Gorachand Dutta

Low-interference washing-free electrochemical immunosensor using glycerol-3-phosphate dehydrogenase as an enzyme label.

In washing-free electrochemical detection, various redox and reactive species cause significant interference. To minimize this interference, we report a washing-free electrochemical immunosensor using flavin adenine dinucleotide (FAD)-dependent glycerol-3-phosphate dehydrogenase (GPDH) and glycerol-3-phosphate (GP) as an enzyme label and its substrate, respectively, because the reaction of FAD-dependent dehydrogenases with dissolved O2 is slow and the level of GP preexisting in blood is low (<0.1 mM). A combination of a low electrocatalytic indium-tin oxide (ITO) electrode and fast electron-mediating Ru(NH3)6(3+) is employed to obtain a high signal-to-background ratio via proximity-dependent electron mediation of Ru(NH3)6(3+) between the ITO electrode and the GPDH label. Electrochemical oxidation of GPDH-generated Ru(NH3)6(2+) is performed at 0.05 V vs Ag/AgCl, at which point the electrochemical interference is very low. When a washing-free immunosensor is applied to cardiac troponin I detection in human serum, the calculated detection limit is approximately 10 pg/mL, indicating that the immunosensor is very sensitive in spite of the use of washing-free detection with a short detection period (10 min for incubation and 100 s for electrochemical measurement). The low-interference washing-free electrochemical immunosensor shows good promise for fast and simple point-of-care testing.

Washing-Free Heterogeneous Immunosensor Using Proximity-Dependent Electron Mediation between an Enzyme Label and an Electrode

Washing processes, essential in most heterogeneous labeled assays, have been a big hurdle in simplifying the detection procedure and reducing assay time. Nevertheless, less attention has been paid to washing-free heterogeneous labeled assays. We report a purely washing-free immunosensor that allows fast, sensitive, and single-step detection of prostate-specific antigen in serum with low interference. Proximity-dependent electron mediation of ferrocenemethanol (Fc) between an indium-tin oxide (ITO) electrode and a glucose-oxidase (GOx) label allows us to discriminate between a bound and an unbound label: a bound label offers faster electron mediation than an unbound one. The electrooxidation of Fc at a low applied potential (0.13 V vs Ag/AgCl) and a low electrocatalytic ITO electrode and the oxidation of l-ascorbic acid by l-ascorbate oxidase minimize the effect of the interfering species. With a high concentration of glucose (200 mM), the signal and background levels are hardly dependent on the glucose-concentration variation in the sample. The washing-free immunosensor can detect a concentration of ca. 1 pg/mL for mouse IgG in phosphate-buffered saline and a concentration of ca. 10 pg/mL for prostate-specific antigen spiked in female serum after an incubation period of 10 min. The concentrations measured with actual clinical serum samples are in good agreement with the concentrations measured with a commercial instrument, which renders the washing-free heterogeneous immunosensor appealing for practical use.