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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

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.

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.

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

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.

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.

Due to the increase in motor vehicle accidents, there is a growing need for high-performance car crash detection systems. The authors of this research propose a car crash detection system that uses both video data and audio data from dashboard cameras in order to improve car crash detection performance. While most existing car crash detection systems depend on single modal data (i.e., video data or audio data only), the proposed car crash detection system uses an ensemble deep learning model based on multimodal data (i.e., both video and audio data), because different types of data extracted from one information source (e.g., dashboard cameras) can be regarded as different views of the same source. These different views complement one another and improve detection performance, because one view may have information that the other view does not contain. In this research, deep learning techniques, gated recurrent unit (GRU) and convolutional neural network (CNN), are used to develop a car crash detection system. A weighted average ensemble is used as an ensemble technique. The proposed car crash detection system, which is based on multiple classifiers that use both video and audio data from dashboard cameras, is validated using a comparison with single classifiers that use video data or audio data only. Car accident YouTube clips are used to validate this research. The experimental results indicate that the proposed car crash detection system performs significantly better than single classifiers. It is expected that the proposed car crash detection system can be used as part of an emergency road call service that recognizes traffic accidents automatically and allows immediate rescue after transmission to emergency recovery agencies.

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.

Graphite blocks are important core components of the high temperature gas-cooled reactor. As these blocks are simply stacked in array, collisions among neighboring components may occur during earthquakes or accidents. Thus, it is important to develop a reliable seismic model of the stacked graphite blocks and have them designed to maintain their structural integrity during the anticipated occurrences. Various aspects involved in modeling and calculating impact-contact dynamics can affect the resulting behavior of the graphite block. These include mesh size, time step, contact behavior, mechanical constraint formulation of impact-contact analysis, etc. This work is dedicated to perform comparative studies and the effects of these parameters will be identified. The insights obtained through these studies will help build a realistic impact-contact model of the graphite block from which a lumped or reduced dynamics model will be developed for the seismic analysis of the reactor including these graphite components.