SEQUENCE-SELECTIVE BIOSENSOR FOR DNA-BASED ON ELECTROACTIVE HYBRIDIZATION INDICATORS

Deoxyribonucleic acid was covalently immobilized onto oxidized glassy carbon electrode surfaces that had been activated using 1-[3-(dimethyl amino)-propyl]-3-ethylcarbodiimide hydrochloride and N-hydroxysulfosuc cinimide. This reaction is selective for immobilization through deoxyg uanosine (dG) residues. Immobilized DNA was detected voltammetrically, using tris(2,2'-bipyridyl)cobalt(III) perchlorate and tris(1,10-phena nthroline)cobalt(III) perchlorate (Co(bpy)3(3+) and Co(phen)3(3+)). Th ese complexes are reversibly electroactive (1e-) and preconcentrate at the electrode surface through association with double-stranded DNA. V oltammetric peak currents obtained with a poly(dG)poly(dC)-modified el ectrode depend on [Co(bpy)3(3+)] and [Co(phen)3(3+)] in a nonlinear fa shion and indicate saturation binding with immobilized DNA. Voltammetr ic peak currents for Co(phen)3(3+) reduction were used to estimate the (constant) local DNA concentration at the modified electrode surface; a binding site size of 5 base pairs and an association constant of 1. 74 x 10(3) M-1 yield 8.6 +/- 0.2 mM base pairs. Cyclic voltammetric pe ak separations indicate that heterogeneous electron transfer is slower at DNA-modified electrodes than at unmodified glassy carbon electrode s. A prototype sequence-selective DNA sensor was constructed by immobi lizing a 20-mer oligo(deoxythymidylic acid) (oligo(dT)20), following i ts enzymatic elongation with dG residues, which yielded the species ol igo(dT)20(dG)98. Cyclic voltammograms of 0.12 mM Co(bpy)3(3+) obtained before and after hybridization with poly(dA) and oligo(dA)20 show inc reased cathodic peaks after hybridization. The single-stranded form is regenerated on the electrode surface by rinsing with hot, deionized w ater. These results demonstrate the use of electroactive hybridization indicators in a reusable sequence-selective biosensor for DNA.