RESUMEN
The selective electrochemical synthesis of 1H-indazoles and their N-oxides and the subsequent C-H functionalization of the 1H-indazole N-oxides are described. The electrochemical outcomes were determined by the nature of the cathode material. When a reticulated vitreous carbon cathode was used, a wide range of 1H-indazole N-oxides were selectively synthesized, and the electrosynthesis products were deoxygenated to N-heteroaromatics, owing to cathodic cleavage of the N-O bond via paired electrolysis, when a Zn cathode was used. The scope of this electrochemical protocol is broad, as both electron-rich and electron-poor substrates were tolerated. The potency of this electrochemical strategy was demonstrated through the late-stage functionalization of various bioactive molecules, making this reaction attractive for the synthesis of 1H-indazole derivatives for pharmaceutical research and development. Detailed mechanistic investigations involving electron paramagnetic resonance spectroscopy and cyclic voltammetry suggested a radical pathway featuring iminoxyl radicals. Owing to the rich reactivity of 1H-indazole N-oxides, diverse C-H functionalization reactions were performed. We demonstrated the synthetic utility of 1H-indazole N-oxides by synthesizing the pharmaceutical molecules lificiguat and YD (3); key intermediates for bendazac, benzydamine, norepinephrine/serotonin reuptake inhibitors, SAM-531, and gamendazole analogues; and a precursor for organic light-emitting diodes.
Asunto(s)
Indazoles , Óxidos , Indazoles/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Preparaciones FarmacéuticasRESUMEN
Various methods have been developed for the detection of Escherichia coli (E. coli); however, they are complex and time-consuming. OmpTâa cell membrane endopeptidase of E. coliâstrongly embedded in the outer membrane of only E. coli, exposed to external solutions, with high proteolytic activity, could be a suitable target molecule for the rapid and straightforward detection of E. coli. Herein, a wash-free, sensitive, and selective amperometric method for E. coli detection, based on rapid and specific proteolytic cleavage by OmpT, has been reported. The method involved (i) rapid proteolytic cleavage of consecutive amino acids, after cleavage by OmpT, linked to an electrochemical species (4-aminophenol, AP), by leucine aminopeptidase (LAP, an exopeptidase), (ii) affinity binding of E. coli on an electrode, and (iii) electrochemical-enzymatic (EN) redox cycling. OmpT cleaved the intermediate peptide bond of a peptide substrate containing alanine-arginine-arginine-leucine-AP (-A-R-R-L-AP), forming R-L-AP, followed by the cleavage of two peptide bonds of R-L-AP sequentially by LAP, to liberate an electroactive AP. Affinity binding and EN redox cycling, in addition to rapid proteolytic cleavage by OmpT and LAP, enabled high electrochemical signal amplification. Two-sequential-cleavage was employed for the first time in protease-based detection. The calculated detection limit for E. coli cells in tap water (approximately 103 CFU/mL after 1 h incubation) was lower than those obtained without affinity binding and EN redox cycling. The detection method was highly selective to E. coli as OmpT is present in only E. coli. High sensitivity, selectivity, and the absence of wash steps make the developed detection method practically promising.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Arginina , Proteínas de la Membrana Bacteriana Externa , Endopeptidasas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Péptido Hidrolasas/metabolismo , Péptidos/metabolismoRESUMEN
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. N3-terminated polylysine was used as the poly-linker for the conjugation between an aptamer probe and multiple redox labels. Approximately 11 redox labels per probe and rapid catalytic NADH oxidation enable high signal amplification. Thrombin in urine could be detected without a wash step with a detection limit of â¼50 pM, which is practically promising for point-of-care testing of proteins.
Asunto(s)
Aptámeros de Nucleótidos , Técnicas Biosensibles , Catálisis , Técnicas Electroquímicas , Electrodos , Electrones , Límite de Detección , Oxidación-ReducciónRESUMEN
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.
Asunto(s)
Periodontitis , Porphyromonas gingivalis , Adhesinas Bacterianas , Cisteína Endopeptidasas , Humanos , Periodontitis/diagnóstico , SalivaRESUMEN
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 ß-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 â¼0.2 pg mL-1, implying that it can be used for highly sensitive electrochemical detection of parathyroid hormone and other biomarkers in human serum.
