cDNA.

J.H. Watterson et al. / Sensors and Actuators B 74 (2001) 27±36 33

it was expected that incremental additions of cDNA and

therefore the mixed cDNA (all four types of cDNA in

equimolar quantities) sample would produce a linear cali-

bration response with respect to cDNA concentration. At the

higher cDNA concentration, it was expected that the mixed

analyte sample would not provide a signal that would fall on

the linear portion of the calibration curves. Hybridization

experiments were done using individual component con-

centrations of 2:5� 10ÿ9 and 2:5� 10ÿ8 M. The mixed

component (all four types of cDNA in equimolar quantities)

hybridization experiments were done using total oligonu-

cleotide concentrations of 1:0� 10ÿ8 and 1:0� 10ÿ7 M.

Experiments using cDNA samples of 2:5� 10ÿ9 M con-

centration regime were done at 28 and 448C. The lower

temperature was chosen such that all types of cDNA were

expected to be totally hybridized with immobilized dT20.

The higher temperature was chosen since the Tm of immo-

bilized d(A19G)-50-¯uorescein:dT20 was found to be 448C,

and this species was expected to have a Tm closest to that of

the fully complementary sequence (Tm � 48�C). Thus,

experiments done at the higher temperature were expected

to show enhanced selectivity relative to those done at the

lower temperature.

Fig. 4 shows the results of the experiments that exposed a

sensor to individual component concentrations of

2:5� 10ÿ9 M, at temperatures of 28 and 448C. The increase

in ¯uorescence intensity observed for each sample (relative

to background, after addition of complementary material

and subsequent washing) was normalized relative to that of

the fully complementary oligonucleotide to correct for small

variations in optical alignment of sensors between sets of

experiments. The results show that at the lower temperature

of 288C, there is little difference in the relative ¯uorescence

yields. This was expected, since the temperature was suf®-

ciently low that almost complete hybridization was expected

with all oligonucleotides used. When the experimental

temperature was increased to 448C, enhancement of the

selectivity of hybridization was observed. Interestingly, the

signal from the partially complementary sequences was not

reduced to <54% of that of the fully complementary

sequence. This is larger than may be expected when con-

sidering differences in Tm for sequences of varying com-

plementarity. This unexpected result likely has two causes.

Firstly, it must be noted that the thermodynamics of hybri-

dization in an interfacial environment are signi®cantly

different than that of bulk solution. In previous papers

[14,15], we have noted that the thermal denaturation transi-

tions of immobilized nucleic acid hybrids are signi®cantly

broader than those of hybrids in bulk solution. The inter-

facial thermal denaturation transitions we have reported

occurred over temperature ranges of 15±208C, while those

observed in bulk solution occurred over temperature ranges

of about 108C. Consequently, there is an increased degree of

overlap of the thermal denaturation pro®les of immobilized

hybrids of varying complementarity relative to analogous

systems in bulk solution. Secondly, when hybridization

occurs at an interface, it does so in competition with the

process of non-selective adsorption. It has been shown with

the systems described here that non-selective adsorption

does occur and cannot be completely eliminated indepen-

dently of hybridization. Thus, non-selective adsorption may

also contribute to the increased signal observed with par-

tially complementary sequences even at the higher tempera-

tures. It must be remembered that the ¯uorescence signal in

these experiments is indicative of the presence of cDNA, but

is not a direct indicator of hybridization.

Examination of Fig. 4 also shows that the response of the

sensor to the mixed analyte sample of 10ÿ8 M total oligo-

nucleotide concentration does not provide a response

equivalent to the sum of the responses to the individual

components. This may seem contradictory to what may be

expected when considering the calibration curves alone, but

Fig. 4. Response of sensor with high dT20 immobilization density to individual solutions of dA20-50-fluorescein, d(A19G)-50-fluorescein, d(A9GA10)-50-fluorescein and d(A9G2A9)-50-fluorescein in 1� PBS buffer (�ssDNA�i � 2:5� 10ÿ9 M), and to a equimolar mixture of these oligonucleotides in 1� PBS

buffer (�ssDNA�Total � 1� 10ÿ8 M), at 28 and 408C. Wash steps were performed after each addition of cDNA.

