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A dicarboxylate-containing and fully substituted ferrocene compound exhibits rapid dissolvability, high solubility, good stability, and moderate along with its high electron-mediation rate, and it was applied to mediated glucose detection.

Direct electron transfer (DET) between a redox label and an electrode has been used for sensitive and selective sandwich-type detection without a wash step. However, applying DET is still highly challenging in protein detection, and a single redox label per probe is insufficient to obtain a high electrochemical signal. Here, we report a wash-free, sandwich-type detection of thrombin using DET and catalytic signal amplification of multiple redox labels. The detection scheme is based on (i) the redox label-catalyzed oxidation of a reductant, (ii) the conjugation of multiple redox labels per probe using a poly-linker, (iii) the low nonspecific adsorption of the conjugated poly-linker due to uncharged, reduced redox labels, and (iv) a facile DET using long, flexible poly-linker and spacer DNA. Amine-reactive phenazine ethosulfate and NADH were used as the redox label and reductant, respectively. N

Indirect detection of Porphyromonas gingivalis in saliva, based on proteolytic cleavage by an Arg-specific gingipain (Arg-gingipain), has traditionally been used for simple, initial diagnosis of periodontitis. To accurately detect P. gingivalis using a point-of-care format, development of a simple biosensor that can measure the exact concentration of P. gingivalis is required. However, electrochemical detection in saliva is challenging due to the presence of various interfering electroactive species in different concentrations. Here, we report a washing- and separation-free electrochemical biosensor for sensitive detection of P. gingivalis in saliva. Glycine-proline-arginine conjugated with 4-aminophenol (AP) was used as an electrochemical substrate for a trypsin-like Arg-gingipain, and glycylglycine was used to increase the Arg-gingipain activity. The electrochemical signal of AP was increased using electrochemical-chemical (EC) redox cycling involving an electrode, AP, and tris(2-carboxyethyl)phosphine, and the electrochemical charge signal was corrected using the initial charge obtained before a 15 min incubation period. The EC redox cycling combined with the matrix-corrected signal facilitated a high and reproducible signal without requiring washing and separation steps. The proteolytic cleavage of the electrochemical substrate was specific to P. gingivalis. The calculated detection limit for P. gingivalis in artificial saliva was 5 × 105 colony-forming units/mL, and the concentration of P. gingivalis in human saliva could be measured. The developed biosensor can be used as an initial diagnosis method to distinguish between healthy people and patients with periodontal diseases.

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.

Bienzymatic systems are often used with electrochemical affinity biosensors to achieve high signal levels and/or low background levels. It is important to select two enzymes whose reactions do not exhibit mutual interference but have similar optimal conditions. Here, we report a sensitive electrochemical immunosensor based on a bienzymatic system consisting of β-galactosidase (Gal, a hydrolase enzyme) and flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH, a redox enzyme). Both enzymes showed high activities at neutral pH, the reactions catalyzed by them did not exhibit mutual interference, and the electrochemical-enzymatic redox cycling based on FAD-GDH coupled with enzymatic amplification by Gal enabled high signal amplification. Among the three amino-hydroxy-naphthalenes and 4-aminophenol (potential Gal products), 4-amino-1-naphthol showed the highest signal amplification. Glucose, as an electro-inactive, stable reducing agent for redox cycling, helped in achieving low background levels. Our bienzymatic system could detect parathyroid hormone at a detection limit of ∼0.2 pg mL-1, implying that it can be used for highly sensitive electrochemical detection of parathyroid hormone and other biomarkers in human serum.