Asunto(s)
Técnicas Biosensibles , Glucosa 1-Deshidrogenasa , Técnicas Electroquímicas , Humanos , Inmunoensayo , Oxidación-Reducción , beta-GalactosidasaRESUMEN
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 (â¼1 pg/mL) in artificial serum. The measured concentrations of PTH in clinical serum samples showed high similarity with those obtained using a commercial instrument.
Asunto(s)
Técnicas Biosensibles/métodos , Formazáns/química , NAD(P)H Deshidrogenasa (Quinona)/química , Hormona Paratiroidea/análisis , Sales de Tetrazolio/química , Catálisis , Técnicas Electroquímicas , Electrodos , Humanos , Oxidación-Reducción , Hormona Paratiroidea/sangreRESUMEN
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 )63+ ), a chemically reversible photoredox catalyst (eosinâ 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 0.3â nm in buffer and 0.4â nm in serum without a washing step.
Asunto(s)
ADN/análisis , Luz , Catálisis , Transporte de Electrón , Límite de Detección , Oxidación-Reducción , Resonancia por Plasmón de SuperficieRESUMEN
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 0.3â pg mL-1 , which indicates the high sensitivity of the immunosensor using the PtNP nanozyme.
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Amoníaco/química , Boranos/química , Ésteres/química , Nanopartículas del Metal/química , Platino (Metal)/química , Hormonas Tiroideas/análisis , Técnicas Biosensibles , Catálisis , Técnicas Electroquímicas , Hidrólisis , Inmunoensayo , Estructura Molecular , Oxidación-Reducción , Propiedades de SuperficieRESUMEN
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.
Asunto(s)
Escherichia coli/química , Vesículas Extracelulares/química , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Biocatálisis , NAD(P)H Deshidrogenasa (Quinona)/química , Monoéster Fosfórico Hidrolasas/químicaRESUMEN
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.
Asunto(s)
Técnicas Biosensibles/métodos , Inmunoensayo/métodos , Límite de Detección , Tirotropina/análisis , Benzoquinonas/química , Electroquímica , Oxidación-ReducciónRESUMEN
Direct electron transfer between a redox label and an electrode requires a short working distance (<1-2 nm), and in general an affinity biosensor based on direct electron transfer requires a finely smoothed Au electrode to support efficient target binding. Here we report that direct electron transfer over a longer working distance is possible between (i) an anionic π-conjugated polyelectrolyte (CPE) label having many redox-active sites and (ii) a readily prepared, thin polymeric monolayer-modified indium-tin oxide electrode. In addition, the long CPE label (â¼18 nm for 10 kDa) can approach the electrode within the working distance after sandwich-type target-specific binding, and fast CPE-mediated oxidation of ammonia borane along the entire CPE backbone affords high signal amplification.
Asunto(s)
ADN/análisis , Polielectrolitos/química , Transporte de ElectrónRESUMEN
Enzyme-like nanocatalytic reactions developed for high signal amplification in biosensors are of limited use because of their low reaction rates and/or unwanted side reactions in aqueous electrolyte solutions containing dissolved O2. Herein, we report a nitrosoreductase-like catalytic reaction, employing 4-nitroso-1-naphthol, Pd nanoparticles, and H3N-BH3, which affords a high reaction rate and minimal side reactions, enabling its use in ultrasensitive electrochemical biosensors. 4-Nitroso-1-naphthol was chosen after five hydroxy-nitro(so)arene compounds were compared in terms of high signal and low background levels. Importantly, the nanocatalytic reaction occurs without the self-hydrolysis and induction period observed in the nanocatalytic reduction of nitroarenes by NaBH4. The high signal level results from (i) fast nanocatalytic 4-nitroso-1-naphthol reduction, (ii) fast electrochemical redox cycling, and (iii) the low influence of dissolved O2. The low background level results from (i) slow direct reaction between 4-nitroso-1-naphthol and H3N-BH3, (ii) slow electrode-mediated reaction between 4-nitroso-1-naphthol and H3N-BH3, and (iii) slow electrooxidation of H3N-BH3 at electrode. When applied to the detection of parathyroid hormone, the detection limit of the newly developed biosensor was â¼0.3 pg/mL. The nitrosoreductase-like nanocatalytic reaction is highly promising for ultrasensitive and stable biosensing.
