Electrochemical-chemical Redox Cycling Research Articles

Wash- and Separation-Free Electrochemical Detection of Proteases

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)

Rapid urinary albumin detection using a simple redox cycling process coupled with a paper-based device

• An electrochemical-chemical (EC) redox cycling process using a mixture of potassium ferricyanide and methylene blue was proposed for sensitive and rapid detection of urinary albumin in 1 min. • A disposable electrochemical paper-based analytical device (ePAD) was proposed as a new detection platform and easily get rid of waste. • The highly sensitive and selective albumin detection was obtained without creatinine correction. • The main features are the use of low-cost devices, rapid detection, and simple procedures. A novel electrochemical-chemical (EC) redox cycling process was proposed for the sensitive, simple, and rapid detection of urinary albumin. This system employs a unique detection scheme based on the combination of ferricyanide ([Fe(CN) 6 ] 3− ) and methylene blue (MB) for signal amplification without the need for any electrode surface modifications. Measurements of bovine serum albumin (BSA) were performed as proof of concept using disposable electrochemical paper-based analytical devices (ePADs) as detection platforms. The quantification of albumin is accomplished by using an indirect detection strategy. In the absence of BSA, electron transfer via EC redox cycling process occurs readily between the redox species and the electrode surface, resulting in a large electrochemical signal. The redox species solution containing BSA, on the other hand, provides a low signal current due to the hindrance of BSA on the electrode surface, which impedes the electron transfer of the proposed redox probe. Under the optimized conditions, the linear range was 1–500 mg dL −1 with a detection limit of 0.072 mg dL −1 . This finding leads to the development of a powerful method for the direct detection of albumin at a low concentration and no requirement for correction by creatinine. The proposed devices were successfully used to detect albumin in real urine samples with satisfactory results, demonstrating good agreement with the standard method and the reliability. Aside from the ease and rapidity, the proposed method is inexpensive, sensitive, and a promising tool for clinical diagnosis of chronic kidney disease and suitable for point-of-care testing in developing countries.

Phenolic Tyrosinase Substrate with a Formal Potential Lower than That of Phenol to Obtain a Sensitive Electrochemical Immunosensor.

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.

Rapid and sensitive detection of Aspergillus niger using permeabilization based on tris buffer containing hydrazine

AbstractIt is challenging to convert redox metabolites present inside fungi into measurable signals via permeabilization because of the fungi's thick and rigid cell walls. We report that Aspergillus niger (A. niger) can be detected via the electrochemical measurement of redox metabolites excreted by a low level of permeabilization of the cell wall of A. niger, when incubated in tris buffer containing hydrazine. Electrochemical signals of redox metabolites are amplified by electrochemical–chemical redox cycling, in which hydrazine acts as a reductant for rapid redox cycling. The detection method is specific to A. niger among the major three fungi causing aspergillosis. The calculated detection limit for A. niger is ~2 × 102 CFU/ml despite the incubation period being only 5 min, indicating that the detection method is highly sensitive and rapid. The detection method does not require a wash step, specific affinity binding, or pretreatment step, indicating that it is a simple method.

Washing- and Separation-Free Electrochemical Detection of Porphyromonas gingivalis in Saliva for Initial Diagnosis of Periodontitis.

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.

Trypsin Detection Using Electrochemical Reduction‐based Redox Cycling

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.

High Electrochemical Signal Amplification Using a Propagating Cascade Reaction and a Redox Cycling Reaction

Propagating cascade reactions based on two enzymes have been used to achieve high signal amplification in biosensors wherein one enzyme continually triggers the activation of the other enzyme that produces signaling species. However, in many cases, biosensors that use cascade reactions are not sensitive enough to allow low detection limits because of the inherent slowness of proteolytic reactions. This paper reports 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 others. The measured concentrations of TSH in clinical serum were found to agree well with those determined using a commercial instrument, indicating that the immunosensor is highly promising for sensitive detection of biomolecules.Keyword: Electrochemical biosensor, Cascade reaction, Redox cycling, Proteolytic reaction

Combined Signal Amplification Using a Propagating Cascade Reaction and a Redox Cycling Reaction for Sensitive Thyroid-Stimulating Hormone Detection

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.

Use of a Phosphatase-Like DT-Diaphorase Label for the Detection of Outer Membrane Vesicles.

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.

Rapid and Sensitive Detection of Aspergillus niger Using a Single-Mediator System Combined with Redox Cycling.

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)63- and menadione is ∼2 × 104 colony-forming unit (CFU)/mL, but the detection limit in the single-mediator system combined with EC redox cycling is ∼2 × 103 CFU/mL, indicating that the newly developed single-mediator system is more sensitive. Importantly, the detection method requires only an incubation period of 10 min and does not require a washing step, an electrode modification step, or a specific probe.

Immunosensor Employing Stable, Solid 1-Amino-2-naphthyl Phosphate and Ammonia-Borane toward Ultrasensitive and Simple Point-of-Care Testing.

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 compounds, including 1A2N, are evaluated to achieve fast EC redox cycling, and four strong reductants, including H3N-BH3, are evaluated to achieve a low background level. The combination of 1A2N and H3N-BH3 allows the achievement of a very high signal-to-background ratio. When the newly developed combination is applied to the detection of creatine kinase-MB (CK-MB), the detection limit for CK-MB is ∼80 fg/mL, indicating that the combination allows ultrasensitive detection. The concentrations of CK-MB in clinical serum samples, determined using the developed system, are in good agreement with the concentrations obtained using a commercial instrument. Thus, the use of stable, solid 1A2N-P and H3N-BH3 along with bare ITO electrodes is highly promising for ultrasensitive and simple point-of-care testing.

