A MIXED BIOSENSING FILM COMPOSED OF OLIGONUCLEOTIDES … · students: Anthony Tavares, Rhys Crasto, Miki Stanikic, and Lori Chong. I must also thank the administrative staff, Carmen
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A MIXED BIOSENSING FILM COMPOSED OF OLIGONUCLEOTIDES AND POLY(2-HYDROXYETHYL
METHACRYLATE) BRUSHES TO ENHANCE SELECTIVITY FOR DETECTION OF SINGLE
NUCLEOTIDE POLYMORPHISMS
by
April Ka Yee Wong
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Graduate Department of Chemistry University of Toronto
Graduate Department of Chemistry University of Toronto
2010
Abstract
This work has explored the capability of a mixed film composed of oligonucleotides and
oligomers to improve the selectivity for the detection of fully complementary oligonucleotide
targets in comparison to partially complementary targets which have one and three base-pair
mismatched sites. The intention was to introduce a “matrix isolation” effect on oligonucleotide
probe molecules by surrounding the probes with oligomers, thereby reducing oligonucleotide-to-
oligonucleotide and/or oligonucleotide-to-surface interactions. This resulted in a more
homogeneous environment for probes, thereby minimizing the dispersity of energetics associated
with formation of double-stranded hybrids. The mixed film was constructed by immobilizing
pre-synthesized oligonucleotides onto a mixed aminosilane layer and then growing the oligomer
portion by surface-initiated atom transfer radical polymerization (ATRP) of 2-hydroxy
methacrylate (PHEMA). The performance of the mixed film was compared to films composed
iii
of only oligonucleotides in a series of hybridization and melt curve experiments. Surface
characterization techniques were used to confirm the growth of the oligomer portion as well as
the presence of both oligonucleotides and oligomer components. Polyatomic bismuth cluster
ions as sources for time-of-flight secondary ion mass spectrometry experiments could detect both
components of the mixed film at a high sensitivity even though the oligomer portion was at least
200-fold in excess.
At the various ionic strengths investigated, the mixed films were found to increase the
selectivity for fully complementary targets over mismatched targets by increasing the sharpness
of melt curves and melting temperature differences (ΔTm) by 2- to 3-fold, and by reducing non-
specific adsorption. This resulted in improved resolution between the melt curves of fully and
partially complementary targets. A fluorescence lifetime investigation of the Cy3 emission
demonstrated that Cy3-labeled oligonucleotide probes experienced a more rigid
microenvironment in the mixed films.
These experiments demonstrated that a mixed film composed of oligonucleotides and
PHEMA can be prepared on silica-based substrates, and that they can improve the selectivity for
SNP discrimination compared to conventional oligonucleotide films.
iv
Acknowledgments I would like to thank Prof. Ulrich J. Krull for being such a positive influence in my life.
He has guided me with enthusiasm and support from the beginning and especially in the end. I
am extremely grateful for all that he has done for me. I wish to also thank Prof. Aaron Wheeler
and Prof. Scott Prosser for being in my advisory committee and giving helpful comments over
the years.
I must give special thanks to Prof. Chris Yip and Dr. Patrick Yang (IBBME, University
of Toronto) for giving me AFM instrument use time. I would also like to express my gratitude to
Dr. Himadri Mandal and Prof. Shirley Teng (University of Waterloo) for collecting the AFM
images. I would also like to thank Dr. Rana Sodhi and Peter Brodersen (Surface Interface
Ontario, University of Toronto) for collecting XPS and TOF-SIMS data and for their expertise.
And finally, I would like to offer many thanks to Dr. Neil Coombs and Ilya Gouverich (Centre
for Nanostructure Imaging, University of Toronto) for giving me SEM training. I would like to
thank Dr. Peter Mitrakos (University of Toronto Mississauga) for assisting me with the operation
of various instruments. I must offer special thanks to Prof. Claudiu Gradinaru and Dr. Denys
Marushchak (University of Toronto Mississauga), for collecting the lifetime and anisotropy data,
which became important pieces of my thesis.
To my colleagues, most notably Taufik Al-Sarraj, Sameer Al-Abdul Wahid, Russ Algar,
Andrew Chan, I-San Chan, Lu Chen, Yevgenia Kratvsova, Melissa Massey, and Kris Wang,
thank you for your friendship and for many years filled with, not only discussions about research,
but laughter, lunches, trips, dinners, and movies. I would like to give special thanks to Russ
Algar for writing the Eslide program, which was a tremendous help for processing the data. I
would like to thank Dr. Neil McKinnon for contributing his expertise on the NMR spectrum of
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polymers. I am grateful for the extra help and the great ideas provided by former summer
students: Anthony Tavares, Rhys Crasto, Miki Stanikic, and Lori Chong. I must also thank the
administrative staff, Carmen Bryson and Anna Liza Villavelez, for being such great help and for
taking care of most matters of Departmental life.
I would like to thank my parents and my sister for their continuous support over the
years. I am also grateful to my new Adorjan family who has embraced me from the start. Last
but not least, I wish to thank my husband, Mike, for being always there when I needed him. I
have thought that Mike was crazy when he said that he is looking forward to his own Ph.D.
defence. His enthusiasm has eventually brought me more confidence to overcome the last hurdle
of this long journey.
vi
Table of Contents Abstract ........................................................................................................................................... ii
Acknowledgments.......................................................................................................................... iv
Table of Contents........................................................................................................................... vi
Symbols and Abbreviations ............................................................................................................ x
List of Tables ................................................................................................................................ xv
List of Figures ............................................................................................................................. xvii
List of Equations and Chemical Reactions ................................................................................ xxiii
List of Appendices ...................................................................................................................... xxv
2.3.9 ATRP of HEMA on Bromoisobutyryl-Immobilized Silicon Wafers ................... 75
viii
2.3.10 Hybridization of Target Oligonucleotides with Probes on Aminosilane Surface or in Mixture with PHEMA on Glass Substrates.................................................. 77
2.3.11 Measurement of Immobilization Efficiency and Hybridization Yield (Chapter 3.2) ........................................................................................................................ 78
2.3.12 Acquisition of Melt Curves from Oligonucleotide Probe Films with and without PHEMA on Glass Surfaces...................................................................... 79
2.3.13 Conjugation of Thiolated-Oligonucleotides to Au Nanoparticles (Chapter 3.3) .. 80
2.3.14 Dissolution of Gold Nanoparticles by KCN and Construction of a Calibration Curve for Calculation of Surface Density of Oligonucleotides on Gold Nanoparticles ........................................................................................................ 81
2.3.15 Hybridization or Adsorption of the Au nps-DNA Conjugates to Fused Silica Surfaces Modified with Oligonucleotide Films or Mixed Films for SEM Analysis (Chapter 3.3) .......................................................................................... 82
3 Results and Discussion............................................................................................................. 83
3.1 Syntheses of Benzaldehyde-Protected Aminosilane, Initiator, and PHEMA for Assembly of Mixed Film .................................................................................................. 83
3.1.2 Synthesis of Benzaldehyde-Protected APTMS (N-[(1Z)-phenylmethylene]-3-(trimethoxysilyl)propan-1-amine)......................................................................... 84
3.1.3 Synthesis of the Bromoisobutyryl NHS Ester Initiator (1-[(2-bromo-2-methylpropanoyl)oxy]pyrrolidine-2,5-dione)....................................................... 86
3.1.4 NMR Characterization of PHEMA....................................................................... 89
3.2 Surfaces for Tuning of Oligonucleotide Biosensing Selectivity Based on Surface-Initiated Atom Transfer Radical Polymerization on Glass and Silicon Substrates .......... 92
3.3.3 XPS Characterization of Mixed Film on Glass Surfaces.................................... 124
3.3.4 Estimation of Density of Surface Immobilized Oligonucleotide Probes by Using Au Nanoparticle-Tagged Complementary Oligonucleotide Targets........ 127
3.3.5 ToF-SIMS Characterization of Mixed Films on Glass Surfaces ........................ 133
3.4 A Mixed film Composed of Oligonucleotides and Poly(2-Hydroxyethyl Methacrylate) Brushes to Enhance Selectivity for Detection of Single Nucleotide Polymorphisms ............................................................................................................... 148
3.4.3 Selectivity of Various Targets on the Mixed films............................................. 153
3.4.4 Fluorescence Lifetime to Identify Different Microenvironments in Oligonucleotide Films vs. Mixed Films.............................................................. 155
3.4.5 Comparison of Selectivity for SNP Detection between Oligonucleotide and Mixed films......................................................................................................... 160
Fig. 2.12: Confocal images (Cy3 channel) of hybridized targets (Sequences 5–7) on a mixed film glass surface and
their fluorescence intensities as temperature was increased. From left to right: 23°C, 49°C, 55°C, 63°C.
2.3.13 Conjugation of Thiolated-Oligonucleotides to Au Nanoparticles (Chapter 3.3)
133.6 µL of 688 nM of the Cy3-labeled thiolated target (Sequence 3, Table 2.1), which
was dissolved in water, was mixed with 450 µL of stock Au nanoparticles (68.1 nM, 5 nm in
diameter) in a microcentrifuge tube. The solution ratio of DNA to Au nps is 3:1. 10 µL of 20
mM of TCEP was added. The mixture was incubated overnight at room temperature. The next
day, 500 µL of 0.1× PBS was added to the mixture and the sample was vortexed briefly. The
mixture was incubated at room temperature for two more days.
The Au-DNA conjugates were centrifuged at 13,000 rpm for 25 min per cycle. A 200-µL
aliquot of the Au-DNA conjugate was centrifuged with 600 µL of 95% ethanol for each cycle. A
faint red oily film was deposited on the side of the microcentrifuge tube and the supernatant was
removed with a pipette. The subsequent aliquots of the sample and ethanol were added to the
same microcentrifuge tube containing the red oily film and the centrifuge process was repeated.