Nanostructured materials offer the potential to drive future developments and applications of electrochemical devices, but are underutilized because their nanoscale cavities can impose mass transfer limitations that constrain electrochemical signal generation. Here, we report a new signal-generating mechanism that employs a molecular redox capacitor to enable nanostructured electrodes to amplify electrochemical signals even without an enhanced reactant mass transfer. The surface-tethered molecular redox capacitor engages diffusible reactants and products in redox-cycling reactions with the electrode. Such redox-cycling reactions are facilitated by the nanostructure that increases the probabilities of both reactant-electrode and product-redox-capacitor encounters (i.e., the nanoconfinement effect), resulting in substantial signal amplification. Using redox-capacitor-tethered Au nanopillar electrodes, we demonstrate improved sensitivity for measuring pyocyanin (bacterial metabolite). This study paves a new way of using nanostructured materials in electrochemical applications by engineering the reaction pathway within the nanoscale cavities of the materials.

The high and selective catalytic activities of tyrosinase (Tyr) have frequently led to its application in sensitive biosensors. However, in affinity-based biosensors, the use of Tyr as a catalytic label is less common compared to horseradish peroxidase and alkaline phosphatase owing to the fact that phenolic Tyr substrates have yet to be investigated in detail. Herein, four phenolic compounds that have lower formal potentials than phenol were examined for their applicability as Tyr substrates, and three reducing agents were examined as potential strong reducing agents for electrochemical-chemical (EC) redox cycling involving an electrode, a Tyr product, and a reducing agent. The combination of 4-methoxyphenol (MP) and ammonia-borane (AB) allows for (i) a high electrochemical signal level owing to rapid EC redox cycling and (ii) a low electrochemical background level owing to the slow oxidation of AB at a low applied potential and no reaction between MP and AB. When this combination was applied to an electrochemical immunosensor for parathyroid hormone (PTH) detection, a detection limit of 2 pg/mL was obtained. This detection limit is significantly lower than that obtained when a combination of phenol and AB was employed (300 pg/mL). It was also found that the developed immunosensor works well in PTH detection in clinical serum samples. This new phenolic substrate could therefore pave the way for Tyr to be more commonly used as a catalytic label in affinity-based biosensors.

Various methods have been developed for the detection of

To obtain a sensitive, wash-free photoelectrochemical biosensor based on electron mediation between an electrode and a photoredox catalyst (PC) label, unavoidable O

The commonly required properties of diffusive electron mediators for point-of-care testing are rapid dissolubility, high stability, and moderate formal potential in aqueous solutions. Inspired by nature, various quinone-containing electron mediators have been developed; however, satisfying all these requirements remains a challenge. Herein, a strategic design toward quinones incorporating sulfonated thioether and nitrogen-containing heteroarene moieties as solubilizing, stabilizing, and formal potential-modulating groups is reported. A systematic investigation reveals that di(thioether sulfonate)-substituted quinoline-1,4-dione (QLS) and quinoxaline-1,4-dione (QXS) display water solubilities of ≈1 m and are rapidly dissoluble. By finely balancing the electron-donating effect of the thioethers and the electron-withdrawing effect of the nitrogen atom, formal potentials suitable for electrochemical biosensors are achieved with QLS and QXS (-0.15 and -0.09 V vs Ag/AgCl, respectively, at pH 7.4). QLS is stable for >1 d in PBS (pH 7.4) and for 1 h in tris buffer (pH 9.0), which is sufficient for point-of-care testing. Furthermore, QLS, with its high electron mediation ability, is successfully used in biosensors for sensitive detection of glucose and parathyroid hormone, demonstrating detection limits of ≈0.3 × 10-3 m and ≈2 pg mL-1 , respectively. This strategy produces organic electron mediators exhibiting rapid dissolution and high stability, and will find broad application beyond quinone-based biosensors.

