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 microC after hybridization and of only 8 +/- 5 microC 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 microC for hybridization to complementary amplicon and of only 2-10 microC after exposure to noncomplementary DNA.
Napier
M E
ME
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290, USA.
Loomis
C R
CR
Sistare
M F
MF
Kim
J
J
Eckhardt
A E
AE
Thorp
H H
HH
D015374:Biosensing Techniques; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D017931:DNA Primers; D015342:DNA Probes; D004563:Electrochemistry / Q000379:methods; D004566:Electrodes; D004579:Electron Transport; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods
D017931:DNA Primers; D015342:DNA Probes; D004247:DNA