| Abstract
| - Identifying infectious organisms, quantitating gene expression, andsequencing genomic DNA on chipsall rely on the detection of nucleic acid hybridization. Describedhere is a novel assay for detection ofthe hybridization of products of the polymerase chain reaction usingelectron transfer from guanineto a transition-metal complex. The hybridization assay was modeledin solution by monitoring thecyclic voltammetry of Ru(bpy)32+ (bpy= 2,2‘-bipyridine) in the presence of a probe strandcontainingonly A, T, and C prior to and after hybridization to a complement thatcontained seven guanines,which led to high catalytic current due to the oxidation of guanine byRu(bpy)33+. To allowrecognitionof all four bases in the target sequence, it was shown that inosine5‘-monophosphate was 3 orders ofmagnitude less reactive than guanosine 5‘-monophosphate, suggestingthat effective hybridizationsensors could be realized by immobilization of probe strands in whichinosine was substituted forguanosine; hybridization to guanosine-containing target strands wouldthen provide high catalyticcurrents. A sensor design was tested in a model system for thedetection of a synthetic 21-meroligonucleotide patterned on the sequence of the ras oncogene, whichgave an increase in chargecollected of 35 ± 5 μC after hybridization and of only 8 ± 5 μCafter exposure to noncomplementaryDNA. Independent quantitation of probe and target by radiolabelingshowed that the hybridizedelectrode contained 3.0 ± 0.3 ng of target. New sensorelectrodes were then prepared for the detectionof PCR-amplified genomic DNA from herpes simplex virus type II, genomicDNA from Clostridiumperfringens, and genomic RNA from humanimmunodeficiency virus and gave an additional charge of35−65 μC for hybridization to complementary amplicon and of only2−10 μC after exposure tononcomplementary DNA.
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