Electrochemical detection of surface hybridization based on highly thermostable DNA monolayers

Over two decades, DNA microarrays and biosensors have witnessed widespread applications in gene expression studies, point mutation/SNP analysis, and genetic disease and cancer diagnostics. However, applications of DNA microarray and biosensor tools to real biological samples containing multiple targets with a broad distribution of sequences and lengths unavoidably involve competitive surface hybridization, which results in a combination of specific hybridization and non-specific hybridization (cross-hybridization). The effects of such combined surface binding complicate and confuse interpretation of data which, in turn, impair reliability of DNA microarrays or biosensors as diagnostic tools.;To address the problem of competitive hybridization at surfaces, an electrochemical method, based on covalently-bound ferrocene labels, was developed for fundamental studies of surface hybridization at elevated temperatures, including processes such as competitive surface hybridization in this thesis. This involves development of both stable immobilization chemistries and stable electroactive labels. To achieve these goals, we first compared five ferrocene derivatives, including two new ones, with respect to bioconjugation reactivity, electrochemical characteristics, and stability at elevated temperatures. From among the five electroactive tags considered, FcFG-NHS provides the best combination of stability against degradation and conjugation yield. Second, we prepared DNA probe monolayers immobilized through thioether bonds to nanometer-thick anchor films of crosslinked poly(mercaptopropyl)methylsiloxane (PMPMS) on gold with excellent hybridization activity toward target strands for hours at temperatures of up to 90 °C. Finally, stability of PMPMS-tethered probe films was exploited to (1) demonstrate measurement of surface melting transitions under reversible conditions and (2) examine effects of temperature on competitive surface hybridization of fully-matched (FM) and singly-mismatched (MM) targets. Surface melting studies showed that PMPMS-immobilized probes exhibited extremely reversible surface melting transitions, that free energies of surface hybridization were suppressed relative to those in solution, and that the mismatch free energy penalty at the surface and in solution was similar. Competitive surface hybridization of FM and MM targets displayed pronounced dependence on temperature, ranging from kinetic freezing at initial FM and MM coverages at room temperatures to rapid equilibration at temperatures sufficiently high to fall within the FM and MM surface melting transitions.