Ultrasensitive Protease Sensors Using Selective Affinity Binding, Selective Proteolytic Reaction, and Proximity-Dependent Electrochemical Reaction.

The development of a fast and ultrasensitive protease detection method is a challenging task. This paper reports ultrasensitive protease sensors exploiting (i) selective affinity binding, (ii) selective proteolytic reaction, and (iii) proximity-dependent electrochemical reaction. The selective affinity binding to capture IgG increases the concentration of the target protease (trypsin as a model protease) near the electrode, and the selective proteolytic reaction by trypsin increases the concentration of the redox-active species near the electrode. The electrochemical reaction, which is more sensitive to the concentration of the redox-active species near the electrode than to its bulk concentration, provides an increased electrochemical signal, which is further amplified by the electrochemical-chemical redox cycling. An indium-tin oxide electrode modified with reduced graphene oxide, avidin, and biotinylated capture IgG is used as the electrode, and p-aminophenol liberated from an oligopeptide is used as the redox-active species. The new sensor scheme using no washing process is compared with the new sensor scheme using washing process, and with the conventional scheme using only proteolytic reaction. The new scheme provides a higher signal-to-background ratio and a lower detection limit. Moreover, the increased electrochemical signal offers a more selective protease detection. Trypsin can be detected in phosphate-buffered saline and in artificial serum containing l-ascorbic acid with a low detection limit of 0.5 pg/mL, over a wide range of concentrations, and with an incubation period of only 30 min without washing process. The washing-free electrochemical protease sensor is highly promising for simple, fast, ultrasensitive, and selective point-of-care testing of low-abundance proteases.

A Tyrosinase-Responsive Nonenzymatic Redox Cycling for Amplified Electrochemical Immunosensing of Protein.

Electrochemical immunoassays with high sensitivity and a wide dynamic range are still a challenge in clinical diagnosis. This study reports a novel tyrosinase (Tyr)-responsive nonenzymatic redox cycling for significantly amplifying the electrochemical signal produced from Tyr-labeled immunoassays. The tyrosinase could be captured on the immunosensor surface by a sandwich immunoreaction. The immunosensor was conveniently prepared by covalently binding capture antibody to a chitosan-coated electrode. Using phenol as a substrate and NADH as reducing agent, which showed negligible background due to low electroactivity of phenol and high oxidation overpotential of NADH, the oxygenation activity of tyrosinase could convert poorly electroactive phenol to highly electroactive catechol to trigger an NADH-related nonenzymatic electrochemical-chemical (EC) catalysis. The EC redox cycling leads to a high signal-to-noise ratio for immunoassays. Using carcinoembryonic antigen (CEA) as an analyte model, the amplified immunoassay showed excellent performance with a detectable range from 1.0 pg/mL to 0.1 μg/mL and a sub-pg/mL-level detection limit. The acceptable accuracy and good reproducibility of the proposed immunoassay method indicated its superior suitability in clinical diagnosis.

A highly sensitive and simply operated protease sensor toward point-of-care testing.

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.

Sensitive and selective trypsin detection using redox cycling in the presence of L-ascorbic acid.

We report a simple, sensitive, and selective electrochemical method for trypsin detection that can cover a wide range of concentrations. The method is based on the proteolytic generation of an electroactive species (P) by trypsin followed by a signal-amplified electrochemical measurement of P using electrochemical-chemical (EC) or electrochemical-chemical-chemical (ECC) redox cycling. The detection is performed using bare indium-tin oxide (ITO) electrodes without washing steps. P is generated by the cleavage of an amide bond between P and oligopeptide (Gly-Pro-Arg) at the C-terminal of Gly-Pro-Arg-P. Four trypsin products including 4-amino-1-naphthol (AN) and their trypsin substrates are investigated to obtain a high signal-to-background ratio in ECC redox cycling. AN and its trypsin substrate produce the highest signal-to-background ratio. The detection limits obtained with ECC redox cycling involving AN (approximately 1 ng mL(-1) and 100 ng mL(-1) with an incubation period of 2 h and 30 min, respectively) in Tris buffer (pH 8.0) are lower than those obtained with EC redox cycling involving AN (approximately 5 ng mL(-1) and 200 ng mL(-1) with an incubation period of 2 h and 30 min, respectively). In trypsin detection using ECC redox cycling, the interference effects of electroactive species such as l-ascorbic acid and uric acid are not significant.

Enzyme-based ultrasensitive electrochemical biosensors

Signal amplification in conventional enzyme-based biosensors is not high enough to achieve the ultrasensitive detection of biomolecules. In recent years, signal amplification has been improved by combining enzymatic reactions with redox cycling or employing multienzyme labels per detection probe. Electrochemical-chemical redox cycling and electrochemical-chemical-chemical redox cycling allow ultrasensitive detection simply by including one or two more chemicals in a solution without the use of an additional enzyme and/or electrode. Multiple horseradish peroxidase labels on magnetic bead carriers provide high signal enhancement along with a multiplex detection possibility. In both cases, the detection procedures are the same as those in conventional enzyme-based electrochemical sensors.