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The entire purification procedure was repeated about four times in total. The deeper red oily
film from the final centrifugation step was dispersed in 0.3× PBS.
2.3.14 Dissolution of Gold Nanoparticles by KCN and Construction of a Calibration Curve for Calculation of Surface Density of Oligonucleotides on Gold Nanoparticles
A typical etching experiment involved dilution of 20 μL of the stock Au nps-DNA
conjugate solution in 60 μL of 0.1× PBS. The fluorescence spectrum of each diluted conjugate
sample (30 μL aliquot) was measured at an excitation wavelength of 520 nm and the emission
was collected from 540–650 nm. 5.00 μL of 40 mM KCN was then added and the fluorescence
spectrum was immediately scanned. The peak fluorescence intensity at 562 nm was used to
calculate the number of immobilized oligonucleotides.
To construct the calibration curve for the determination of oligonucleotides adsorbed
onto Au nps, thiolated oligonucleotides of various concentrations (62.5 nM, 125.0 nM, 250.0
nM, and 500.0 nM) of the SMN sequence labelled with Cy3 were mixed with 30.00 μL of stock
Au nps (68.1 nM), 5.00 μL of 40 mM KCN, and 0.1× PBS to make up a total volume of 105 μL.
The blank solution contained all reagents except for the oligonucleotides. The fluorescence
intensity of each DNA concentration was measured at an excitation wavelength of 520 nm and
the emission was collected from 540–650 nm. The peak fluorescence intensity at 562 nm was
plotted against the oligonucleotide concentration.
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2.3.15 Hybridization or Adsorption of the Au nps-DNA Conjugates to Fused Silica Surfaces Modified with Oligonucleotide Films or Mixed Films for SEM Analysis (Chapter 3.3)
An aliquot of 20 μL of the Au nps-DNA conjugate solution was diluted in 60 μL of 0.1×
PBS. The diluted solution was first heated at 60–70°C for 5 min and was smeared across
surfaces with a cover slip on PHEMA-covered surfaces with and without oligonucleotide probes.
The surfaces were then rinsed with water and dried with a stream of nitrogen.
83
Chapter 3
3 Results and Discussion
3.1 Syntheses of Benzaldehyde-Protected Aminosilane, Initiator, and PHEMA for Assembly of Mixed Film
3.1.1 Introduction
Covalent linkages were chosen for immobilization of the mixed film. The silane
coupling agent APTMS was first immobilized onto silica-based substrates by way of
alkoxysilane condensation [89, 90]. The free amine can then be used for immobilization of
thiolated oligonucleotides onto silica-based substrates via the heterobifunctional linker, sulfo-
SMCC. However, instead of using APTMS alone, half of the volume of APTMS used for the
silane immobilization reaction was first reacted with benzaldehyde (BZ) to protect the primary
amine group. This protection step reduces the number of crosslinkages between neighbouring
APTMS molecules [99], allows spacing and control of orientation of APTMS with respect to the
surface [101, 108], and temporarily saves amine sites for immobilizing the second component,
i.e. the PHEMA oligomer.
To grow PHEMA, surface-initiated ATRP was used, which required the immobilization
of an initiator. The chosen initiator compound is bromoisobutyryl bromide. This compound and
its derivatives are in common use for initiation of ATRP [123, 145, 146]. To immobilize the
initiator, it was first activated by coupling it to N-hydroxysuccinimide (NHS). Activation with
NHS enhances the rate at which the primary amine can react with the carbonyl group in
bromoisobutyryl because NHS is a good leaving group [147], making bromoisobutyrate a more
active electrophile [123]. Once the remaining amine sites were deprotected by hydrolysis, they
were free to react with the activated bromoisobutyrate initiator. Hence, immobilization of the
84
initiator to aminosilanized surfaces can be achieved. BZ-APTMS and the bromoisobutyryl NHS
ester were characterized by 1H and 13C NMR.
3.1.2 Synthesis of Benzaldehyde-Protected APTMS (N-[(1Z)-phenylmethylene]-3-(trimethoxysilyl)propan-1-amine)
Fig. 3.1 shows the structure of BZ-APTMS and its 1H NMR spectrum. The peak at ~ 0.7
ppm is consistent with the methylene protons adjacent to silicon (Protons a). The peak at 1.8
ppm was that of the methylene protons that are one carbon away from silicon (Protons b). At 3.5
ppm, protons from the methoxy groups (Protons c) were in a matrix that was highly crosslinked,
hence a broader singlet was observed. Protons d were not observed and they may be buried in
the broad methoxy proton peak since they were reported to be located at 3.6 ppm [101]. From 7–
8 ppm, the two peaks represented the two different groups of benzylic protons (Protons e and f).
The allylic proton (Proton g) at the carbon adjacent to the nitrogen was at 8.2 ppm, which again
agreed with Hicks and Jones [101]. The peak at 2.4 ppm was due to toluene, which was used as
the solvent in the reaction [148]. All the peaks found in the appropriate regions of the spectrum
agreed with the chemical shifts listed by Hicks and Jones [101], and the peak integration
provided results that were consistent with the structure of BZ-APTMS.
85
δ, ppm
Fig. 3.1: 1H NMR spectrum of BZ-APTMS in CDCl3. The inset contains the structure of BZ-APTMS. Different
types of protons are labeled.
86
3.1.3 Synthesis of the Bromoisobutyryl NHS Ester Initiator (1-[(2-bromo-2-methylpropanoyl)oxy]pyrrolidine-2,5-dione)
Fig. 3.2 shows the structure of the activated bromoisobutyryl NHS ester and its 1H NMR
spectrum. The spectrum contained two singlets, one for the methyl groups in the
bromoisobutyryl portion and one for the methylene groups in the NHS portion. The singlet
representing the methyl groups was at 2.1 ppm. Since the methyl groups were on the same
carbon and experienced a similar environment, they only appeared as a single peak. The
methylene protons should be more deshielded than the methyl protons due to the neighbouring
electron withdrawing carbonyl groups; hence the peak was found at a slightly more downfield
chemical shift, which was at 2.9 ppm. These NMR data agreed with those presented by Lou and
He [123].
The integration ratio between the singlet at 2.1 ppm and the one at 2.9 ppm accounted for
the number of protons in each group. The ratio was 1.7:1.0 (peak at 2.1 ppm: peak at 2.8 ppm
[123], which scaled to a ratio of 6:3.5. This was consistent with expectations because the peak at
2.1 ppm should have 6 protons (from two methyl groups) and the singlet at 2.9 ppm should have
4 protons (two methylene groups). The peak at 7.3 was the residual solvent peak from
chloroform [148]. Thus, the 1H NMR spectrum indicated that the bromoisobutyryl bromide was
successfully activated into its NHS form.
The 13C NMR spectrum of the initiator was also obtained and it matched well with the
results presented by Lou and He (Fig. 3.3) [123]. The assignment of the different carbons is also
shown in the 13C NMR spectrum. The corresponding chemical shifts are listed in Table 3.1. The
expected peaks should be located at 25.9, 30.9, 51.4, 166.2, and 168.9 ppm [123], which were in
agreement with the data in the table. The peak at 77 ppm is that of DMSO [148]. The NMR
data indicate that a pure form of the activated initiator was synthesized.
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δ (ppm)
Fig. 3.2: 1H NMR spectrum of the activated bromoisobutyryl NHS ester initiator in CDCl3. The inset shows the
structure of the bromoisobutyryl NHS ester. The spectroscopically different protons are labeled.
88
δ, ppm
(a)
δ, ppm
(b)
Fig. 3.3: (a) 13C NMR (500 MHz) spectrum of the activated bromoisobutyryl NHS ester initiator in CDCl3. The
inset shows the structure of the bromoisobutyryl NHS ester. The spectroscopically different carbon atoms are
labeled; (b) high resolution data showing detail of the downfield region.
89
Table 3.1: Carbon peak positions of the bromoisobutyryl NHS ester.
Carbons δ (ppm)
a 25.5
b 30.4
c 50.8
d 167.4
e 168.5
3.1.4 NMR Characterization of PHEMA
To demonstrate that the polymerization procedure was functional, a solution-based ATRP
reaction was done. Figure 3.4 shows the 1H NMR spectrum of the resulting product in DMSO-d6
and the anticipated structure of the product. Table 3.2 lists each group of protons and their
chemical shifts. The chemical shifts and integrations were in accord with published data [149-
151]. The methyl peak (a) contained three protons and the hydroxyl (f) contained one. The
remainder of the peaks represented methylene groups and all were integrated to two protons.
The methyl protons (a) gave a doublet due to the tacticity of the polymer, as was also observed
for the methyl group in the carbon NMR spectra reported by Roman et al. and Verhoeven et al.
[152, 153]. The methylene protons (b) were located on the backbone of a polymer and were
expected to give a broad peak at 1.8 ppm. These NMR data showed that the ATRP reaction was
effective in solution, and provided the confidence that the procedure could be applied to surface-
initiated polymerization.
90
δ, ppm
Fig. 3.4: 1H NMR of PHEMA in DMSO-d6. Structure of PHEMA. The inset shows different types of protons,
which are labeled.
91
Table 3.2: Proton peak positions of PHEMA.
Protons δ (ppm)
a 0.8, 1.0
b 1.8
c 3.6
d 3.9
e 4.8
Acetone/Acetonitrile 2.1[148]
DMSO 2.5[148]
Water 3.4[148]
3.1.5 Conclusions
1H and 13C NMR demonstrated that BZ-APTMS, the bromoisobutyryl NHS ester
initiator, and PHEMA were successfully synthesized. BZ-APTMS would be mixed with
APTMS and used to immobilize the oligomer portion of the mixed film. The bromoisobutyryl
NHS ester would be immobilized to the remaining primary amine sites and used to initiate the
ATRP reaction. The successful synthetic scheme to produce PHEMA in solution was then
adapted for application for deposition onto surfaces.
