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 electronmediating Ru(NH3)63+ is employed to obtain a high signal-to-background ratio via proximitydependent electron mediation of Ru(NH3)63+ between the ITO electrode and the GPDH label. Electrochemical oxidation of GPDH-generated Ru(NH3)62+ 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. [ABSTRACT FROM AUTHOR]