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Strategies to build mixed DNA and PEG films on silica Strategies to build mixed DNA and PEG films on silica surfaces to achieve molecularly uniform biosensing surfaces to achieve molecularly uniform biosensing
conditionsconditions
Acknowledgements
Conclusion
It is evident at this point that mixed films of different proportions of oligonucleotides and polymers can be immobilized on glass surfaces by mixing different molar ratios of oligonucleotides to polymers using the same chemistry. The first two steps in the surface ATRP reaction have been achieved. However, surface characterization of these mixed films must be done to confirm the presence of the polymer and to correlate different doping amounts of polymer with film thicknesses and wettability.
Future Directions• Characterize mixed films by ellipsometry, wettability and ToF-SIMS• Use chemical force microscopy to determine the spatial distribution of oligonucleotides and polymers• Collect melt curves using different molar ratios, targets and stringency
Abstract
A standard protocol for creating a DNA sensing layer is to covalently immobilize short single-stranded oligonucleotide probes onto a surface. However, the selectivity of DNA hybridization is very sensitive to the local environmental conditions including pH, ionic strength and molecular interactions [1]. The local interfacial environment changes as soon as the first hybridization events occur because of the increase in surface negative charges and increase in rigidity. A new design to create sensing layers with improved dynamic range and selectivity is being developed by immobilizing low molecular weight polyethylene glycol (PEG) derivatives to surround oligonucleotide probes. Such mixed films are generated by mixing amine-terminated PEG with amine-terminated oligonucleotides of 20-mer length, and immobilizing the binary mixture onto epoxy-functionalized silica surfaces. Atom transfer radical polymerization of EG methacrylate monomers on amine-functionalized silica surfaces is also being attempted [2].
Introduction
Experimental Procedures
Selected References[1] Paul A.E. Piunno, James H. Watterson, Christopher C. Kotoris, Ulrich J. Krull, Anal. Chim. Acta, 534, 53-61 (2005).[2] X. Lou and L. He, Langmuir, 22 (2006) 2640.
• University of Toronto UTF Conference Travel Grant• Natural Sciences and Engineering Research Council of Canada (NSERC)
April K. Y. Wong, Julie Lukkarila, Paul A. E. Piunno, Michael Georges, and Ulrich. J. KrullChemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto at Mississauga,
3359 Mississauga Rd N, Mississauga ON, Canada L5L 1C6E-mail: [email protected]
Figure 9.
Presence of non-nucleic acid species (EGp) altered interfacial environment, which caused change in thermodynamic stability of DNA hybrids [1].
ssDNA only ssDNA + ethylene glycol phosphate (EGp) oligomers
0.1 × PBS
0.5 × PBS
1.0 × PBS
Case 1
DNA film only – random orientations of ssDNA probe; ‘multiphasic’.
Case 2
Mixed film system containing oligomer/polymer and DNA – control orientation of each ssDNA probe and eliminate nearest-neighbour and strand-to-surface interactions.
Will it be possible to achieve sharper melt curves and improve selectivity between fully matched and partially matched targets?
≡ SiOMe + H2O ≡ SiOH + MeOH
≡ SiOH + ≡ SiOH ≡ Si-O-Si ≡ + H2O≡ SiOH + ≡ SiOMe ≡ Si-O-Si ≡ + MeOH
Hydrolysis
Condensation
Chemically cleaned glass slides were silanized with 3-glycidoxypropyltrimethoxysilane (GOPS) (Figure 1) [1]. The epoxide groups that resulted were cleaved by spotting a mixture of amine-modified oligonucleotides and oligomers (H2N-R) dissolved in 1× PBS on the glass slide, resulting in covalently immobilized oligonucleotides and polymers (Figure 2).
Figure 1.
Figure 2.
