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

Abstract

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

Redox cycling amplified electrochemical detection of DNA hybridization: Application to pathogen E. coli bacterial RNA

“…Generally, redox cycling can be achieved electrochemically [18,19], enzymatically [20–23], or chemically [15,24–26]. In the model of electrochemical redox cycling, the electroactive species oxidized at one electrode are reduced back at the second electrode, so two working electrodes or an interdigitated array electrode are needed, while it conflicts with the requirement of simpleness and cost-effectiveness for DNA sensor.…”

“…In the model of electrochemical redox cycling, the electroactive species oxidized at one electrode are reduced back at the second electrode, so two working electrodes or an interdigitated array electrode are needed, while it conflicts with the requirement of simpleness and cost-effectiveness for DNA sensor. Enzymatic redox cycling provides a relatively simple way for redox cycling amplification [14,20,23]. However, in this model multiple enzymes are required and the redox cycling efficiency is highly dependent on the enzyme kinetics.…”

Recent advances in electrochemical DNA hybridization sensors

“…Overall, this enzymatic process enhanced the analytical signal by two orders of magnitude. 51 Park and co-workers synthesized a biotin tagged anthraquinone containing molecule that intercalated into double stranded helices immobilized on a gold electrode. The resultant hybrids were then exposed to a modified streptavidin-peroxidase polymer.…”

DT-Diaphorase as a Bifunctional Enzyme Label That Allows Rapid Enzymatic Amplification and Electrochemical Redox Cycling

“…DT-diaphorase [NAD­(P)H dehydrogenase (quinone) (EC 1.6.99.2, currently transferred to EC 1.6.5.2)] (DT-D) is a redox enzyme that catalyzes a two-electron reduction of quinone in the presence of NADH or NADPH, , and can reduce nitro and nitroso groups to amine groups even under aerobic conditions. , Importantly, DT-D from Bacillus stearothermophilus (EC 1.6.99.-) has a low molecular weight (30 kDa) and high thermal stability. , Because of its unique characteristics along with a high catalytic performance, DT-D has been used to obtain amplified signals via electrochemical-enzymatic (EN) redox cycling of the enzyme product produced by enzyme labels, such as ALP , and β-galactosidase . Nevertheless, DT-D has never been employed as an enzyme label in ELISAs.…”

“…10,14 Because of its unique characteristics along with a high catalytic performance, DT-D has been used to obtain amplified signals via electrochemicalenzymatic (EN) redox cycling 2 of the enzyme product produced by enzyme labels, such as ALP 15,16 and βgalactosidase. 17 Nevertheless, DT-D has never been employed as an enzyme label in ELISAs.…”