Enzyme-induced seedless Ag deposition is useful for selective Ag deposition and subsequent electrochemical Ag oxidation; however, a washing step is required after the deposition and before the electrochemical oxidation as the enzyme substrate can be oxidized during the electrochemical oxidation. Here, we report a fast Ag deposition method using a redox enzyme and quinone substrate that does not require a washing step. We found that the quinone substrate is reduced by a redox enzyme label, which is later oxidized to its original form via the reduction of Ag+ to Ag. Moreover, the quinone substrate is not electrochemically oxidized during the electrochemical Ag oxidation. We selected one diaphorase and 1,4-naphthoquinone from among seven redox enzymes (four diaphorases and three glucose-oxidizing enzymes) and six quinones, respectively. We applied this Ag deposition method for the detection of thyroid-stimulating hormone (TSH) over a dynamic range from 100 fg/mL to 100 ng/mL and found that TSH could be detected at concentrations as low as approximately 100 fg/mL in artificial serum. Therefore, the Ag deposition strategy developed in this study exhibits promising potential for ultrasensitive clinical applications.

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.

DT-diaphorase (DT-D) is known to mainly catalyze the two-electron reduction of quinones and nitro(so) compounds. Detection of Gram-negative bacterial outer membrane vesicles (OMVs) that contain pyrogenic lipopolysaccharides (LPSs, also called endotoxins) is required for evaluating the toxic effects of analytical samples. Here, we report that DT-D has a high dephosphorylation activity: DT-D catalyzes reductive dephosphorylation of a phosphate-containing substrate in the presence of NADH. We also report that sensitive and simple OMV detection is possible with a sandwich-type electrochemical immunosensor using DT-D and two identical LPS-binding antibodies as a catalytic label and two sandwich probes, respectively. The absorbance change in a solution containing 4-nitrophenyl phosphate indicates that dephosphorylation occurs in the presence of both DT-D and NADH. Among the three phosphate-containing substrates [4-aminophenyl phosphate, ascorbic acid phosphate, and 1-amino-2-naphthyl phosphate (ANP)] that can be converted into electrochemically active products after dephosphorylation, ANP shows the highest electrochemical signal-to-background ratio, because (i) the dephosphorylation of ANP by DT-D is fast, (ii) the electrochemical oxidation of the dephosphorylated product (1-amino-2-naphthol, AN) is rapid, even at a bare indium-tin oxide electrode, and (iii) two redox cycling processes significantly increase the electrochemical signal. The two redox cycling processes include an electrochemical-enzymatic redox cycling and an electrochemical-chemical redox cycling. The electrochemical signal in a neutral buffer (tris buffer, pH 7.5) is comparable to that in a basic buffer (tris buffer, pH 9.5). When the immunosensor is applied to the detection of OMV from Escherichia coli, the detection limit is found to be 8 ng/mL. This detection strategy is highly promising for the detection of biomaterials, including other extracellular vesicles.

Proteases play crucial roles in biological systems, including digestion, catabolism, and cell signaling. Indirect detection of Porphyromonas gingivalis in saliva, based on proteolytic cleavage by an Arg-specific gingipain (Arg-gingipain) protease, has traditionally been used for simple, initial diagnosis of periodontitis. To accurately detect P. gingivalis using a point-of-care format, development of a simple biosensor that can measure the exact concentration of P. gingivalis is required. However, electrochemical detection in saliva is challenging due to the presence of various interfering electroactive species in different concentrations. Here, we report a washing- and separation-free electrochemical biosensor for sensitive detection of P. gingivalis in saliva. Glycine–proline–arginine conjugated with 4-aminophenol (AP) was used as an electrochemical substrate for a trypsin-like Arg-gingipain, and glycylglycine was used to increase the Arg-gingipain activity. The electrochemical signal of AP was increased using electrochemical–chemical (EC) redox cycling involving an electrode, AP, and tris(2-carboxyethyl)phosphine, and the electrochemical charge signal was corrected using the initial charge obtained before a 15 min incubation period. The EC redox cycling combined with the matrix-corrected signal facilitated a high and reproducible signal without requiring washing and separation steps. The proteolytic cleavage of the electrochemical substrate was specific to P. gingivalis. The calculated detection limit for P. gingivalis in artificial saliva was 5 × 105 colony-forming units/mL, and the concentration of P. gingivalis in human saliva could be measured. The developed biosensor can be used as an initial diagnosis method to distinguish between healthy people and patients with periodontal diseases. References S. Park, K. Park, H. S. Na, J. Jung, H. Yang, Anal. Chem. 93, 5644 (2021) S. Park, J. Shin, J. Kwon, W. Lee, J. Kim, G. Kim, J. M. Joo, H. Yang, ACS Sens. 6, 1305 (2021) S. Park, G. Kim, J. Seo, H. Yang, Anal. Chem. 88, 11995 (2016) S. Park, Y. M. Shin, J. Seo, J.-J. Song, H. Yang, Analyst 141, 2481 (2016) S. Park, Y. Shin, J.-J. Song, H. Yang, Biosens. Bioelectron. 72, 211 (2015) S. Park, H. Yang, Analyst, 139, 4051 (2014)