DNA sequences used: 5’-NH2-C12-dA20-Cy3-3’ (probe), length ~ 92.5 Å
Domino effect. The local surface charge density is dynamic as more and more hybridization events occur at the solid-liquid biosensing interface. Once the surface charge density changes, the surface free energy and consequently the orientation of the single-stranded (ss) DNA strand with respect to the surface changes. This may dramatically influence the efficiency, rate and selectivity of DNA hybridizations.
Can the local environment be controlled by isolating each DNA probe with a non-nucleic acid oligomer or polymer?
Sur
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cha
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Sur
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fre
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ssD
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Effi
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1. Mixing NH2-PEG derivatives or NH2-PAA with NH2-dA20-Cy3 in solution before immobilization onto epoxy-functionalized surfaces
Figure 4. Polymer # 1: O-(2-aminoethyl)-O’-methylpolyethylene glycol (NH2-PEG 2000),
Mn ~2000, length ~ 137 Å
The ultimate goal will be to collect melt curves and compare the melting temperature (Tm) in different mixed film systems at different ionic strengths. This can be achieved on optical fibers in an automated flow-cell system and by monitoring the emission of the fluorophore attached to the target strand during denaturation as shown by Piunno et al. (see Figure 9) [1].
# of NH2-dA20-Cy3 probes/cm2 immobilized as [NPEG 2000] increased
0.00E+00
2.00E+10
4.00E+10
6.00E+10
8.00E+10
1.00E+11
1.20E+11
1.40E+11
1.60E+11
0 5 10 15 20 25 30 35 40 45
[NPEG 2000] /uM
pro
bes
/cm
2
13Feb
# of NH2-dA20-Cy3 probes/cm2 as [PAA] increased
0.00E+00
5.00E+10
1.00E+11
1.50E+11
2.00E+11
2.50E+11
0 5 10 15 20 25 30 35 40 45
[PAA] /uM
pro
be
s/c
m2
March 27 PAA sonicated
# of NH2-dA20-Cy3 probes/cm2 immobilized as [PEG 2000] increased
0.00E+00
5.00E+10
1.00E+11
1.50E+11
2.00E+11
2.50E+11
3.00E+11
0 5 10 15 20 25 30 35 40 45
[PEG 2000] /uMp
rob
es/c
m2
2. Atom Radical Transfer Polymerization (ATRP)Reaction scheme:
Change in film thickness and wettability following immobilization of initiator for surface ATRP rxn
0.0
10.0
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30.0
40.0
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60.0
70.0
80.0
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1 2 3 4
Sample #
Th
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ess
(an
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s) o
r W
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bil
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(deg
rees
)
APTMS-Si
initiator-NH-Si
WettabilityAPTMS
Wettabilityinitiator
Figure 3. Control experiment: HO-(CH2-CH2-O)n-H or PEG 2000
There was a steady number of DNA probes immobilized on the surface as [PEG 2000] increased. No adsorption of PEG 2000 was evident to cause steric hindrance which would lower the efficiency of the covalent immobilization of the DNA probes.
As [NPEG 2000] increased, a decrease in immobilized DNA probes was observed. Therefore, immobilization of NPEG 2000 may have occurred.
Polymer 2: polyacrylic acid (PAA) with NH2-functionalized group (synthesized by Julie Lukkarila)
The same trend was observed for the PAA sample. Less DNA probes were immobilized as the [PAA] increased, which may also signify successful immobilization of the PAA.
Synthesis details available upon request
PAA
Product 1 confirmed by 1H-NMR and ESI-MS (data not shown)
Amine-functionalized silicon wafer produced via silanization with 3-(aminopropyltrimethoxy)silane (APTMS) using the same procedure described for GOPS
Future work
Evidence of immobilized initiator as shown by ellipsometry and wettability data (see below)
Figure 8. There was an average increase of 20 ± 9.8 Å in immobilized film thickness and 15 ± 7 º in water drop shadow angle from the previous NH2-functionalized silicon wafers. Therefore, the initiator has immobilized onto the amine-modified surfaces.
Figure 5. Reaction scheme for synthesis of PAA.
Figure 6.
Figure 7.