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3.2 Surfaces for Tuning of Oligonucleotide Biosensing Selectivity Based on Surface-Initiated Atom Transfer Radical Polymerization on Glass and Silicon Substrates
April K. Y. Wong and Ulrich J. Krull
Chemical Sensors Group, University of Toronto Mississauga, 3359 Mississauga Rd. N.,
Mississauga, ON, Canada L5L 1C6
Contributions: April Wong performed all measurements (except for XPS, ToF-SIMS, and AFM)
and data analysis and interpretation under the guidance of Ulrich Krull.
Previously published in Analytica Chimica Acta, 639 (2009) 1-12. Supplementary melt curve
data were added in this chapter for completeness.
93
3.2.1 Abstract
Covalently immobilized mixed films of oligonucleotide and oligomer components on
glass and silicon surfaces are reported. This work has investigated how such films can improve
selectivity for the detection of multiple base-pair mismatches. The intention was to introduce a
“matrix isolation” effect on oligonucleotide probe molecules by surrounding the probes with
a One trial represents one glass slide with all the usable spots (out of 8 spots) that exhibited a sigmoidal function with an R2 > 0.95 on the same slide. At least 3 replicates were used, unless otherwise noted.
b SMN probes were immobilized on a 1:1 ratio of APTMS and BZ-APTMS as depicted in Figure 2.5
c Mixed film composed of oligonucleotides and PHEMA immobilized on APTMS/BZ-APTMS-modified surfaces as depicted in Figure 2.10.
d The solution melt curve was collected in 0.5× PBS.
e The confidence interval (C.I.) at 95% was constructed for the difference in Tm values between fully complementary and 3 bpm targets.
± Standard deviations were reported. Avg: Average values. The averages of all experiments were reported with their standard errors.
118
An examination of the slopes of the denaturation profiles of the fully complementary
SMN targets on mixed film surfaces compared to films composed only of oligonucleotides
confirmed the sharper melting transition observed on surfaces that had undergone ATRP (Table
3.6). A smaller dx value corresponds to a sharper slope because dx represents the change in x
(temperature) for the largest change in y (fraction of ssDNA). The 3-fold increase in steepness
for the melt curves collected from mixed films that were hybridized with fully complementary
targets is very clear; it may suggest that the local environment of oligonucleotide probes is more
controlled and homogeneous for the mixed film surfaces. Another example of the increase in
steepness of slope, possibly due to PHEMA, is further reflected in comparisons of slope values
for the 3 bpm targets as seen in Table 3.6. There was a 1.5-fold increase in sharpness observed
for the 3 bpm targets when the melt curves collected from mixed films were compared to the
oligonucleotide films. The dx values obtained from the mixed films were equal to or smaller, i.e.
sharper melting transitions, than those obtained from bulk solution. The oligonucleotide films
had much greater variability in the dx values than the mixed films, particularly for melt curves of
fully complementary targets.
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Table 3.6: dx values of fully complementary and 3 bpm SMN targets on mixed films and oligonucleotide films
immobilized on glass slides.
Triala dx valuee of fully complementary targets
(ºC)
dx value of 3 bpm targets (ºC)
Oligonucleotideb film only
1 5.8 ± 0.6 3.4 ± 0.8
2 7.5 ± 1.1 3.0 ± 1.3
Avg 6.6 ± 0.8 3.2 ± 0.2
Mixed filmc 1 2.3 ± 0.4 1.2 ± 0.5
2 2.5 ± 0.8 1.5 ± 0.1
3 2.8 ± 0.7 2.2 ± 0.6
4 2.1 ± 0.4 3.1 ± 0.6
5 4.2 ± 1.6 2.5 ± 0.4
Avg 2.8 ± 0.4 2.1 ± 0.3
Bulk solutiond 1 2.9 ± 0.1 3.0 ± 0.1
a One trial represents one glass slide with all the usable spots (out of 8 spots) that exhibited a sigmoidal function with an R2 > 0.95 on the same slide. At least 3 replicates were used, unless otherwise noted.
b SMN probes were immobilized on a 1:1 ratio of APTMS and BZ-APTMS as depicted in Fig. 2.5.
c Mixed film composed of oligonucleotides and PHEMA immobilized on APTMS/BZ-APTMS-modified surfaces as depicted in Fig. 2.10.
d The solution melt curve was collected in 0.5× PBS.
e dx value (obtained from sigmoidal curve fitting) represents the change in temperature for the greatest change in the fraction of ssDNA
± Standard deviations were reported. The averages of all experiments were reported with their standard errors.
Avg: Average values
Overall, the melting temperature and slope data both suggest that the melt curves reflect
similar general energetics for hybridization, but a narrower distribution of energetics for the case
of the mixed film coating. This may be due to the effectiveness of PHEMA coating in
120
controlling the local environment of oligonucleotide probes. These data are certainly very
encouraging, suggesting greater selectivity than observed for films that are composed of only
immobilized probes. It appears that the PHEMA matrix exerts a destabilizing force towards
targets that contain mismatches.
3.2.7 Conclusions
The use of a series of surface analysis methods revealed the success of each
immobilization step including silane and initiator immobilization and surface-initiated ATRP.
The use of BZ-APTMS was superior in the control of the reproducibility of the polymerization
rate. It provided a smoother surface for polymerization as well as served as a temporary
protecting group for immobilized amines for subsequent reaction steps. HEMA was shown to be
the most useful material for development of biosensing surfaces. It dramatically reduced non-
specific adsorption, allowed reusability, sharpened the melt curves for both fully complementary
and 3 bpm targets, and increased resolution of such targets. Further experiments will be done to
explore effects of solution ionic strength, mismatch location and tuning for SNP analysis. The
results of this work support the hypothesis that selectivity of hybridization can be improved by
narrowing the distribution of energetics experienced by immobilized DNA hybrids.
121
3.3 Bin+ Cluster Ion Sources for Investigation of a Covalently-Immobilized Mixed Film Composed of Oligonucleotides and Poly(2-hydroxyethyl methacrylate) Brushes
April K.Y.Wonga, R.N.S. Sodhib, and Ulrich J. Krulla*
aChemical Sensors Group, University of Toronto Mississauga, 3359 Mississauga Rd. N.,
Mississauga, ON, Canada L5L 1C6
bSurface Interface Ontario, Department of Chemical Engineering and Applied Chemistry,
University of Toronto, On, Canada M5S 3E5
Contributions: April Wong performed all measurements and data analysis and interpretation
under the direction of Ulrich Krull. Dr. Rana Sodhi and Peter Brodersen of Surface Interface
Ontario collected the XPS and ToF-SIMS data.
Submitted part of this chapter to Surface and Interface Analysis, special conference proceedings
issue for the SIMS XVII conference (September 2009).
122
3.3.1 Abstract
The covalent co-immobilization of short non-nucleic acid oligomers of poly(2-
hydroxyethyl methacrylate) (PHEMA), which are grown by surface-initiated atom transfer
radical polymerization, with oligonucleotides on glass surfaces has been demonstrated to
significantly improve selectivity of nucleic acid hybridization for applications involving
microarrays and biosensors [178]. Physical characterization is required to examine how the two
components localize within the same area on a glass surface. Glass slides coated with
immobilized spots of oligonucleotide probes of 20mer length at a density of ~3.7 × 1011
molecules cm-2, and PHEMA of nominal thickness of less than 10 nm, were analyzed using static
time-of-flight secondary ion mass spectrometry (TOF-SIMS). The samples were subjected to a
polyatomic bismuth cluster ion source for the TOF-SIMS experiment. In the positive ion
spectra, fragments of PHEMA and nucleosides were observed. In the negative ion spectra,
PHEMA as well as signature fragments from nitrogenous bases, phosphate and phosphite groups
were identified. Comparison of negative ion spectra indicated that the Bi5+ source provided
details that were either not apparent or at much reduced sensitivity when using Bi3+ and Bi+
sources. Bi3+ provided higher secondary ion yields for positive ion fragments, and more than
200-fold selectivity for ionization of oligonucleotide in comparison to PHEMA when in mixture.
3.3.2 Introduction
A reduction in the ensemble of degrees of freedom of immobilized oligonucleotide
probes can be achieved by forming a mixed film composed of oligonucleotide probes and
poly(2-hydroxyethyl methacrylate) (PHEMA) brushes, which are co-immobilized and grown
from the surface to maximize the surface density [178]. Melt curve data based on fully
123
complementary and three base-pair mismatched targets have demonstrated that the presence of
PHEMA can enhance selectivity, as evident from the sharpness of the DNA melting transition
and increases of the difference between the Tm values [178].
X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry
(TOF-SIMS), atomic force microscopy (AFM) and ellipsometry have previously been used to
ensure that the surface-initiated polymerization reaction of PHEMA had proceeded on silicon
wafers [178]. However, structural details about how oligonucleotides and PHEMA co-exist on
surfaces are lacking. Such data would be useful to improve the design of immobilization, and
therefore to optimize selectivity. As one step towards this goal we have used TOF-SIMS to
investigate oligonucleotides that were co-immobilized with PHEMA brushes. An ion source that
produced monoatomic and polyatomic bismuth was investigated based on the ability of such a
system to generate increased secondary molecular and fragment ion yields [179, 180].
Furthermore, data from this TOF-SIMS experiment was contrasted with data from XPS. SEM
was used to estimate the density of immobilized oligonucleotide probes using oligonucleotide
targets which were labelled with Au nanoparticles (nps).
