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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.

Various methods have been developed for the detection of

Escherichia coli (E. coli) is recognized as an indicator of fecal bacterial contamination, and the induction of intracellular β-galactosidase (Gal) is commonly used for sensitive and selective E. coli detection. However, this method requires a permeabilization process to ensure that the Gal substrate and product are highly permeable through the E. coli membrane, making it unsuitable for simple point-of-care detection. Here, we report a sensitive electrochemical method for E. coli detection that does not require a permeabilization process. Intracellular Gal expression is increased via induction by isopropyl-β- d -thiogalactopyranoside (IPTG). 4-Methoxyphenyl-β- d -galactopyranoside (MPGP) and 4-methoxyphenol (MP) are used as the highly cell-permeable Gal substrate and product, respectively. Externally-added tyrosinase (Tyr) converts MP into electrochemically active 4-methoxycatechol, which is then electrochemically oxidized via electrochemical-chemical redox cycling involving an electrode, 4-methoxycatechol, and tris(2-carboxyethyl)phosphine. The presence of MPGP during the induction of intracellular Gal led to amplified MP production. The results of four different detection methods using three Gal substrates (MPGP, phenyl-β- d -galactopyranoside, and 4-aminophenyl-β- d -galactopyranoside) were compared. The detection limit for both drinking water and tap water spiked with E. coli, obtained following a 2-h-long IPTG treatment and 5-min-long Tyr reaction, was ∼2 × 103 colony-forming units (CFU)/mL, indicating that this method can be applied to monitoring E. coli contamination in water samples. Importantly, the present method does not require the electrode modification, affinity binding, washing, and filtration steps.

Rapid and sensitive mold detection is becoming increasingly important, especially in indoor environments. Common mold detection methods based on double-mediated electron transfer between an electrode and molds are not highly sensitive and reproducible, although they are rapid and simple. Here, we report a sensitive and reproducible detection method specific to Aspergillus niger ( A. niger), based on a single-mediator system combined with electrochemical-chemical (EC) redox cycling. Intracellular NAD(P)H-oxidizing enzymes in molds can convert electro-inactive hydroxy-nitro(so)arenes into electro-active hydroxy-aminoarenes. Since the membrane and wall of A. niger is well permeable to both a substrate (4-nitro-1-naphthol) and a reduced product (4-amino-1-naphthol) in tris buffer (pH 7.5) solution, the electrochemical signal is increased in the presence of A. niger due to two reactions: (i) enzymatic reduction of the substrate to the reduced product and (ii) electrochemical oxidation of the reduced product to an oxidized product. When a reducing agent (NADH) is present in the solution, the oxidized product is reduced back to the reduced product and then electrochemically reoxidized. This EC redox cycling significantly amplifies the electrochemical signal. Moreover, the background level is low and highly reproducible because the substrate and the reducing agent are electro-inactive at an applied potential of 0.20 V. The calculated detection limit for A. niger in a common double-mediator system consisting of Fe(CN)