34 J.H. Watterson et al. / Sensors and Actuators B 74 (2001) 27±36

may be understood when the thermal denaturation pro®les

are considered. In a previous paper [15], we reported that

®bers with high oligonucleotide immobilization density

(where nearest neighbour interactions between immobilized

oligonucleotides become signi®cant) have reduced Tm

values relative to analogous systems with lower oligonu-

cleotide immobilization densities, where the probability of

nearest neighbour interactions is lower. At higher packing

densities, the formation of hybrids at the surface increases

the charge density at the interface and therefore affects the

nearest neighbour interactions, as observed in the calibration

curves presented in Fig. 3. Non-selective adsorption also

affects the surface charge distribution, and cannot be entirely

eliminated independently of hybridization. These effects

may combine to cause further reductions in the stability

of the partially complementary hybrids at the temperatures

used herein, resulting in smaller contributions to the net

signal than would be expected on the basis of the calibration

curves of Fig. 3.

Experiments were done using individual component

concentrations of 2:5� 10ÿ8 M, using the temperature of

448C to enhance selectivity. These concentrations corre-

spond to a region on the calibration curves where deviations

from linearity were observed. Consequently, there was

little or no signi®cant change in ¯uorescence yield as

the complementarity of the oligonucleotides in solution

was adjusted. It was observed that the mixed analyte sample

showed only a very small increase in ¯uorescence yield

relative to the individual component samples, also as

expected. These results are consistent with the correspond-

ing concentration response range of the calibration curves of

Fig. 3, and serve as further con®rmation of the limited

dynamic range of the sensors with high oligonucleotide

packing density.

3.6. Conclusions and analytical consequences

The results that are presented here suggest that the control

of the density of nucleic acid ®lms may impart some control

of sensitivity and selectivity for a given hybridization assay

when optimization is done concurrently by adjustment of

other experimental conditions such as temperature and

solution ionic strength. Careful attention must be given to

activation of the ®lm for hybridization. Once activated, it

was found that higher immobilization densities provided

increased sensitivity and selectivity. Enhancements in sen-

sitivity and selectivity afforded by increasing the oligonu-

cleotide immobilization density may be realized only in

lower analyte concentration regimes, since higher oligonu-

cleotide immobilization density resulted in a smaller

dynamic range for such sensors. Consequently, it is of

fundamental importance to characterize the calibration

and thermal denaturation behavior of any nucleic acid sensor

before use with a given sample. It has been shown that

selectivity cannot be predicted from thermal denaturation

experiments done in bulk solution, nor can it be predicted

on the basis of Tm considerations alone, even if the Tm

values that are associated with speci®c sensors are mea-

sured. Non-selective adsorption occurs concurrently with

hybridization, and the dynamic response of the sensors due

to incomplete activation, surface occlusion and due to the

changing surface environment must be accounted for in each

experiment.

Acknowledgements

We are grateful to the Natural Sciences and Engineering

Research Council of Canada, and to FONA Technologies,

Inc., for ®nancial support of this research.

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Biographies

Ulrich J. Krull received his PhD from the University of Toronto in 1983.

He is appointed as Professor of Analytical Chemistry at the University of

Toronto, where he holds the position of Astra Zeneca Chair in

Biotechnology. His current research interests are in the development of

nucleic acid biosensors based on optical fiber technology and also bilayer

lipid membrane electrochemistry.

J.H. Watterson et al. / Sensors and Actuators B 74 (2001) 27±36 35

Paul A.E. Piunno received his PhD in Analytical Chemistry from the

University of Toronto in 1999. He currently holds the position of Director of

Research at FONA Technologies, Inc., in Mississauga, Canada. His research

interests are in the development of fiber optic nucleic acid biosensors.

Christopher C. Wust received his BSc in Chemistry from the University of

Toronto in 1995. He is currently serving as a Product Development

Scientist at FONA Technologies, Inc., in Mississauga, Canada. His

research interests are in the development of fiber optic nucleic acid

biosensors.

James H. Watterson received his MSc in Analytical Chemistry from the

University of Toronto in 1999, where he is currently working toward the

completion of his PhD. His research interests are focussed on investigation

of thermodynamic and kinetic aspects of nucleic acid hybridization at

interfaces for the development of fiber optic nucleic acid biosensors.

36 J.H. Watterson et al. / Sensors and Actuators B 74 (2001) 27±36