Aqueous electron mediators for point-of-care testing require a variety of properties, such as rapid dissolubility, high stability, and moderate formal potential (near 0 V). Using the redox-active properties of quinone derivatives, various quinone-containing electron mediators have been developed for electrochemical sensors. However, most quinone derivatives exhibit very low solubility in aqueous solutions and instability in the presence of nucleophilic species. Herein, we report a strategic design for quinones incorporating sulfonated thioether and nitrogen-containing heteroarene moieties as solubilizing, stabilizing, and formal potential-modulating groups. Systematic investigations found that di(thioether sulfonate)-substituted quinoline-1,4-dione (QLS) and quinoxaline-1,4-dione (QXS) displayed high water solubility and rapid dissolubility. By finely balancing the electron-donating effect of the thioethers and the electron-withdrawing effect of the nitrogen atom, formal potentials suitable for electrochemical biosensors are achieved with QLS and QXS (−0.15 and −0.09 V vs Ag/AgCl, respectively, at pH 7.4). QLS is stable for >1 d in PBS (pH 7.4) and for 1 h in tris buffer (pH 9.0), which is sufficient for point-of-care testing. Furthermore, QLS, with its high electron mediation ability, is successfully used in biosensors for sensitive detection of glucose and parathyroid hormone. This strategy provides organic electron mediators that exhibit rapid dissolution and high stability and will find broad applications beyond quinone-based biosensors. Figure 1

Indirect detection of

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.

Horseradish peroxidase (HRP)-based electrochemical immunoassays are considered promising techniques for point-of-care clinical diagnostics, but the necessary addition of unstable H2O2 in the enzymatic system may hinder their practical application. Although glucose oxidase (GOx) has been widely explored for in situ generation of H2O2 in HRP-based immunoassay, the GOx-catalyzed reduction of oxidized peroxidase substrate may limit the immunosensing performance. Here, we report a sensitive electrochemical immunosensor based on a choline oxidase (ChOx)-HRP cascade reaction. In this design, ChOx catalyzes the oxidation of choline, during which H2O2 is generated in situ and thus oxidizes acetaminophen (APAP) in the presence of HRP. The electrochemical behavior of APAP in the ChOx-HRP cascade was compared with that of the commonly used GOx-HRP cascade, which confirmed that ChOx could be a superior preceding enzyme for sensitive immunoassay based on the bienzymatic cascade. The developed ChOx-HRP cascade was also further explored for a sandwich-type electrochemical immunoassay of parathyroid hormone in artificial and clinical serum. The calculated detection limit was ~3 pg/mL, indicating that the ChOx-HRP cascade is especially promising for highly sensitive electrochemical immunoassays when APAP is used as the peroxidase substrate.

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.

