In this study, neutravidin-coated screen-printed carbon sensors were fully characterized and further used for the amperometric detection of specific DNA sequences of human cytomegalovirus (HCMV DNA). For this purpose, we took advantage of an earlier established relationship between the amount of HRP affinity immobilized on the surface of the electrode and the steady-state current recorded in the presence of H(2)O(2) as substrate and the single electron donor [Os(III)(bpy)(2)pyCl](2+) as cosubstrate. After incubating a saturating concentration of biotinylated horseradish peroxidase (Bio-HRP) onto the neutravidin-modified sensors, a surface concentration of active HRP of 3.6 pmol cm(-2) was calculated from the measurement of the electrocatalytic plateau current value. This result indicates that monolayers of neutravidin were adsorbed on the screen-printed carbon sensors. These neutravidin-covered platforms were then used to immobilize biotinylated nucleic acid targets. After hybridization with a complementary digoxigenin-labeled detection probe, the extent of hybrids formed was determined with an anti-digoxigenin HRP conjugate. The biosensor assay was applied to the detection of a synthetic oligonucleotide target, and then to the determination of an amplified viral DNA sequence. Monolayers of HRP-labeled oligonucleotide hybrids were immobilized onto the sensing surface whereas one third of the surface was covered with HCMV DNA hybrids. On the other hand, detection limits of 200 pM and 1 nM were obtained for the short oligonucleotide and the longer DNA targets, respectively. Finally, we demonstrated that the sensitivity of the electrochemical assay could be significantly improved by using high concentrations of the reduced form of the mediator [Os(II)(bpy)(2)pyCl](+), thus allowing one to detect as low as 30 pM of amplified HCMV DNA fragment.
To cite this version:Murielle Rochelet-Dequaire, Naima Djellouli, Benoit Limoges, Pierre Brossier. Bienzymatic-based electrochemical DNA biosensors: a way to lower the detection limit of hybridization assays. The Analyst, JSTOR, 2009, 134, pp.349-353. 10 The use of the alkaline phosphatase (AP) as 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 10 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 paminophenol (PAP) product. The intensity of the cyclic voltammetric anodic peak current resulting from the oxidation of PAP into pquinoneimine (PQI) was related to the number of viral amplified DNA targets present in the sample, and a detection limit of 10 pM was 15 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 bienzymatic amplification scheme enabled a 100-fold lowering of the HCMV DNA detection limit obtained with the monoenzymatic system. 20
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