The XPS and TOF-SIMS experiments used surfaces containing APTMS (Surface A),
oligonucleotides only (Surface B), mixed oligonucleotides and PHEMA (Surface C), and
PHEMA only (Surface D). Oligonucleotides were deposited as 2 mm spots on predetermined
areas on Surfaces B and C. The success of the immobilization of oligonucleotides was verified
by detecting the fluorescence emission from Cy3-labeled oligonucleotides using a confocal
scanner. PHEMA was grown from the surface on Surfaces C and D. Surface-initiated
polymerization was confirmed using ellipsometry, which indicated an increase in the apparent
thickness on silicon wafer samples that were exposed to the ATRP reaction mixture. The total
124
change in thickness was between 3 to 6 nm (determined by ellipsometry). For characterization
by SEM, the Au np-oligonucleotide conjugates and PHEMA were immobilized across fused
silica surfaces.
3.3.3 XPS Characterization of Mixed Film on Glass Surfaces
The four sample surfaces were investigated by angularly-resolved XPS using 30° as the
take-off angle. It is known that the smaller the angle (<45°), the more sensitive the XPS
instrument is in terms of detection of electrons from species of the uppermost sample layer [181].
Figure 3.13 shows the high resolution C 1s spectra of all four surfaces. The C 1s signatures of
PHEMA are not present in Fig. 3.13a-b but are very evident in the spectra shown in Fig. 3.13c-d.
The peaks at 289.0 eV, 287 eV, and 286 eV are representative of the ester carbon, carbon-
oxygen, and carbon bonded to a carbonyl, respectively [182]. These peaks are only present on
the mixed and PHEMA-modified surfaces (Surfaces C and D), demonstrating that PHEMA was
grown from the surface via the ATRP reaction. These high resolution spectra agreed with
previously collected spectra of PHEMA films alone [178].
125
(a) b)
(c) (d)
Fig. 3.13: High resolution C 1s spectra of all surfaces (A-D) investigated; (a) Surface A: aminosilanes only; (b) Surface B:
Table 3.8 shows the selectivity of Bi3+ as a primary ion beam to ionize fragments
associated with oligonucleotides and PHEMA on different surfaces. Fragments unique to
oligonucleotides were observed on Surfaces B and C and those specific to PHEMA were
141
detected from Surfaces C and D at an intensity that was one to two orders of magnitude greater
than the respective control samples. The peak intensities of most secondary ion fragments
associated with oligonucleotides decreased when collected from Surface C (mixed film), as
opposed to Surfaces B (positive control). The relative intensities of the PHEMA fragments
stayed statistically similar between the mixed film and positive control surfaces (C and D). This
demonstrates that TOF-SIMS equipped with a bismuth cluster ion can selectively and
simultaneously detect ion fragments that are unique to two different components which co-exist
on the same surface.
Bi+ as a primary ion source was found to be inefficient in generating high secondary ion
yields (i.e. peak intensity) for fragments that are unique to oligonucleotides. Polyatomic bismuth
cluster ion sources (Bi3+ and Bi5
+) provided the highest secondary ion intensities for both
negative and positive ion species, which agrees with literature [179, 180, 189, 190]. The
selectivity for each fragment between the mixed film surface and the negative control surfaces
was also higher when the polyatomic bismuth cluster ion sources were used. Polyatomic cluster
ion sources have a higher probability of providing higher secondary ion yields because of
increased number of collision cascades near the surface [179], and this might translate into
increased secondary ion intensities. The secondary ion yield, which is defined as the number of
secondary ions divided by the number of primary ions, is in turn dependent on the amount of
primary ion energy which is deposited near the surface, and this relies on the mass and size of
the primary ion projectile [179, 189]. This enhancement is typically observed for substrates that
have insulating properties such as Si as reported by Bernhardt et al. [194], and this condition
might be expected for glass substrates as used in this work. Silicon surfaces have were found to
have higher scattering ion yields of antimony and bismuth cluster ions compared to conductive
materials such as gold and graphite [194]. These surfaces have a higher work function, hence
142
there is a high probability for cluster ions to be neutralized on the surface [194]. This in turn
reduces the number of cluster ions that can ionize surface species. However, the collision energy
used in this work was almost 200-fold higher than that used by Bernhardt et al. [194]. Another
contributing factor for the increase in secondary ion yield would be the nonlinear effects of
polyatomic cluster ion sources [195], besides the effects caused by the increase in mass of the
projectile [189]. Hellweg et al. have found a total enhancement factor of 31.1 and 10.4 per Bi
atom for the secondary ion yield of PO3- when the Bi3
+ cluster ion was used (compared to the Bi+
ion) for the analysis of an oligonucleotide-immobilized surface [189]. This nonlinear increase
was also observed for Bi52+ and Bi2
+ primary ion sources [189].
For the analysis of negative ion fragments related to oligonucleotides, only the Bi5+ ion
source was able to substantially increase the ion intensities from the low oligonucleotide density
of Surface C. The Bi3+ ion source generated higher secondary ion intensities from Surface B but
it was not sensitive enough to generate the same secondary ion fragments from Surface C.
However, the converse was true for the positive ion fragments. The Bi3+ ion source yielded
higher ion intensities of positive ion fragments that originated from both oligonucleotides and
PHEMA than the Bi5+ ion source. Therefore, the mechanism of dissociation for each primary
cluster ion and its interaction with surface species must be fundamentally different.
Nevertheless, these results support the fact that there is an increase in secondary ion intensities
when polyatomic cluster ion sources are used as primary ion sources [179].
Although some unexpected results were obtained for some secondary ion species when
using Bi5+ bombardment, it can be concluded that in general the secondary ion intensities were
maximized for both polarities when Bi3+ was used. The secondary ion yield was generally
maximized for both positive and negative ion cases when Bi3+ was used. The Bi3
+ ion source
143
was also observed to produce higher secondary ion yield than Bi5+ in the study by Touboul and
co-workers [180]. They pointed out that the velocities of various sizes of cluster ions, at the
same given kinetic energy, are inversely related to the square root of the number of substituents
in the cluster ion [180]. The decreased velocity may lead to lowered secondary ion generation
[179, 180].
One interesting observation worth noting is that the negatively charged oligonucleotide
fragments such as PO2- and PO3
- had comparable magnitudes in peak intensities as the negatively
charged PHEMA fragments (see Fig. 3.20 and Table 3.8), and only an order of magnitude lower
for positively charged oligonucleotide fragment when compared to positively charged PHEMA
fragments (see Fig. 3.21 and Table 3.8). The number of repeating units in oligonucleotides was
19 bases and PHEMA was about 20 to 40 units of monomer, with each repeating unit providing
one PO3- or C4H5O2
-, respectively. However, the relative densities on the surface was about
1:200 oligonucleotides:PHEMA, assuming that there were as many as 7 × 1013 polymer chains
cm-2 [136] on the surface and the density of oligonucleotides was determined to be about 3.7 ×
1011 molecules cm-2. The similarity in the magnitudes of the observed signal intensities for the
characteristic fragments suggests a significant selectivity for ionization of oligonucleotides, and
points to an unusual matrix effect.
144
(a)
C2H5O_n=3 C4H5O _n=3 C2H5O_n=5 C4H5O _n=5 0.0
1.0x105
2.0x105
3.0x105
4.0x105
5.0x105
6.0x105
7.0x105
8.0x105
9.0x105
1.0x106
1.1x106
Peak
inte
nsity
(cou
nts)
PHEMA Fragment and Bin+ (n=1, 3, 5) cluster ion used
Control conc1 conc2 conc3
(b)
C4H5N4_n=3 C6H7N2O_n=3 C4H5N4_n=5 C6H7N2O_n=5 0.0
5.0x104
1.0x105
1.5x105
2.0x105
2.5x105
3.0x105
Pea
k in
tens
ity (c
ount
s)
Oligonucleotide fragment and Bin+ (n=1, 3, 5) cluster ion used
Control conc1 conc2 conc3
Fig. 3.22: Comparison of peak intensities of selected positive fragments from PHEMA and oligonucleotides
between the Bi3+ and Bi5
+ primary ion sources at various concentrations; (a) C2H5O+ and C4H5O+ fragments from
PHEMA; (b) C4H5N4+ and C6H7N2O+ from oligonucleotides. The control was a mixed BZ-APTMS and APTMS;
conc1 was 0.10 μM, conc2 was 0.48 μM, and conc3 was 1.02 μM (loading concentrations).
145
In a separate experiment, a mixed film sample was prepared containing three different
surface densities of oligonucleotides, while keeping that of PHEMA constant. Figure 3.22 shows
the peak intensities of selected fragments from PHEMA and oligonucleotides at various PHEMA
to oligonucleotide ratio. The fragments C4H5N4+ (m/z = 109) and C6H7N2O+ (m/z = 111)
originated from adenine and thymine [183]. The Bi3+ cluster ion source provided higher peak
intensities than the Bi5+ source, which agreed with the first set of TOF-SIMS data (Fig. 3.20 and
3.21) and with those observed by Touboul et al. [180]. The density of the immobilized
oligonucleotide probes was at least 3.9 × 1011 (± 19%) probes·cm-2 by using a loading
concentration of 0.10 μM. This resulted in a PHEMA:oligonucleotide ratio of 181:1 by using a
density value of 7 × 1013 polymer chains·cm-2 for PHEMA. However, the peak intensities of
fragments associated with oligonucleotides were still high, and reached magnitudes about 2 to 7-
fold less than the peak intensities of PHEMA-related fragments.
Moreover, changing the oligonucleotide loading concentration from 0.1 μM to 0.48 μM
and 1.02 μM did not result in any changes in the peak intensities of all fragments. The
difference in surface density between the three loading concentrations was confirmed by
obtaining a confocal image of the surface. The integrated fluorescence intensity ratio collected
from each Cy3-labelled oligonucleotide-spotted area of a different loading concentration was
1:5:10 (0.1 μM:0.48 μM:1.02 μM). Therefore, the relative ratios of the number of immobilized
oligonucleotide probes from each area were the same as that of the loading concentrations. Since
changing the number of immobilized probes did not change the secondary ion intensities of
fragments from both PHEMA and oligonucleotides components, the formation of fragments may
have reached a saturated level. Another observation was that fragments that originated from the
nucleobases were detected this time but not the fragments from the phosphate backbone.
