Studies of Surface Hybridization In a Model System Ping Gong, Damion Irving, Dongbiao Ge, Rastislav Levicky Polytechnic Institute of New York University This research was supported by the National Institutes of Health (HG & HG004512) and by National Science Foundation (DMR ).

Noncompetitive Hybridization HO-(CH 2 ) 3 -SH - easy - highly reproducible - preserves probe activity * Herne T.M. & Tarlov M.J. JACS 1997 a “DNA brush” Gold support DNA-SH 20mer Probe: 5’-TTTTAAATTCTGCAAGTGAT – (CH 2 ) 3 -S-S-(CH 2 ) 3 -OH -3’ 18mer Target: 3’-AATTTAAGACGTTCACTA-5’ 7 probe Coverages (S 0 ) 5 ionic strengths (C B ) thiol or disulfide Surface Hybridization Regimes C T = 100 nM

x = S D /S 0 S 0 (10 12 cm -2 ) L: Langmuir Gong, Levicky PNAS 2008 Irving, Gong, Levicky JPCB 2010 Gong, Wang, Liu, Shepard, Levicky JACS 2010 Surface Hybridization Regimes

x = S D /S 0 S 0 (10 12 cm -2 ) PL: Pseudo-Langmuir surface  G S o = -44,000 kJ/mol vs -100,000 kJ/mol in solution (reference state 1 M Na +, 22 C) Gong, Levicky PNAS 2008 Irving, Gong, Levicky JPCB 2010 Gong, Wang, Liu, Shepard, Levicky JACS 2010 Surface Hybridization Regimes

x = S D /S 0 S 0 (10 12 cm -2 ) Gong, Levicky PNAS 2008 Irving, Gong, Levicky JPCB 2010 Gong, Wang, Liu, Shepard, Levicky JACS 2010 SH-E: Electrostatically suppressed hybridization Surface Hybridization Regimes

x = S D /S 0 S 0 (10 12 cm -2 ) Gong, Levicky PNAS 2008 Irving, Gong, Levicky JPCB 2010 Gong, Wang, Liu, Shepard, Levicky JACS 2010 SH-P: “Packing” suppressed hybridization C B = 0.63 mol L -1 Surface Hybridization Regimes

Competitive Hybridization 20mer Probe: 5' HS-(CH 2 ) 6 -CAG GCA CAA ACA TGA ACC TC 3‘ 20mer Perfect Match (PM) Target: 5' GAG GTT CAT GTT TGT GCC TG-(CH 2 ) 3 -NH 2 3‘ 20mer Mismatch (MM) Target: 5' GAG GTT CGT GTT TGT GCC TG 3' DNA 0.2 M PB pH :1 MM:PM range of temperatures Thermally-stable Surface Immobilization

Surface Melt Transition (*) Perfect match:  H PM o,  S PM o Mismatch:  H MM o,  S MM o Solution Melt Transitions ( o )  Mismatch Penalty in Solution:  H o =  H MM o -  H PM o  S o =  S MM o -  S PM o Perfect match:  H PM *,  S PM *  assume mismatch penalty same as in solution:  H MM *=  H PM * +  H o  S MM *=  S PM * +  S o PM

20mer Probe: 5' HS-(CH 2 ) 6 -CAG GCA CAA ACA TGA ACC TC 3‘ 20mer Perfect Match (PM) Target: 5' GAG GTT CAT GTT TGT GCC TG-(CH 2 ) 3 -NH 2 3‘ 20mer Mismatch (MM) Target: 5' GAG GTT CGT GTT TGT GCC TG 3' 0.2 M PB pH :1 MM:PM ● max discrimination ~ 20 C below solution T M for PM ● suppression of PM signal at lower temperatures ● enhancement of PM signal at higher temperatures