Distance-dependent electromagnetic radiation and electron transfer have been commonly employed in washing-free fluorescence and electrochemical bioassays, respectively. In this study, we combined the two distance-dependent phenomena for sensitive washing-free DNA detection. A distance-dependent surface plasmonic field induces rapid photoredox catalysis of surface-bound catalytic labels, and distance-dependent mediated electron transfer allows for rapid electron transfer from the surface-bound labels to the electrode. An optimal system consists of a chemically reversible acceptor (Ru(NH3 )6 3+ ), a chemically reversible photoredox catalyst (eosin Y), and a chemically irreversible donor (triethanolamine). Side reactions with O2 do not significantly decrease the efficiency of photoredox catalysis. Energy transfer quenching between the electrode and the label can be lowered by increasing the distance between them. Washing-free DNA detection had a detection limit of approximately 0.3 nm in buffer and 0.4 nm in serum without a washing step.

Catalytic precipitation and subsequent electrochemical oxidation or reduction of a redox-active precipitate has been widely used in electrochemical biosensors. However, such biosensors often do not allow for low detection limits due to a low rate of precipitation, nonspecific precipitation, loose binding of the precipitate to the electrode surface, and insulating behavior of the precipitate within a normal potential window. Here, we report an ultrasensitive electrochemical immunosensor for parathyroid hormone (PTH) detection based on DT-diaphorase (DT-D)-catalyzed formation of an organic precipitate and electrochemical oxidation of the precipitate. In the present study we found that DT-D can be used as a catalytic label in precipitation-based affinity biosensors because DT-D catalyzes fast reduction of 3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to MTT-formazan precipitate; the MTT reduction does not occur in the absence of DT-D; and a high electrochemical signal is obtained at low potentials during electrodissolution of MTT-formazan precipitate. The immunosensor is fabricated using a silane copolymer-modified ITO electrode surface that is suitable for both efficient and strong adsorption of MTT-formazan precipitate. When the enzymatic MTT-formazan precipitation and subsequent MTT-formazan electrodissolution is applied to a sandwich-type immunosensor, PTH can be detected over a wide range of concentrations with a very low detection limit (∼1 pg/mL) in artificial serum. The measured concentrations of PTH in clinical serum samples showed high similarity with those obtained using a commercial instrument.

Simple and sensitive competitive immunosensors for small molecules are difficult to obtain, especially in serum containing numerous interfering species (ISs) with different concentrations. Herein, we report a washing-free and sensitive (competitive) displacement immunosensor for cortisol in human serum, based on electron mediation of Os(bpy)2Cl2 between an electrode and a redox label [oxygen-insensitive diaphorase (DI)] (i.e., electrochemical–enzymatic redox cycling). The anticortisol IgG–DI conjugate bound to a cortisol-immobilized electrode is displaced by competitive binding of cortisol in serum and diffuses away from the electrode during incubation; therefore, the concentration of the displaced conjugate near the electrode becomes very low, even without washing. Electrochemically interfering ascorbic acid is converted to a redox-inactive species by ascorbate oxidase during incubation. The remaining bound conjugate mainly contributes to electrochemical currents. Compared with ferrocene methanol, Fe(CN)...

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.

Direct electron transfer between a redox label and an electrode requires a short working distance (

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.

Biosensors for ultrasensitive point-of-care testing require dried reagents with long-term stability and a high signal-to-background ratio. Although ortho-substituted diaromatic dihydroxy and aminohydroxy compounds undergo fast redox reactions, they are not used as electrochemical signaling species because they are readily oxidized and polymerized by dissolved oxygen. In this report, stable, solid 1-amino-2-naphthyl phosphate (1A2N-P) and ammonia-borane (H3N-BH3) are respectively employed as a substrate for alkaline phosphatase (ALP) and a reductant for electrochemical-chemical (EC) redox cycling. ALP converts 1A2N-P to 1-amino-2-naphthol (1A2N), which is then employed in EC redox cycling using H3N-BH3. The oxidation and polymerization of 1A2N by dissolved oxygen is significantly prevented in the presence of H3N-BH3. The electrochemical measurement is performed without modification of indium–tin oxide (ITO) electrodes with electrocatalytic materials. For comparison, nine aromatic dihydroxy and aminohydroxy...

