Bienzymatic-based electrochemical DNA biosensors: a way to lower the detection limit of hybridization assays

The use of the alkaline phosphatase (AP) as an enzyme label and the amplification of its analytical response with a diaphorase (DI) secondary enzyme were investigated in an electrochemical hybridization assay involving arrays of carbon screen-printed DNA biosensors for the sensitive quantification of an amplified 406-base pair human cytomegalovirus DNA sequence (HCMV DNA). For this purpose, PCR-amplified biotinylated HCMV DNA targets were simultaneously bound to a monolayer of neutravidin irreversibly adsorbed on the surface of the electrodes and hybridized to complementary digoxigenin-labeled detection probes. The amount of hybrids immobilized on the electrode surface was labeled with an anti-digoxigenin AP conjugate and quantified electrochemically by measuring the activity of the AP label through the hydrolysis of the electroinactive p-aminophenylphosphate (PAPP) substrate into the p-aminophenol (PAP) product. The intensity of the cyclic voltammetric anodic peak current resulting from the oxidation of PAP into p-quinoneimine (PQI) was related to the number of viral amplified DNA targets present in the sample, and a detection limit of 10 pM was thus achieved. The electrochemical response of the AP label product was further enhanced by adding the diaphorase enzymatic amplifier in the solution. In the presence of the auxiliary enzyme DI, the PQI was reduced back to PAP and the resulting oxidized form of DI was finally regenerated in its reduced native state by its natural substrate, NADH. Such a bienzymatic amplification scheme enabled a 100-fold lowering of the HCMV DNA detection limit obtained with the monoenzymatic system.