146
Besides fragments from thymine and adenine, fragments from cytosine, C4H6N3O+ (m/z = 112),
and guanine, C5H3N4O+ (m/z = 135) [183], were also readily identified in the positive ion mass
spectra. This demonstrates that any slight changes in the matrix may influence the yield of
particular fragments.
The comparison of data collected from both XPS and TOF-SIMS demonstrated the
superior sensitivity of TOF-SIMS equipped with a polyatomic bismuth cluster primary ion to
detect fragments from DNA (present at a low density) within a polymer matrix. Although XPS
provides chemical information of surface species such as oxidation states, type of bonding, and
environment of each element, it is known not to be as sensitive as SIMS [196]. The detection of
fragment ions from oligonucleotides (19 mer) at a low density within a matrix of PHEMA of less
than 10 nm in theoretical length, reiterates the superior capability of TOF-SIMS to investigate
such a unique surface.
3.3.6 Conclusions
A novel biosensing surface composed of oligonucleotides and poly(2-hydroxyethyl
methacrylate) (PHEMA) brushes that co-existed as a thin mixed film on silica substrates was
investigated by XPS, SEM, and TOF-SIMS. XPS was not able to detect oligonucleotides but
could detect PHEMA readily. This was in part due to the lower sensitivity of XPS and a low
density of oligonucleotides immobilized on the surface. TOF-SIMS with a bismuth cluster ion
source could selectively detect fragments unique to each component of the mixture. The
secondary ion intensities were generally maximized for both positive and negative ion fragments
when Bi3+ was used, which can be related to a higher velocity compared to a larger cluster ion at
the same kinetic energy, besides the well known enhancement in secondary ion yield provided
147
by cluster ions and their non-linear effects. There is evidence of a selective ionization for
oligonucleotides since the secondary ion intensities of ion fragments coming from both
components were similar in magnitudes even though the ratio of the density of oligonucleotides
to that of PHEMA was calculated to be 1:200. Therefore, the use of polyatomic bismuth cluster
ion sources in TOF-SIMS offered a sensitive and selective tool to detect oligonucleotides in a
polymeric environment.
148
3.4 A Mixed film Composed of Oligonucleotides and Poly(2-Hydroxyethyl Methacrylate) Brushes to Enhance Selectivity for Detection of Single Nucleotide Polymorphisms
April K.Y.Wonga,c, Denys O. Marushchakb,c, Claudiu C. Gradinarub,c and Ulrich J. Krulla,c*
aDepartment of Chemistry, University of Toronto, Toronto, Canada
bDepartment of Physics, Institute for Optical Sciences, University of Toronto, Toronto, Canada
cDepartment of Chemical and Physical Sciences, University of Toronto Mississauga, 3359
Mississauga Rd. N., Mississauga, ON, Canada L5L 1C6
Contributions: April Wong performed all measurements, except for the lifetime data, which
were contributed by Denys Maruschchak and Claudiu Gradinaru. All other data analysis and
interpretation were done by April Wong under the guidance of Ulrich Krull.
Accepted in Anal. Chim. Acta (2009), doi:10.1016/j.aca.2009.12.001
149
3.4.1 Abstract
Preliminary studies of mixed films composed of oligonucleotides and poly(2-
hydroxyethyl methacrylate) (PHEMA) have recently been shown to enhance the selectivity for
detection of 3 base-pair mismatched (3 bpm) oligonucleotide targets. Evaluation of selectivity
for detection of single nucleotide polymorphisms (SNP) using such mixed films has now been
completed. The selectivity was quantitatively determined by considering the sharpness of melt
curves and melting temperature differences (ΔTm) for fully complementary targets and SNPs.
Stringency conditions were investigated, and it was determined that the selectivity was
maximized when a moderate ionic strength was used (0.1 to 0.6 M). Increases of ΔTm observed
when using mixed films were up to 3-fold larger compared to surfaces containing only
immobilized oligonucleotide probes. Concurrently, increases in sharpness of melt curves for 1
bpm targets were observed to be up to 2-fold greater for mixed films. The co-immobilization of
PHEMA resulted in a more homogeneous distribution of oligonucleotide probes on surfaces.
Lifetime measurements of fluorescence emission from immobilized oligonucleotide probes
labeled with Cy3 dye indicated the difference in microenvironment of immobilized
oligonucleotides in the presence of PHEMA.
3.4.2 Introduction
Single nucleotide polymorphisms (SNPs) are sites found in deoxyribonucleic acid (DNA)
which contain a single base-pair mismatch [9]. It is defined as such when the more common
sequence variant occurs less than 99% of the time [197]. In the human genome, a SNP occurs on
average once in every 1000 bases [197]. As the most abundant human genetic variation, SNPs
are used as a genetic marker which can be traced from one generation to another [9].
150
Furthermore, the study of how SNPs can lead to the onset of diseases is also of great interest [9].
Rapid, selective, and sensitive tools are needed for the identification and detection of SNPs.
Established molecular biology techniques to screen SNPs involve amplification and separation
steps [7]. A number of sensor and microarray detection strategies have been reported based on
colourimetric [125, 128], electrochemical [126, 130, 198], and optical [7, 199] detection. Some
of these involve large scale parallel analysis of samples for SNPs, and there is also interest in
dedicated analysis to detect specific SNPs.
A common feature of all biosensors, regardless of the transducer type, is the obligatory
immobilization of single-stranded oligonucleotides onto a solid substrate [18]. Ultimately, these
oligonucleotide probes are exposed to DNA targets, which may or may not hybridize to the
probes depending on sequence complementarity. The key parameters that govern stringency of
DNA hybridization and denaturation at a surface are temperature, ionic strength, pH, and density
of oligonucleotides. Temperature, pH, and ionic strength affect interactions that keep the DNA
duplex stable, such as hydrogen bonding, charge repulsion of the phosphate backbones, and base
stacking [28, 29]. The extent, rate, and selectivity of hybridization are heavily dependent on the
environment that is experienced [18]. For interfacial hybridization and denaturation, factors that
affect the environment include oligonucleotide distribution and density, the presence of nearest
neighbour interactions, and of interactions between probes and the surface [18]. Density,
however, is also a dynamic function of the degree of hybridization [18]. Ideally, to control
density, one must control the structural environment around each oligonucleotide probe. This
would then provide similar energetics for each probe molecule, resulting in improved selectivity
for the detection of SNPs.
151
Melt curves are commonly used to evaluate selectivity (stability) of binding by
considering differences in melting temperature (ΔTm) between a fully complementary (FC) and a
SNP target [7, 125, 200]. In bulk solution, the ΔTm can be 4–5°C for a 20-mer oligonucleotide
[16, 201]. This difference may be smaller for interfacial denaturation [7]. Moreover, melt
curves collected from surfaces tend to be broader, reflecting the diversity of energetics at an
interface and providing even lower selectivity for SNP detection [7]. A higher selectivity is
indicated by a greater difference in melting temperature as well as a sharper melting transition.
An increase in ΔTm helps to distinguish between FC and SNP target populations at a given
temperature and ionic strength [18]. An example of sharpening of melting transitions has
previously been demonstrated by Taton et al. by implementation of gold nanoparticles [125].
Control of orientation and nearest-neighbour interactions of oligonucleotide probes and
hybridized DNA is desirable to improve selectivity of hybridization at interfaces of
electrochemical sensors. The application of electric fields on conductive substrates has been
used to alter orientation of immobilized probes [202] to improve the discrimination of SNPs
[130], and to enhance hybridization kinetics [203]. However, electrostatic “combing” is not
possible with glass and silica-based substrates. A “matrix isolation” design was proposed by
Piunno et al. which involved the co-immobilization of non-nucleic acid oligomers with
oligonucleotide probes [18]. They have shown that a mixed film composed of oligonucleotide
probes and ethylene glycol phosphate can lower the Tm by 5°C and that SNP detection was still
possible [18]. Another example of inclusion of non-nucleic acid oligomers was demonstrated by
Boozer et al. [81]. The conformation of immobilized oligonucleotides was controlled by self-
assembling thiolated oligoethylene glycol and oligonucleotides on a gold surface, resulting in
improved hybridization efficiency [81]. This experiment showed that the orientation of
152
oligonucleotide probes could be controlled to maximize hybridization efficiency. Polymeric
coatings have frequently been used on surfaces to prevent adsorption of biomolecules. Yalçin et
al. have further explored this concept to improve DNA hybridization efficiency by 50%
(compared to silanized surfaces) by taking advantage of the swelling properties to create a 3-D
polymeric platform for the immobilization of oligonucleotides in microarray applications [204].
Urakawa et al. have also used a gel-coated microarray to optimize the discrimination of SNPs
[205]. The use of polymers is becoming more widespread to optimize sensor designs.
