Amperometric Detection of Nucleic Acid at Femtomolar Levels with a Nucleic Acid/Electrochemical Activator Bilayer on Gold Electrode

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Abstract
Cationic redox polymers containing osmium−bipyridine complexes strongly interact with anionic enzymes, such as glucose oxidase and peroxidases, and electrochemically “activate” the enzymes. On the basis of these observations, attempts were made to develop an ultrasensitive nucleic acid biosensor. A mixed monolayer of single-stranded oligonucleotide capture probe and 16-mercaptohexadecanoic acid was formed on a gold electrode through self-assembly. Following hybridization with a complementary nucleic acid and glucose oxidase labeled oligonucleotide detection probe, a cationic redox polymer (electrochemical activator) overcoating was applied to the electrode through layer-by-layer electrostatic self-assembly. The formation of an anionic−cationic bilayer brought the glucose oxidase in electrical contact with the redox polymer, making the bilayer an electrocatalyst for the oxidation of glucose. Thus, nucleic acid molecules were quantified amperometrically at femtomolar levels. The effect of experimental variables on the amperometric response was investigated and optimized to maximize the sensitivity and speed up the assay time. A detection limit of 1.0 fmol/L in 1.0-μL droplets and a linear current−concentration relationship up to 800 fmol/L were attained following a 30-min hybridization. The biosensor was applied to the detection of the 16S gene in a mixture of Escherichia coli 16S + 32S rRNA and a full-length rat housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), of a RT-PCR product.