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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.

Propagating cascade reactions based on two proteases are promising for obtaining high signal amplification. However, in many cases, biosensors that use cascade reactions do not have low detection limits because of the inherent slowness of proteolytic reactions. Here, we report a sensitive electrochemical immunosensor using a high-signal-amplification method that combines a propagating cascade reaction and a redox cycling reaction. The cascade reaction uses ecarin and prothrombin: the ecarin label proteolytically converts inactive prothrombin into active thrombin, which then proteolytically liberates electroactive p-aminophenol (AP) from an AP-conjugated peptide. The liberated AP is electrochemically oxidized to p-benzoquinone imine (QI), regenerated by the reduction of QI by NADH, and then electrochemically reoxidized. This electrochemical-chemical (EC) redox cycling reaction significantly increases the electrochemical signal. The developed immunosensor is also compared with an immunosensor that uses only a propagating cascade reaction and an immunosensor that uses a single proteolytic reaction and an EC redox cycling reaction. The detection limits for thyroid-stimulating hormone (TSH) obtained using the three immunosensors are 3 pg/mL, 2 ng/mL, and 4 ng/mL, respectively, indicating that the newly developed immunosensor is more sensitive than the other two. The measured concentrations of TSH in clinical serum are found to agree well with those determined using a commercial instrument.

Recombinase polymerase amplification (RPA) is considered one of the best amplification methods for realizing a miniaturized diagnostic instrument; however, it is notably challenging to obtain low detection limits in solid-phase RPA. To overcome these difficulties, we combined solid-phase RPA with electrochemical detection and used a new concentration combination of three primers (surface-bound forward primer, solution reverse primer, and an extremely low concentration of solution forward primer). When solid-phase RPA was performed on an indium tin oxide (ITO) electrode modified with a surface-bound forward primer in a solution containing a biotin-terminated solution reverse primer, an extremely low concentration of a solution forward primer, and a template DNA or genomic DNA for a target gene of hepatitis B virus (HBV), amplification occurred mainly in solution until all the solution forward primers were consumed. Subsequently, DNA amplicons produced in solution participated in solid-phase amplification involving surface-bound forward primer and solution reverse primer. Afterward, neutravidin-conjugated DT-diaphorase (DT-D) was attached to a biotin-terminated DNA amplicon on the ITO electrode. Finally, chronocoulometric charges were measured using electrochemical-enzymatic redox cycling involving the ITO electrode, 1,4-naphthoquinone, DT-D, and reduced β-nicotinamide adenine dinucleotide. The detection limit for HBV was measured using microfabricated electrodes and was found to be approximately 0.1 fM. This proposed method demonstrated better amplification efficiency for HBV genomic DNA than solid-phase RPA without using additional solution primer and asymmetric solid-phase RPA.

Metal nanoparticle surfaces are used for peroxidase- and oxidase-like nanozymes but not for esterase-like nanozymes. It is challenging to obtain rapid catalytic hydrolysis on a metal surface and even more so without a catalytically labile substrate. Here, we report that metal nanoparticle surfaces rapidly catalyze non-redox ester hydrolysis in the presence of redox H3 N-BH3 (AB). Metal hydrides are readily generated on a Pt nanoparticle (PtNP) from AB, and as a result the PtNP becomes electron-rich, which might assist nucleophilic attack of H2 O on the carbonyl group of an ester. The nanozyme system based on PtNP, AB, and 4-aminonaphthalene-1-yl acetate provides an electrochemical signal-to-background ratio much higher than natural enzymes, due to the rapid ester hydrolysis and redox cycling involving the hydrolysis product. The nanozyme system is applied in a sensitive electrochemical immunosensor for thyroid-stimulating hormone detection. The calculated detection limit is approximately 0.3 pg mL-1 , which indicates the high sensitivity of the immunosensor using the PtNP nanozyme.

Spectroscopic and microscopic techniques are not suitable for the rapid monitoring of time-dependent dissolution behavior (particularly, pinhole changes) of a layer deposited by atomic layer deposition (ALD). Here, we present a simple electrochemical method that provides information on the dissolution mechanism including pinhole generation and thickness change. Because indium tin oxide (ITO) electrodes exhibit flat capacitive currents and good (electro)chemical stability, they are selected as ideal underlying substrates for the electrochemical monitoring of the ALD layers even under harsh conditions. Two ALD layers (Al2O3 and TiO2 layers) that exhibit opposite dissolution behaviors are chosen as model layers because the as-deposited Al2O3 layers are pinhole-free but unstable in aqueous solutions, whereas the as-deposited TiO2 layers are not pinhole-free but stable in aqueous solutions. The combination of capacitive current level (in an electrolyte solution containing no redox-active species) and electrochemical blocking behavior (in an electrolyte solution containing a redox-active species such as Ru(NH3)63+ and ferrocenemethanol) obtained from cyclic voltammograms enables us to verify whether the dissolution of an ALD layer occurs, to evaluate the dissolution rate, and to identify the plausible dissolution mechanism. The electrochemical results reveal that the Al2O3 layers are dissolved in biological buffers, along with pinhole generation, and that the TiO2 layers are stable with no pinhole generation. The difference in electrochemical blocking behavior between Ru(NH3)63+ and ferrocenemethanol provides information on the approximate size of the pinholes. The present method is appealing for practical use because even an ALD layer with a thickness of only a few nanometers can be tested to monitor the dissolution behavior and because any ALD layer that can be readily deposited on ITO electrodes can be easily examined using this method.