The polymeric coating used by Urakawa and co-workers is usually deposited as a layer
before oligonucleotides are immobilized. In contrast, the mixed oligomer and oligonucleotide
film used in our work is similar to that described in Piunno et al. [18], and comprises
interspersed oligomer brushes with oligonucleotides. This new method involves the building of
oligomer brushes from the surface via surface-initiated atom transfer radical polymerization
(ATRP) [178]. ATRP is based on exchanges of halogen atoms between dormant species and
metal catalysts to control the dynamics of radical polymerization [117-120]. It is a controlled/
“living” polymerization, providing controlled molecular weight and narrow polydispersity [117-
120]. Other advantages include mild conditions, availability of aqueous-based ATRP and a wide
choice of acrylate and methacrylate-based monomers [116]. Surface-initiated ATRP also allows
the growth of dense brushes without issues of steric hindrance [112]. In a previous investigation,
DNA hybridization and denaturation were found to be functional when mixed into films of
poly(2-hydroxyethyl methacrylate, PHEMA) grown onto silicon and glass substrates [178]. It
was demonstrated that the selectivity for the detection of 3 base-pair mismatch (3 bpm) can be
improved by physically controlling the environment surrounding the oligonucleotide probes
using PHEMA brushes [178]. Not only were the mixed films reusable for a number of cycles,
153
but the melt curves were 3-fold sharper, and the melting temperature differences (ΔTm) increased
by 30% in comparison to films composed only of oligonucleotides [178].
This new report provides a detailed examination of conditions required to control the
sharpness of the melting transitions and ΔTm values to distinguish between SNP and FC targets
for PHEMA-oligonucleotide coatings on glass surfaces. Fluorescence intensity measurements
were used to reflect the extent of hybridization. Fluorescence lifetime data provided a further
means to evaluate differences in the environments experienced by probe oligonucleotides in the
PHEMA-oligonucleotides mixed films, and films composed only of oligonucleotides.
3.4.3 Selectivity of Various Targets on the Mixed films
The mixed films were first investigated for selectivity towards FC (Sequence 5, Table
2.1) and 3 bpm targets (Sequence 7, Table 2.1) versus non-complementary (NC) targets
(Sequence 4, Table 2.1). Figure 3.23 shows that the relative integrated fluorescence intensities
for the FC:3bpm:NC targets were 30.5:4.6:1.0. These results suggested that hybridization
occurred, and that the signals were not dominated by non-specific adsorption. Figure 3.24
indicates that the FC targets adsorbed minimally to the 1× PBS-treated area in the absence of
probes, whereas the signal intensities at the probe-deposited areas were 1000-fold higher. The
various targets (Sequences 5–7 of Table 2.1) hybridized to the areas that contained probes, and
resulted in a relative fluorescence intensity ratio of 15.5:5.2:1.0 for FC:1bpm:3bpm.
154
Fig. 3.23: Integrated fluorescence intensities of various targets introduced to oligonucleotide probes (Sequence 1).
Standard deviations are shown. FC = fully complementary (Sequence 4), number of trials (n) = 8. 3 bpm = 3 base-
pair mismatch (Sequence 6), n = 8. NC = non-complementary (Sequence 3), n = 3
Figure 3.24: Integrated fluorescence intensities of various targets introduced to areas with and without immobilized
oligonucleotide probes. Standard deviations are shown. The “no probe” areas were spotted with 1× PBS. n (No
probe+ FC) = 4, n (probe + all other targets) = 8
155
3.4.4 Fluorescence Lifetime to Identify Different Microenvironments in Oligonucleotide Films vs. Mixed Films
The excited state decays of Cy3 dyes that were end-labeled to single-stranded
oligonucleotides (Sequences 2, Table 2.1) were measured for samples in which the probes were
immobilized on surfaces with and without PHEMA. Table 3.9 lists the lifetimes and their
relative amplitudes obtained from bi-exponential fitting applied to both types of surfaces. Figure
3.25 shows the raw data and the fitting curves for some fluorescence lifetime experiments. The
instrument response was very fast (~50 ps rise time) and had a negligible influence on fitting the
decay, so that no deconvolution routine was applied. In both cases, a monoexponential decay
was not sufficient to adequately fit the decay, as judged from the chi-square value and the profile
of the fitting residuals.
The fast component of Cy3 fluorescence decay in oligonucleotide films had a lifetime of
0.8 ns, while the slower component had a lifetime of 2.5 ns. The shorter lifetime had twice the
weight of the longer lifetime (66% vs. 34%). In the mixed film the fast decay time was 1.5 ns,
while the slow decay time was 3.8 ns. Again, the fast component was dominant, which was 82%
vs. 18% for the slower component. Overall, it is evident that the lifetime of Cy3 was
significantly longer in the mixed film than that measured from the oligonucleotide film. In
addition, the intensity time trajectories show that the fluorescence of the PHEMA film was bright
and stable, while the signal from the oligonucleotide film was less intense and photobleached
rapidly.
156
Table 3.9: Fluorescence lifetime and steady-state anisotropy values obtained from intensity time trajectories
measured on a confocal microscope.
τ1 (ns) α1 (%) τ2 (ns) α2 (%) χ2 r
Oligonucleotide filma 0.80±0.03 66 2.54±0.05 34 1.60 0.23±0.03
The lifetime fitting program uses the Levenberg-Marquardt algorithm and a χ2 value close to 1 represents a good fit. a –fluorescence acquired in the first 20s of every time series and b – fluorescence from the entire time series (300s).
Figure 3.25: Fluorescence intensity decay of Cy3-ssDNA in different environments: upper decay corresponds to
the mixed film and the lower one to the oligonucleotide film. Raw data in black, bi-exponential fit curves in red and
green. See table 2 for the numeric results of the fitting analysis.
157
Free Cy3 dyes in solution have a very short lifetime value of 180 ± 10 ps as reported by
Sanborn et al.[206]. This is because cyanine dyes are known to photoisomerize, i.e. undergo
trans-cis isomerisation through the polymethine chain, from the first excited singlet state to the
ground state (see Fig. 3.26). This process is in competition with fluorescence [206]. The
activation energy barrier for this process is strongly dependent on the rigidity of the local
environment which the dye experiences [206]. The study by Sanborn et al. compared the
photoisomerization efficiency, fluorescence quantum yields and lifetimes of Cy3 with Cy3B, the
latter having a rigid backbone which prevented isomerisation [206]. Cy3B had a much longer
lifetime of 2.70 ± 0.01 ns than Cy3. Therefore, the rigidity of the environment was a
determinant of these parameters [206]. Without a flexible linker, isomerisation was impeded.
Consequently, the fluorescence process dominated, and the fluorescence quantum yield and
lifetime were increased.
Fig. 3.26: The structure of 5′-Cy3-labeled oligonucleotides. Cy3 can be photoisomerized from the trans to cis
conformation through the methine bridge. R is the oligonucleotide sequence
158
When Cy3 is covalently attached to the 5′-end of ssDNA, both quantum yield and
lifetime increase in magnitude [206]. This increase is associated with base-stacking of the dye,
which can occur with two to three terminal nucleobases [206, 207]. The decay of 3′-Cy3-ssDNA
in solution was previously measured and fitted to a sum of two exponentials, 1.2 ns (84%) and
2.1 ns (16%) on our instrument (Gradinaru, unpublished results). The two populations of probe
molecules with covalently attached Cy3 dyes are proposed to be systems with Cy3 stacked with
oligonucleotide (longer component), and systems without stacking which yielded a lower
activation barrier for photoisomerization to occur (shorter component) [206, 208]. Single-
molecule anisotropy data acquired in our lab supports this hypothesis (Gradinaru, unpublished
results). The longer lifetimes detected in the mixed films compared to oligonucleotide films
indicate a more rigid environment for the Cy3 dye in both the stacked and unstacked
conformations in a PHEMA matrix. Fig. 3.27 shows the two possible stacked and unstacked
interactions of Cy3 with oligonucleotides in an oligonucleotide film vs. a mixed film.
Fig. 3.27: Proposed stacked and unstacked interactions of Cy3 with immobilized oligonucleotide probes (a) in an
oligonucleotide film; (b) in a mixed film.
159
The presence of PHEMA provided a much more homogeneous spatial distribution of
Cy3-labeled oligonucleotides than observed from films composed of only oligonucleotides.
Figure 3.28 shows two fluorescence images of Cy3-oligonucleotides immobilized on glass, with
and without PHEMA. The oligonucleotide film showed a highly non-uniform surface
distribution, with variations on a micrometer scale, whereas the mixed film showed a
homogeneous intensity across tens of micrometers or more. In addition, when embedded in the
PHEMA matrix the Cy3 fluorescence was much more intense and photobleached much more
slowly than in the absence of the polymer. The combination of the fluorescence lifetime and
imaging are consistent with the hypothesis that the oligonucleotide probes were surrounded by
PHEMA brushes in the mixed film.
a) b)
Fig. 3.28: Fluorescence images of (a) oligonucleotide-only film, and (b) PHEMA film on glass cover slips. The
intensity variations in the PHEMA image are mostly due to imperfect flattening of the excitation field in a wide-
field microscope.
160
3.4.5 Comparison of Selectivity for SNP Detection between Oligonucleotide and Mixed films
Interfacial melt curves were collected from oligonucleotide-modified glass surfaces with
and without co-immobilized PHEMA. The extent of hybridization was determined using the
integrated fluorescence intensity from Cy3-labeled FC and SNP targets (Sequences 5 and 7,
Table 2.1) collected at each temperature during each melt curve experiment. The modification
of oligonucleotides with fluorophores such as Cy3 at the 5′-end has been shown by Moreira and
co-workers to increase Tm by 1.4 °C [209]. However, Cy3 does not have an influence on the
width of the melting transition and slope [209]. The same dye was used on both types of
surfaces. The melt curves which were collected from solution used unlabeled oligonucleotides.
These latter sets of data were intended as benchmarks for ΔTm and dx values.
The melt curves were sigmoidal in nature with a single, cooperative transition similar to
those obtained by Piunno et al. under equilibrium conditions [18]. To compare the effectiveness
of the mixed film relative to the oligonucleotide film to discriminate SNP from FC targets, the dx
and Tm values were obtained by fitting each melt curve with the sigmoidal function:
dxxx
e
AAAy0
1
)( 212 −
+
−+= (3.4)
where A1 and A2 are the lower and upper baselines, x is temperature, y is the fraction of single-
stranded DNA (fssDNA), x0 is the melting temperature, and dx is the change in temperature for the
greatest change in fssDNA. Each melt curve experiment used slides that contained at least 8
hybridized spots for each of the two targets, hence yielding 16 melt curves per run. Large ΔTm
and a small dx values are desired for a highly selective surface. A small dx value signifies a
161
steep slope. Only melt curves which exhibited a good sigmoidal fit with R2 ≥ 0.95 were included
in the data analysis.