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Nanopartículas del Metal/química , Naftoles/química , Compuestos Nitrosos/química , Hormona Paratiroidea/análisis , Anticuerpos/inmunología , Técnicas Biosensibles/métodos , Catálisis , Técnicas Electroquímicas/métodos , Humanos , Límite de Detección , Oxidación-Reducción , Paladio/química , Hormona Paratiroidea/inmunologíaRESUMEN
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)64-, and Ru(NH3)63+, the electrochemical and redox cycling behaviors of Os(bpy)2Cl2 are influenced significantly less by ISs in serum. Comparative studies reveal that washing-free displacement assay shows better cortisol-induced signal change than three other assays. The surface concentration of cortisol immobilized on the electrode is optimized, because the electrochemical signal is highly dependent on the surface concentration. When the washing-free displacement immunosensor is applied for the detection of cortisol in artificial serum, cortisol is measured with a detection limit of â¼30 pM within 12 min. The cortisol concentrations measured in clinical serum samples agree well with those obtained using a commercial instrument. The new immunosensor is highly promising for the simple, sensitive, and rapid point-of-care detection of small molecules.
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Técnicas Biosensibles/instrumentación , Hidrocortisona/sangre , Técnicas para Inmunoenzimas/instrumentación , Técnicas Electroquímicas/instrumentación , Electrodos , Diseño de Equipo , Geobacillus stearothermophilus/enzimología , Humanos , Inmunoconjugados/química , Límite de Detección , Sistemas de Atención de PuntoRESUMEN
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 â¼2 × 104 colony-forming unit (CFU)/mL, but the detection limit in the single-mediator system combined with EC redox cycling is â¼2 × 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.
Asunto(s)
Aspergillus niger/aislamiento & purificación , Técnicas Electroquímicas/métodos , Técnicas Microbiológicas/métodos , Aspergillus niger/enzimología , Humanos , Límite de Detección , NADPH Oxidasas/química , Naftoles/química , Nitrocompuestos/química , Oxidación-Reducción , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Recombinase polymerase amplification (RPA) has been combined with electrochemical detection for simple and rapid point-of-care testing. However, there are two major hindrances to this simple and rapid testing: (i) washing or purification steps are required to remove unbound labeled probes and interfering species in the sample; (ii) it is difficult to quantify double-stranded DNA (dsDNA) electrochemically by using biospecific affinity binding without dsDNA denaturation. In the present study, we describe a wash-free and rapid electrochemical method to detect RPA-amplified dsDNAs using a zinc finger protein, Zif268. Electrochemical detection is achieved using proximity-dependent electron mediation of ferrocenemethanol between a glucose-oxidase (GOx) label and an electrode, which differentiates the specifically electrode-bound and -unbound labels without a washing or purification step. RPA-amplified dsDNA containing a biotin-terminated forward primer is specifically bound to a neutravidin-modified electrode, and GOx-conjugated Zif268 is specifically bound to the dsDNA. The whole detection is performed within 17 min (15 min for the RPA reaction and <2 min for the electrochemical measurement). Electrochemical detection is carried out without an additional incubation period, because the specific binding between Zif268 and the dsDNA occurs during the RPA reaction. The detection method could discriminate between target template DNA of Piscirickettsia salmonis and nontarget DNAs (random sequence and calf thymus DNA). The detection limit for the target DNA is approximately 300 copies in 13.2 µL, indicating that the detection method is ultrasensitive. We believe that the method could offer a promising solution for simple and rapid point-of-care testing.
