1. Strategies to build mixed DNA and PEG films on silica surfaces to achieve molecularly uniform biosensing conditions 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. Krull Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto at Mississauga, 3359 Mississauga Rd N, Mississauga ON, Canada L5L 1C6 E-mail: ukrull@utm.utoronto.ca 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 + H 2 O  ≡ SiOH + MeOH ≡ SiOH + ≡ SiOH  ≡ Si-O-Si ≡ + H 2 O ≡ 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 (H 2 N-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’-NH 2 -C 12 -dA 20 -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? Surface charge density Surface free energy ssDNA orientation Efficiency, rate and selectivity of DNA hybridizations 1. Mixing NH 2 -PEG derivatives or NH 2 -PAA with NH 2 - dA 20 -Cy3 in solution before immobilization onto epoxy- functionalized surfaces Figure 4. Polymer # 1: O-(2-aminoethyl)-O’-methylpolyethylene glycol (NH 2 -PEG 2000), M n ~2000, length ~ 137 Å The ultimate goal will be to collect melt curves and compare the melting temperature (T m ) 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]. 2. Atom Radical Transfer Polymerization (ATRP) Reaction scheme: Figure 3. Control experiment: HO-(CH 2 -CH 2 -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 NH 2 -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 1 H-NMR and ESI-MS (data not shown) Amine-functionalized silicon wafer produced via silanization with 3- (aminopropyltrimeth oxy)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 NH 2 - 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.