In general, interfacial hybridization and denaturation are very different energetically
compared to these processes done in bulk solution [160, 210]. The Tm values collected from
solid-liquid interfaces are often lower than those observed from bulk solution because the probes
are anchored to a solid substrate, hence lowering the stability of duplexes [129]. Melt curves
from bulk solution have been reported to have melting transition ranges of about 10 °C [129]
while interfacial melt curve were reported to have melting transition that spanned from 12–20 °C
[129, 199]. In this investigation, melting transitions of oligonucleotide films were as small as
10°C, whereas the mixed PHEMA-oligonucleotide films showed transition ranges as small as
5°C. This is greatly improved from previous studies [129, 199]. A more accurate variable to
describe the steepness of the melting transition is to use the dx value. Table 3.10 lists the dx
values obtained from both oligonucleotide and mixed film surfaces in all ionic strengths. For
SNP targets in 0.5× PBS, the values ranged from 1.1–3.0 °C for the mixed films compared to
3.6–5.7°C for the oligonucleotide films, indicating that overall, the mixed films provided sharper
melting transitions. The average of dx values found for the mixed films for both targets were
statistically the same as those obtained from bulk solution (1.9°C).
162
Table 3.10: Comparison of dx values in all ionic strengths studied.
Avg: average values. ± The standard deviations of at least 3 replicates were reported. The averages of all experiments were reported with their standard errors.
a The confidence interval (C.I.) at 95% was constructed for the difference in Tm values between FC and SNP targets.
b Two replicates.
The total ionic strength for each salt condition was: 0.11 M (0.1× PBS), 0.56 M (0.5× PBS), and 1.12 M (1.0× PBS).
164
The ΔTm values collected from oligonucleotide films and mixed films in all ionic
strengths are listed in Table 3.11. In 0.5× PBS, the ΔTm values observed for each trial from the
oligonucleotide surface are much lower than the differences observed for the mixed film (at a
confidence level of 95%). The largest ΔTm found for oligonucleotide films was 4.7 ± 2.6 ºC. At
the same ionic strength, the largest ΔTm found for mixed films was 15.7 ± 1.7 ºC, and exceeded
the performance observed for the bulk solution experiment by 6.5 ºC. The average ΔTm for
mixed films was more than three times as large as the average ΔTm found for the oligonucleotide
films. The 2- to 3-fold increase in ΔTm seen when using the mixed films is due to a decrease in
the Tm for the SNP targets and an increase in the Tm of FC targets; i.e. the mixed film structure
stabilizes FC hybrids and destabilizes SNPs, relative to films composed of only oligonucleotides.
In general, the melting temperatures decreased as the salt concentration decreased and the
SNP targets had lower Tm than the FC targets, as was expected. The coefficient of variation
(CV) (%) for the Tm values in each experiment was under 10%. The CV between experiments
for the same target was under 7%. The CVs of the dx values for each experiment were larger due
to the magnitudes of the standard deviations with respect to the data values. The dx values
between experiments were generally reproducible within 1–2 °C.
The increase in the sharpness of melt curves was also evident in 1× and 0.1× PBS. The
former ionic strength yielded dx values for the SNP targets ranging from 2.0–2.7 °C for the
mixed films versus 2.3–5.5 °C for the oligonucleotide films. Using FC targets, the mixed films
showed a more consistent pattern of sharper melting transitions than the transitions seen for
oligonucleotide films. In 0.1× PBS, the dx values for the SNP targets collected from the mixed
films were twice as sharp as those from oligonucleotide films and bulk solution. The dx values
165
for the FC targets between the two surfaces were not significantly different. The average ΔTm
value associated with the mixed films in 0.1× PBS was more than twice as large as that from the
oligonucleotide films. For the 1× PBS case, the average ΔTm value was generally similar
between the mixed films and the oligonucleotide films at the 95% confidence level. Figure 3.29
is a collection of representative melt curves that reflect the trends shown in Tables 3.10–3.11.
The mixed films provided better resolution between the FC and SNP targets compared to the
oligonucleotide films 0.1× and 0.5× PBS (Figures 3.29a–d). The mixed films either had sharper
melting transitions and/or larger ΔTm values to achieve SNP discrimination, whereas the
oligonucleotide films had significant overlapping populations of FC and SNP targets at any
given temperature and ionic strength. The melt curves of Figs. 3.29b and 3.29d indicate the
advantage for SNP analysis by using the PHEMA-oligonucleotide mixed film in combination
with moderate ionic strength.
OLIGONUCLEOTIDE FILMS MIXED FILMS
(a) (b)
166
(c) (d)
(e) (f)
Figure 3.29: Individual melt curves of (•) SNP targets and (■) FC targets that represent average Tm and dx values
collected from oligonucleotide (left column) and mixed films (right column) in increasing PBS strengths (0.1, 0.5,
The increase in sharpness of melt curves associated with the mixed films appears most
prominently for mismatched duplexes, and this is the case for SNPs as well as previously
reported 3 bpm targets [178]. The sharpness observed for mismatched systems may be due to
167
the sterics associated with “breathing” events at terminal ends and mismatched sites. Fig. 3.30
illustrates this concept. The “breathing” mechanism is well known in which segments in these
regions repeatedly denature and re-anneal [27]. The PHEMA brushes tend to magnify the
sharpness of the melting transition more so for the 3 bpm targets [178] compared to the SNP
targets, as would be expected if the interactions were dominated by physical occupancy
considerations. The hanging pendant groups of 2-hydroxyethyl may achieve higher chain
mobility as temperature increases. The mismatched sites could then be destabilized by these side
chains.
Furthermore, the data shows that the destabilization effect is related to salt concentration,
suggesting an electrostatic contribution is also significant. The local salt concentration may be
amplified due to molecular crowding by PHEMA brushes (see Fig. 3.30). At 1× PBS, there was
a large amount of salt to counteract repulsion arising from negative charges of the DNA
backbone and destabilizing forces from PHEMA brushes; all Tm values for both FC and SNP
were relatively high. At 0.1× PBS, there may have been insufficient salt to suppress the
repulsion, hence both FC and SNP targets denatured at a lower temperature. At 0.5× PBS, the
effect of uncharged PHEMA played a more dominant role in the destabilization of the SNP
targets, but did not compromise the stability of the FC targets. The trend in destabilization of the
duplexes can also be explained by the swelling behaviour trend of PHEMA. It was found by
D’Agostino et al. that the swelling ratio decreases with increasing NaCl concentration [211].
The physical occupancy of PHEMA and hence steric repulsion would play more of a dominant
role at lower ionic strengths.
168
Fig. 3.30: Interpretation of melting transition width and Tm differences between different targets hybridized to the
mixed film.
A further consideration about the origins of the changes of duplex stability is related to
water activity in the presence of PHEMA. Spink and Chaires found that increasing the
concentration of polyethylene glycol (PEG) 3400 increased the melting temperature of duplexes
in bulk solution [212]. Here we observed some stabilization for FC targets for immobilized
PHEMA-oligonucleotide films. However, the effects of immobilized PHEMA tended to
destabilize base mismatches. Spink and Chaires reasoned that the presence of PEG caused a
change in the osmotic environment, which can affect the stability of DNA and its helical
structure by changing the number of water molecules that can bind to DNA [212]. It is known
that the amount of water that is uniquely bound to DNA can influence DNA stability because
bound water is thermodynamically different from bulk water [212]. Water molecules first adsorb
to the sugar-phosphate regions but it is also known that some are associated with base-pairs (4–5
water molecules per base-pair) [212]. Such bound water can be released during denaturation,
causing changes in molar volume and entropy. The addition of co-solutes such as PHEMA with
its pendant sidegroups can affect water activity, particularly when duplex disruption due to a
169
mismatch is present. The co-immobilization of PHEMA brushes should be able to contribute
both stabilizing and destabilizing effects on double stranded DNA, depending on the presence of
mismatches.
The broad nature of melt curves collected from oligonucleotide films can also be
attributed to adsorbed oligonucleotides and/or other intermediate structures [160]. The
minimization of adsorption of oligonucleotides on the mixed films contributed to the sharpness
of the melting transition [178]. The signal-to-background ratio was about 2:1 for oligonucleotide
films but was 100:1 for the mixed films. The mixed film apparently blocked adsorption-prone
sites by interspersing of PHEMA chains. One additional point worth noting is that the high
signal-to-background ratio obtained from mixed films was not the result of stringent washing
conditions such as using formamide and SDS washes, and sonication, as is typically done in
hybridization experiments [205].
3.4.6 Conclusions
A comparison has been made of the selectivity and physical environment of
oligonucleotide films with mixed films of oligonucleotides and PHEMA brushes. The resolution
to distinguish between SNP and FC targets by means of temperature was clearly enhanced for
the mixed films. A greater separation of Tm values by as much as 3-fold was observed, and a
sharpening of melting transitions reached as much as 2-fold. The fluorescence of Cy3 attached
to single-stranded oligonucleotide probes showed a bi-exponential decay on the nanosecond
timescale. Both fast and slow lifetime values increased substantially in the mixed films
compared to oligonucleotide films. Cy3 is known to be a molecular rotor and our results indicate
that in the presence of PHEMA the dye experiences a much more rigid environment. The
170
oligonucleotide probes are most likely surrounded by PHEMA brushes at a molecular level,
which is instrumental in improving the selectivity for SNP detection.