Asunto(s)
ADN/análisis , Técnicas Electroquímicas , ADN/genética , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Recombinasas/metabolismoRESUMEN
The most common enzyme labels in enzyme-linked immunosorbent assays are alkaline phosphatase and horseradish peroxidase, which, however, have some limitations for use in electrochemical immunosensors. This Article reports that the small and thermostable DT-diaphorase (DT-D) and electrochemically inactive 4-nitroso-1-naphthol (4-NO-1-N) can be used as a bifunctional enzyme label and a rapidly reacting substrate, respectively, for electrochemical immunosensors. This enzyme-substrate combination allows high signal amplification via rapid enzymatic amplification and electrochemical redox cycling. DT-D can convert an electrochemically inactive nitroso or nitro compound into an electrochemically active amine compound, which can then be involved in electrochemical-chemical (EC) and electrochemical-enzymatic (EN) redox cycling. Six nitroso and nitro compounds are tested in terms of signal-to-background ratio. Among them, 4-NO-1-N exhibits the highest signal-to-background ratio. The electrochemical immunosensor using DT-D and 4-NO-1-N detects parathyroid hormone (PTH) in phosphate-buffered saline containing bovine serum albumin over a wide range of concentrations with a low detection limit of 2 pg/mL. When the PTH concentration in clinical serum samples is measured using the developed immunosensor, the calculated concentrations are in good agreement with the concentrations obtained using a commercial instrument. Thus, the use of DT-D as an enzyme label is highly promising for sensitive electrochemical detection and point-of-care testing.
Asunto(s)
Técnicas Biosensibles/métodos , Técnicas Electroquímicas , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Hormona Paratiroidea/análisis , Electrodos , Humanos , Inmunoensayo , Límite de Detección , NAD(P)H Deshidrogenasa (Quinona)/química , Naftoles/química , Oxidación-Reducción , Hormona Paratiroidea/sangre , Sistemas de Atención de Punto , Especificidad por SustratoRESUMEN
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.
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Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Técnicas Electroquímicas/métodos , MicroARNs/análisis , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Dedos de Zinc , Fosfatasa Alcalina/química , Técnicas Electroquímicas/normas , Electrodos , Humanos , Límite de Detección , Hibridación de Ácido NucleicoRESUMEN
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.
Asunto(s)
Técnicas Electroquímicas/métodos , Tripsina/análisis , Aminofenoles/química , Aminofenoles/metabolismo , Electrodos , Grafito/química , Inmunoglobulina G/inmunología , Límite de Detección , Oligopéptidos/química , Oligopéptidos/metabolismo , Oxidación-Reducción , Óxidos/química , Proteolisis , Compuestos de Estaño/química , Tripsina/sangre , Tripsina/metabolismoRESUMEN
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.
Asunto(s)
Técnicas Biosensibles/métodos , Toxinas Botulínicas/análisis , Endopeptidasas/metabolismo , Exopeptidasas/metabolismo , Pruebas en el Punto de Atención , Adsorción , Secuencia de Aminoácidos , Aminofenoles/química , Técnicas Biosensibles/instrumentación , Toxinas Botulínicas/química , Toxinas Botulínicas/metabolismo , Ditiotreitol/química , Electroquímica , Electrodos , Límite de Detección , Proteolisis , Compuestos de Estaño/químicaRESUMEN
In washing-free electrochemical detection, various redox and reactive species cause significant interference. To minimize this interference, we report a washing-free electrochemical immunosensor using flavin adenine dinucleotide (FAD)-dependent glycerol-3-phosphate dehydrogenase (GPDH) and glycerol-3-phosphate (GP) as an enzyme label and its substrate, respectively, because the reaction of FAD-dependent dehydrogenases with dissolved O2 is slow and the level of GP preexisting in blood is low (<0.1 mM). A combination of a low electrocatalytic indium-tin oxide (ITO) electrode and fast electron-mediating Ru(NH3)6(3+) is employed to obtain a high signal-to-background ratio via proximity-dependent electron mediation of Ru(NH3)6(3+) between the ITO electrode and the GPDH label. Electrochemical oxidation of GPDH-generated Ru(NH3)6(2+) is performed at 0.05 V vs Ag/AgCl, at which point the electrochemical interference is very low. When a washing-free immunosensor is applied to cardiac troponin I detection in human serum, the calculated detection limit is approximately 10 pg/mL, indicating that the immunosensor is very sensitive in spite of the use of washing-free detection with a short detection period (10 min for incubation and 100 s for electrochemical measurement). The low-interference washing-free electrochemical immunosensor shows good promise for fast and simple point-of-care testing.