Both high sensitivity and high specificity are crucial for detection of miRNAs that have emerged as important clinical biomarkers. Just Another Zinc finger proteins (JAZ, ZNF346) bind preferably (but nonsequence-specifically) to DNA-RNA hybrids over single-stranded RNAs, single-stranded DNAs, and double-stranded DNAs. We present an ultrasensitive and highly specific electrochemical method for miRNA-21 detection based on the selective binding of JAZ to the DNA-RNA hybrid formed between a DNA capture probe and a target miRNA-21. This enables us to use chemically stable DNA as a capture probe instead of RNA as well as to apply a standard sandwich-type assay format to miRNA detection. High signal amplification is obtained by (i) enzymatic amplification by alkaline phosphatase (ALP) coupled with (ii) electrochemical-chemical-chemical (ECC) redox cycling involving an ALP product (hydroquinone). Low nonspecific adsorption of ALP-conjugated JAZ is obtained using a polymeric self-assembled-monolayer-modified and casein-treated indium-tin oxide electrode. The detection method can discriminate between target miRNA-21 and nontarget nucleic acids (DNA-DNA hybrid, single-stranded DNA, miRNA-125b, miRNA-155, single-base mismatched miRNA, and three-base mismatched miRNA). The detection limits for miRNA-21 in buffer and 10-fold diluted serum are approximately 2 and 30 fM, respectively, indicating that the detection method is ultrasensitive. This detection method can be readily extended to multiplex detection of miRNAs with only one ALP-conjugated JAZ probe due to its nonsequence-specific binding character. We also believe that the method could offer a promising solution for point-of-care testing of miRNAs in body fluids.

In order to detect a protease that cleaves the peptide bond between two specific amino acids via fluorescence, a synthetic peptide modified with two labels (a donor and an acceptor) for fluorescence resonance energy transfer (FRET) is commonly used. However, the preparation and optimization of a peptide for the sensitive and selective detection of a target protease are time-consuming. In this study, we report a simple method for fluorescence protease detection using a readily prepared, one label-based peptide. The fluorescence detection of botulinum neurotoxin type E light chain (BoNT/E-LC) is based on a two-step proteolytic cleavage that involves the use of BoNT/E-LC and an externally supplemented L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves the specific peptide bond between arginine and isoleucine within C-terminally 7-amino-4-methylcoumarin (AMC)-labeled oligopeptide, leaving fragmented isoleucine–AMC. Subsequently, LAP cleaves the peptide bond between isoleucine and AMC, liberating fluorescent AMC. This method does not require two label-modified peptides. Capping the oligopeptide with the D-form of tyrosine does not result in better performance in terms of detection limit, although a higher concentration of LAP can be used. The detection limit for BoNT/E-LC in both phosphate-buffered saline and commercial bottled water is 2 ng mL−1 for an incubation period of 1 h. The fluorescence detection is selective for BoNT/E-LC among the four tested BoNTs. Fluorescence detection using one label can be readily applied to any type of proteases without using FRET.

Polyenzymes have been commonly employed as catalytic labels to obtain high signal amplification in immunoassays, but they are mainly constructed using horseradish peroxidase that requires the use of H2O2, whose storage is difficult. Here, we present a new polyenzyme label based on diaphorase (DI), a dehydrogenase redox enzyme with high catalytic activity and long-term stability. The polyenzyme label is constructed using a complex of a biotinylated DI and an avidin analog. Among the five commercially available DIs, the one that shows the highest signal amplification using electrochemical-enzymatic redox cycling involving an electron mediator, a DI, and NADH is chosen. Among the three avidin analogs (avidin, neutravidin, and streptavidin), neutravidin provides the lowest nonspecific binding of the polyenzyme label. The polyenzyme label shows higher signal amplification and lower nonspecific binding than a single enzyme label of DI. Comparative studies reveal that the detection limit for the detection of interleukin-8 (IL-8) in saliva using the polyenzyme label (∼1 pg/mL) is one order of magnitude lower than that using a single enzyme label (∼10 pg/mL). IL-8 concentrations measured in clinical saliva samples agree well with those measured using a conventional microplate immunoassay. Thus, the polyenzyme label can be applied for the noninvasive detection of other clinically relevant proteins in saliva.