171
Chapter 4
4 Summary This investigation demonstrated that a mixed film composed of oligonucleotides and
PHEMA oligomers can be immobilized on silica-based substrates. The use of a mixed BZ-
APTMS and APTMS led to a smoother surface, as shown by AFM data, compared to a surface
derivatized with APTMS alone. The surface-initiated growth of HEMA monomers via ATRP on
silica-based substrates was demonstrated. The initial mixed silane layer provided similar ATRP
reaction rates as on the APTMS film, as shown by ellipsometry data, but the rates were more
reproducible on the mixed aminosilane surface. The smoother mixed aminosilane surface
became an excellent base film layer for further immobilization of both oligonucleotides and
PHEMA to yield reproducible and functional biosensing surfaces.
Hybridization events on the mixed film were functional as proven by using Cy3-labeled
targets of various degrees of complementarity. Comparison of adsorption levels and melt curve
data was made between mixed films and oligonucleotide films. There was an increase in signal-
to-background ratio of 4-fold for mixed films compared to the oligonucleotide films. The mixed
film was also reusable for at least 5 cycles of use. Melt curve data using 3 bpm targets
demonstrated a 2-fold increase in ΔTm and 3-fold increase in sharpness of melting transitions
when PHEMA was co-immobilized with oligonucleotide probes.
A comparison of the ability of XPS and ToF-SIMS equipped with a bismuth cluster ion
source to detect both oligonucleotide and PHEMA components on a glass substrate was done.
XPS could not detect atomic signatures of oligonucleotides but ToF-SIMS could. The bismuth
cluster ion source could also enhance the signal even though the oligonucleotide probes were
172
surrounded by an excess of PHEMA brushes. The oligonucleotide density was estimated by
fluorescence data and a gold nanoparticle-labeling strategy to be on the order of 7×1010 to
3.7×1011 oligonucleotide probes cm-2. ToF-SIMS was determined to be the more sensitive
surface analysis technique to characterize the mixed oligonucleotide and PHEMA films.
The enhancement of detection of SNP targets by the mixed film compared to the
oligonucleotide film was shown. The signal-to-noise ratio was found to be 100-fold higher for
the mixed film. Numerous melt curves were collected from each surface at three different ionic
strengths to determine the effects of salt on the ability of PHEMA brushes to change ΔTm and
steepness of the melting transition. Moderate ionic strength was found to allow PHEMA brushes
to increase ΔTm by 3-fold and steepness of melt curve slope by 2-fold. Increases in ΔTm are due
to an increase in stability for fully complementary targets and a decrease in stability for partially
matched targets. The sharpness was improved due to the isolation effect of PHEMA, leading to
a reduction of intermediate states that often broaden the melting transition ranges. This effect
was amplified when there are base-pair mismatches because there is a lower number of degrees
of freedom for melting, hence increasing the cooperativity for melting. At moderate ionic
strength, the increase in stability for FC targets may have been due to effective charge screening,
but this screening was not adequate when there were base-pair mismatches due to the
destabilizing effect of the steric repulsion of PHEMA and the increased bulkiness of mismatched
duplexes. The lifetimes of Cy3 emission from Cy3-labeled oligonucleotide probes on surfaces
with PHEMA were about twice as long as those immobilized on surfaces without PHEMA. The
increase in lifetime values indicated an increase in rigidity of the mixed film environment for the
oligonucleotide probes compared to the oligonucleotide film. This was consistent with the
intention that the oligonucleotide probes should be surrounded by PHEMA brushes, resulting in
173
a narrower distribution of energetics of DNA hybrids observed in the melt curves collected from
mixed films.
These collective results verified that the mixed aminosilane base layer and the ATRP-
based growth of the oligomer portion were successful in producing the mixed film. This mixed
film strategy has proven to be practical in enhancing signal-to-noise ratio by reducing adsorption
and reducing the degrees of freedom favourable to hybridization. Furthermore, the increased
ability of the mixed film at low to moderate ionic strength to discriminate SNP targets from fully
complementary targets by narrowing the melting transition width and increasing the melting
temperature differences makes it an excellent sensing layer candidate for numerous biosensor
and microarray applications.
174
Chapter 5
5 Future Directions Thus far, this work has only focused on the performance of mixed films which were
composed of one neutral monomer, and which was polymerized from a 1:1 mixed protected and
unprotected (3-aminopropyl)trimethoxysilane (APTMS). Further changes in the experimental
parameters can be examined to understand how to increase the steepness of the melting transition
slope and difference in melting temperatures. For example, a negatively charged monomer such
as 3-sulfopropyl methacrylate can be used to examine the effect of a constant net negative charge
around oligonucleotide probes. The ratio of protected and unprotected APTMS can be varied,
which should theoretically change the oligonucleotide-to-oligomer ratio. Alternatively, the
loading concentration of oligonucleotides can be varied to change the oligonucleotide-to-
oligomer ratio. Furthermore, different oligomer and oligonucleotide lengths can be used. One
important experiment that should be done is to test the mixed films on other sequences to
determine if it can also decrease the melting transition range and increase the melting
temperature differences. The location of the SNP site can be varied, and the most pronounced
effect would probably be observed for mismatched sites which are centrally located or closer to
the surface, but not at the terminus. Not only does ionic strength matters but the choice of buffer
ion types for use in the denaturation process might affect how PHEMA brushes could change the
melting transition width and melting temperature differences. For example, ions of different
sizes and charges of various degrees of polarizability should be studied since different ion
properties may affect the stability of DNA to a different extent [213]. Lastly, real samples
should be tested to see the mixed film surface can detect complementary sequences from a
175
complex matrix. These changes may affect the overall performance of the mixed film to
discriminate fully complementary targets from targets containing a one base-pair mismatch.
The lifetime studies of Cy3 fluorescence were promising in that data from such
experiments can be used to probe the dynamics of the mixed film environment. A more detailed
examination may encompass fluorescence anisotropy, i.e. the rotational freedom of fluorophores
by following the intensities of the perpendicularly and parallel polarized fluorescence emission.
However, a different fluorophore may have to be chosen because Cy3 labels on oligonucleotides
are too sensitive to temperature and local rigidity, and have been shown to provide high steady-
state anisotropy values [206]. It would be more prudent to collect time-resolved anisotropy data
instead and/or choose another dye [206]. Fluorophores that are categorized as “molecular rotors”
such as meso-substituted boron-dipyrrin (BODIPY) dyes are very sensitive to the local viscosity
[214]. Alexa and fluorescein dyes have also been used in anisotropy measurements [215]. If
Cy3 is still chosen as the physical probe, according to Sanborn and co-workers, single-stranded
DNA are better probes for studying lifetime and anisotropy data because isomerization of Cy3 is
suppressed [206]. It has been shown that single-stranded DNA-Cy3 stacking interactions are
reduced when they undergo hybridization, hence resulting in a lower barrier for Cy3
isomerization [206]. Besides using single-stranded probes, one can change the flexibility of the
probe by changing the sequence. For example, an oligonucleotide composed of just thymine is
known to be very flexible as opposed to one composed of just adenine, which is very rigid due to
the opportunity for more base-stacking interactions between the purine bases [216]. The use of a
single-stranded, flexible oligonucleotide sequences may give more information about the
dynamics of the mixed biosensing environment.
176
The vision of an ideal DNA biosensing surface is to achieve minimal adsorption, high
reusability, a high selectivity, and a high sensitivity as well as being reagentless and labeless.
The mixed film as a novel DNA biosensing surface was shown to be reusable and selective with
minimal adsorption levels, but it must be further modified to become an “ideal biosensor”. An
idea for the next step to improve sensitivity would be to immobilize the mixed oligonucleotide-
polymer film on a 3-D polymeric coating as demonstrated in the work by Yalçin and co-workers
[204]. This coating was shown to increase binding sites and lower steric hindrance, which led to
a higher hybridization efficiency compared to silanized surfaces [204]. The swellable property
of this polymer provides more rotational freedom and less steric hindrance for the immobilized
probes because they are lifted away from the surface [204]. The co-immobilization of oligomers
would still serve to reduce nearest neighbour interactions, and to reduce interactions between
probes and the surface.
The intention to create the mixed films on silica-based substrates is to apply this
biosensing surface to optical fiber surfaces such that TIR can be used to excite surface-localized
fluorophores. The first detection scheme may involve just the detection for Cy3-labeled targets,
for example. The dye can be attached to the target strands once they have been isolated and
amplified from a real complex sample. The use of fluorescence to transduce DNA
hybridizations means that the use of labeled reagents cannot be avoided, but labels can also be
incorporated in such a way that the sample itself need not be labeled. For example, Wang and
Krull have demonstrated a self-contained optical fiber biosensor by attaching an intercalator,
thiazole orange, to the immobilized probes. When DNA hybridization occurs with unlabeled
target sequences, the dye emission was enhanced due to intercalation [46]. The sensor was
regenerable as well [46]. Alternatively, the dye can be covalently attached to PHEMA (either to
the monomer or polymer) so that it can be permanently attached to the mixed films. Lastly, one
177
can use a sandwich assay in which reporter strands containing a fluorophore are used; they can
bind to immobilized probes on optical fibers after the unlabeled target sequences have bound to
immobilized probes, as described in Algar and Krull [47]. The strategy for immobilizing the
mixed films would have to be altered. The initiator for the surface-initiated polymerization
would have to be attached to biotin such that it can be immobilized onto a neutravidin-coated
surface. Another way would be to mix thiolated oligonucleotides with thiolated-initiator such
that there are more initiators immobilized onto the quantum dot-coated surface, without using
neutravidin. The fluorophores attached to these reporter strands should experience a “matrix
isolation” environment in the mixed films to yield melt curves with narrow distribution of
energetics and increased melting temperature differences between fully and SNP targets.
178
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