It is more difficult to obtain high signal‐to‐background ratios in biosensors using electrochemical reduction than using electrochemical oxidation. Here, we present a method for trypsin detection using electrochemical reduction‐based redox cycling. Electrochemical‐enzymatic (EN) redox cycling and electrochemical‐chemical (EC) redox cycling for trypsin detection were tested and compared. Trypsin cleaves a peptide bond in an electrochemically inactive p‐aminophenol (AP)‐conjugated oligopeptide, and this cleavage results in the release of electrochemically active AP, which is involved in EN and EC redox‐cycling reactions. Horseradish peroxidase and cytochrome c (Cyt c) were tested as redox enzymes for EN redox cycling involving a redox enzyme and H2O2. Cyt c was better than horseradish peroxidase, as its use resulted in lower background levels. The trypsin detection based on the EN redox cycling involving Cyt c and H2O2 (~50 ng/mL) exhibited lower detection limits than the detection based on EC redox cycling involving IO3− (~100 ng/mL), because of higher signal levels.

The duplex detection of both total and active enzyme concentrations without interferences at a single working electrode is challenging, especially when two different assays are combined. It is also challenging to obtain two different redox-cycling reactions without interference. Here, we present a simple but sensitive combined assay that is based on two redox-cycling reactions using two incubation periods and applied potentials at a single electrode. The assay combines an immunoassay for the determination of the total enzyme (total prostate-specific antigen, tPSA) concentration with a protease assay for the determination of the active enzyme (free PSA, fPSA) concentration. The immunoassay label and fPSA that are affinity-bound to the electrode are used for high sensitivity and specificity in the protease assay as well as the immunoassay. In the immunoassay, electrochemical-enzymatic (EN) redox cycling involving ferrocenemethanol is obtained at 0.1 V versus Ag/AgCl without incubation before the proteolytically released 4-amino-1-naphthol is generated. In the protease assay, EN redox cycling involving 4-amino-1-naphthol is obtained at 0.0 V after 30 min of incubation without ferrocenemethanol electro-oxidation. The detection procedure is almost the same as common electrochemical sandwich-type immunoassays, although the two different assays are combined. The duplex detection in buffer and serum is highly interference-free, specific, and sensitive. The detection limits for tPSA and fPSA are approximately 10 and 1 pg/mL, respectively.

Rapid and sensitive electrochemical detection of NADH, NADH‐dependent enzymes, and relevant metabolites requires a redox enzyme along with an electron mediator. Preferably, the redox enzyme should be immobilized on electrode rather than dissolved in solution. In this study, to simply immobilize a redox enzyme on electrode and maintain its enzymatic activity for long time, thermostable DT‐diaphorase (DT‐D) is immobilized on an avidin‐modified indium tin oxide (ITO) electrode. Electrochemical‐enzymatic (EN) redox cycling involving ITO electrode, ferrocenemethanol (FcMeOH), DT‐D, and NADH is employed for NADH detection. Electrochemical‐enzymatic‐enzymatic (ENN) redox cycling involving ITO electrode, FcMeOH, DT‐D, NAD+, lactate dehydrogenase (LDH), and lactate is employed for LDH detection. In both cases, a new combination of a redox enzyme and an electron mediator (DT‐D and FcMeOH) is used. The detection limits for NADH and LDH in artificial serum obtained without an incubation period are approximately 0.2 μM and 8 ng/mL, respectively. When an incubation period of 10 min is employed, the detection limit for LDH is approximately 5 ng/mL. Because the ENN redox cycling is very fast, the two detection limits are similar irrespective of incubation period. The enzymatic activity of DT‐D on ITO electrode is maintained for one month without deactivation.