In the electrochemical detection method for pesticides that measures their inhibitory effects on acetylcholinesterases (AChEs), the direct electrooxidation of the enzyme product (thiocholine, SCh) is slow at conventional electrodes. To overcome this limitation, an electron mediator is required to lower the applied potential and facilitate the transfer of electrons between the enzyme product and electrode. In this study, [Ru(NH3)5py]3+ is introduced as an electron mediator in inhibition-based pesticide detection. To obtain a better signal-to-background ratio, [Ru(NH3)5py]3+, which undergoes a fast outer-sphere reaction, is combined with low-electrocatalytic indium-tin-oxide (ITO) electrodes at which many interfering species undergo slow redox reactions. AChE is immobilized onto an avidin-modified ITO electrode via the direct adsorption of avidin onto ITO followed by the biospecific binding of biotinylated AChE to the avidin. SCh is generated from acetylthiocholine by AChE. Subsequently, SCh converts [Ru(NH3)5py]3+ to [Ru(NH3)5py]2+, which is then oxidized at the ITO electrode. This procedure allows the sensitive detection of carbaryl at a low applied potential of 0.15 V vs Ag/AgCl. The calculated detection limit for carbaryl is approximately 0.3 pM. This simple and sensitive pesticide sensor is thus very promising and should be extendable to the onsite environmental monitoring of other pesticides.

Protease sensors for point-of-care testing (POCT) require simple operation, a detection period of less than 20 minutes, and a detection limit of less than 1 ng mL(-1). However, it is difficult to meet these requirements with protease sensors that are based on proteolytic cleavage. This paper reports a highly reproducible protease sensor that allows the sensitive and simple electrochemical detection of the botulinum neurotoxin type E light chain (BoNT/E-LC), which is obtained using (i) low nonspecific adsorption, (ii) high signal-to-background ratio, and (iii) one-step solution treatment. The BoNT/E-LC detection is based on two-step proteolytic cleavage using BoNT/E-LC (endopeptidase) and l-leucine-aminopeptidase (LAP, exopeptidase). Indium-tin oxide (ITO) electrodes are modified partially with reduced graphene oxide (rGO) to increase their electrocatalytic activities. Avidin is then adsorbed on the electrodes to minimize the nonspecific adsorption of proteases. Low nonspecific adsorption allows a highly reproducible sensor response. Electrochemical-chemical (EC) redox cycling involving p-aminophenol (AP) and dithiothreitol (DTT) is performed to obtain a high signal-to-background ratio. After adding a C-terminally AP-labeled oligopeptide, DTT, and LAP simultaneously to a sample solution, no further treatment of the solution is necessary during detection. The detection limits of BoNT/E-LC in phosphate-buffered saline are 0.1 ng mL(-1) for an incubation period of 15 min and 5 fg mL(-1) for an incubation period of 4 h. The detection limit in commercial bottled water is 1 ng mL(-1) for an incubation period of 15 min. The developed sensor is selective to BoNT/E-LC among the four types of BoNTs tested. These results indicate that the protease sensor meets the requirements for POCT.

