Probing biomolecule recognition with electron transfer: Electrochemical sensors for DNA hybridization

Identifying infectious organisms, quantitating gene expression, and sequencing genomic DNA on chips all rely on the detection of nucleic acid hybridization, Described here is a novel assay for detection of the hybridization of products of the polymerase chain reaction using electron transfer from guanine to a transition-metal complex, The hybridization assay was modeled in solution by monitoring the cyclic voltammetry of Ru(bpy)(3)(2+) (bpy = 2,2'-bipyridine) in the presence of a probe strand containing only A, T, and C prior to and after hybridization to a complement that contained seven guanines, which led to high catalytic current due to the oxidation of guanine by Ru(bpy)(3)(3+). To allow recognition of all four bases in the target sequence, it was shown that inosine 5'-monophosphate was 3 orders of magnitude less reactive than guanosine 5'-monophosphate, suggesting that effective hybridization sensors could be realized by immobilization of probe strands in which inosine was substituted for guanosine; hybridization to guanosine-containing target strands would then provide high catalytic currents. A sensor design was tested in a model system for the detection of a synthetic 21-mer oligonucleotide patterned on the sequence of the ras oncogene, which gave an increase in charge collected of 35 +/- 5 mu C after hybridization and of only 8 +/- 5 mu C after exposure to noncomplementary DNA. independent quantitation of probe and target by radiolabeling showed that the hybridized electrode contained 3.0 +/- 0.3 ng of target. New sensor electrodes were then prepared for the detection of PCR-amplified genomic DNA from herpes simplex virus type II, genomic DNA from Clostridium perfringens, and genomic RNA from human immunodeficiency virus and gave an additional charge of 35-65 mu C for hybridization to complementary amplicon and of only 2-10 mu C after exposure to noncomplementary DNA.