The most common enzyme labels in biosensors are alkaline phosphatase (ALP) and horseradish peroxidase (HRP), which, however, have some limitations for use in electrochemical immunosensors. DT-diaphorase (DT-D) is a redox enzyme that catalyzes a two-electron reduction of quinone in the presence of NADH or NADPH. Importantly, DT-D from Bacillus stearothermophilus (EC 1.6.99.-) has a low molecular weight (30 kDa) and high thermal and long-term stability. Nevertheless, DT-D has never been employed as a catalytic label in biosensors. In this presentation, we report that DT-D can be used as a bifunctional enzyme label for rapid and sensitive electrochemical immunosensors: DT-D can convert an electrochemically inactive substrate into an electrochemically active product and DT-D can participate in electrochemical-chemical (EC) and electrochemical-enzymatic (EN) redox cycling induced by the product. We found that DT-D has high catalytic activities for nitroso reduction, phosphoester hydrolysis, and carboxyl ester hydrolysis as well as quinone reduction in the presence of NADH. 4-Nitroso-1-naphthol, 1-amino-2-naphthyl phosphate, and 4-aminonaphthalene-1-yl acetate are used as electrochemically inactive substrates for corresponding enzymatic reactions, which are converted into electrochemically active aminohydroxy-naphthalenes. This combination of DT-D and the substrates allows for high electrochemical signal-to-background ratios, because (i) the catalytic reaction by DT-D is fast, (ii) the electrochemical oxidation of the aminohydroxy-naphthalenes is rapid, even at an indium-tin oxide electrode, and (iii) two redox cycling processes significantly increase the electrochemical signal. The electrochemical immunosensor using DT-D and 4-nitroso-1-naphthol detects parathyroid hormone with a low detection limit of 2 pg/mL, the immunosensor using DT-D and 1-amino-2-naphthyl phosphate detects outer membrane vesicles from Escherichia coli with a detection limit of 8 ng/mL, and the immunosensor using DT-D and 4-aminonaphthalene-1-yl acetate detects thyroid-stimulating hormone with a detection limit of ~2 pg/mL. DT-D may be used as a catalytic label in sensitive and stable bioassays instead of common ALP and HRP. References 1. P. Nandhakumar, A. M. Ichzan, N.-S. Lee, Y. H. Yoon, S. Ma, S. Kim, H. Yang, ACS Sens. in press. 2. A. Bhatia, P. Nandhakumar, G. Kim, J. Kim, N.-S. Lee, Y. H. Yoon, H. Yang, ACS Sens. 4, 1641 (2019) 3. A. M. Ichzan, S. Lee, C. S. Fang, P. Nandhakumar, H. Ha, J. M. Joo, K. Kim, H. Yang, Anal. Chem. 91, 4680 (2019) 4. P. Nandhakumar, A.-M. J. Haque, N.-S. Lee, Y. H. Yoon, H. Yang, Anal. Chem. 90, 10982 (2018) 5. C. Kang, J. Kang, N.-S. Lee, Y. H. Yoon, H. Yang, Anal. Chem. 89, 7974 (2017)

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.

In horseradish peroxidase (HRP)-based electrochemical immunosensing, an appropriate HRP substrate needs to be chosen to obtain a high electrochemical signal-to-background ratio. This is limited by the unwanted electrochemical reduction of H2O2, oxidation of the substrate, and the slow electrochemical reduction of the product. Herein, we report acetaminophen (AMP) as a new HRP substrate that allows for highly sensitive immunosensing. Electrochemical behavior and immunosensing performance using AMP are compared with those using the most popular HRP substrate, hydroquinone (HQ). To maintain a high electrocatalytic activity even at an electrode modified with an immunosensing layer, an indium tin oxide electrode partially modified with reduced graphene oxide is employed. AMP allows for a higher signal-to-background ratio than HQ, because the formal potential of AMP is 0.28 V higher than that of HQ and the redox reaction of AMP is as reversible as that of HQ, resulting in a lower detection limit in a sandwich-type immunoassay using AMP for thyroid-stimulating hormone detection. The calculated detection limit is ~0.2 pg/mL. The use of AMP as an HRP substrate is especially promising for highly sensitive electrochemical immunoassays.