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Based on the signal transduction with a horseradish peroxidase (HRP)-functionalized gold nanoprobe, this work develops a new electrochemical aptasensing method for kanamycin (Kana) detection. The aptasensor was constructed through the hybridization of biotinylated Kana-aptamer at its complementary oligonucleotide strand-modified electrode, followed by intercalating methylene blue (MB) into the formed double-stranded DNA (dsDNA). Then, the streptavidin and high-content HRP functionalized gold nanoparticle probes were bound onto the aptasensor. As the MB-mediated HRP-catalytic reaction could produce sensitive electrochemical signal and the aptamer-biorecognition toward Kana could cause quantitative decrease of MB intercalation and nanoprobe capture, convenient electrochemical signal transduction was achieved. Both the enzymatic reaction and nanoprobe signal amplification greatly enhance the electrochemical signal, offering ultrahigh sensitivity of the method. The MB intercalation into dsDNA not only provides necessary electron mediator for the enzymatic reaction but also simplifies the electrochemical measurement. Under optimal conditions, this method showed a wide linear range over four-order of magnitude with a low detection limit of 0.88 pg/mL. In addition, the aptasensor features high specificity, excellent repeatability and stability as well as satisfactory reliability. Thus it possesses valuable application potentials. [ABSTRACT FROM AUTHOR]

In this work, a sandwich-type electrochemical biosensing platform is developed based on carbon sphere-MoS 2 (CS-MoS 2 ) and target recycling amplification of catalyzed hairpin assembly (CHA) strategy for ultrasensitive determination of microRNA (miRNA). The CS-MoS 2 is prepared and used as sensing substrate, which shows high specific surface area, enhanced stability and good dispersibility. The capture probe is first modified on Au nanoparticles/CS-MoS 2 modified glassy carbon electrode through Au-S band, and hairpin DNA1 (H1) then reacts with it and unfolds its loop domain by hybridizing with target miRNA-21. The opened H1 subsequently assembles with biotin labeled hairpin DNA 2 (H2) to displace the target. The liberated miRNA-21 is free and backs to the original solution to proceed next circulation, resulting in the generation of large amounts of H1–H2 duplex. Massive signal indicators horseradish peroxidase are then immobilized on electrode, and catalyze hydrogenperoxide + hydroquinone (H 2 O 2  + HQ) system to produce a strong electrochemical response. The proposed assay benefits from advantages of sandwich-type structure, favorable electrochemical properties of CS-MoS 2 and CHA target recycling amplification strategy. Therefore, it exhibits a wide linear relation between 1 × 10 −16 and 1 × 10 −10  M with a low detection limit of 1.6 × 10 −17  M for miRNA-21 detection. [ABSTRACT FROM AUTHOR]

Porous Co 3 O 4 hexagonal nanosheets (PCHNSs) with a well-defined morphology have been synthesized successfully via a hydrothermal process and followed by calcinations. Co 3 O 4 nanosheets immobilizing horseradish peroxidase (HRP), together with reduced graphene oxide, have been used to modify glassy carbon electrode to fabricate a mediator-free biosensor. The morphology and structure of PCHNSs are studied by XRD, FESEM, and HRTEM techniques. Spectroscopic and electrochemical results reveal that Co 3 O 4 /rGO composite film is an excellent immobilization matrix with biocompatibility for enzymes, affording good enzymes’ bioactivity and stability. Meanwhile, due to the unique structure of the modified electrode, the direct electron transfer of HRP is facilitated and the prepared nitrite biosensors display good performance—a wide linear range of 1–5400 μM, an extremely low limit of detection (LOD) as 0.21 μM and a limit of quantification as (LOQ) as 0.58 μM. This work suggests that Co 3 O 4 /rGO composite film is a potential matrix for enzymes’ immobilization and biosensors’ preparation. [ABSTRACT FROM AUTHOR]

Abstract: Neurotoxic organophosphorus (OP) compounds are commonly used as chemical warfare agents and pesticides. Due to their high toxicity, rapid and sensitive field detection of these compounds has been an ongoing topic of interest. Biosensors made with organophosphate hydrolase enzyme (OPH) are generally designed to either amperometrically detect an electroactive leaving group produced following enzymatic cleavage, or to potenitometrically detect the pH change that occurs during cleavage. Since OPs are more likely to have phenolic leaving groups as compared to electroactive leaving groups, we have developed a new amperometric dual enzyme electrochemical assay that enables the detection of a broad class of OP compounds using the OPH enzyme combined with horseradish peroxidase (HRP). The assay has been applied to the detection of dichlofenthion, which does not have an electroactive leaving group and is not a commonly investigated OPH substrate. Using reverse phase HPLC, we have determined the Michaelis–Menten kinetic parameters of an engineered OPH enzyme to be K M =0.11±0.02mM and k cat =0.046±0.003s−1 with dichlofenthion as the substrate. Detection of the phenolic leaving group from the OPH enzyme reaction using the HRP electrode is carried out at −50mV vs. Ag/AgCl where the noise and background are low and interferences are negligible. After optimization of the solution pH, the dual enzyme biosensor was found to have a limit of detection (LOD) of 24μM (7.6ppm), and a sensitivity of 0.095±0.024nA/μM for dichlorofenthion. By detecting the phenolic leaving groups from the OP targets using the HRP electrode, biosensors made using this new platform have the potential to detect a broad range of important OP compounds. [Copyright &y& Elsevier]

Abstract: In this work, a novel label-free amperometric immunosensor has been constructed for detecting α-1-fetoprotein (AFP) based on nanocomposite of horseradish peroxidase (HRP) labeled carbon nanotubes (CNTs). First, the gold nanoparticles (AuNPs) were electrodeposited on the surface of the glass carbon electrode by electrochemical reduction of gold chloride tetrahydrate (HAuCl4) to immobilize horseradish peroxidase labeled carbon nanotubes (HRP-CNTs). Then HRP-CNTs bioconjugate was immobilized on the surface of the electrodeposited AuNPs layer by the combination of forces (coordination and electrostatic force). Subsequently, it was immersed into gold colloidal nanoparticles (GNPs) solution, which was used to immobilize antibody biomolecules (anti-AFP). Enhanced sensitivity was obtained by using bioconjugates featuring HRP labeled (HRP-CNTs), which had lager specific surface area and good electronic catalysis (current response signal) compared to carbon nanotubes. Under optimized conditions, the linear ranges were from 0.2 to 200ngmL−1 with a detection limit of 0.067ngmL−1 (at an S/N of 3). The proposed immunosenor showed good precision, acceptable stability and reproducibility and could be used for the detection AFP in normal human serum, which provided a potential alternative tool for the detection of protein in clinical diagnosis. [Copyright &y& Elsevier]

Abstract: A novel chemiluminescence immunosensor using horseradish peroxidase (HRP)-functionalized mesoporous silica nanoparticles (MSN) as labels was developed, which increases the sensitivity of the chemiluminescence immunoassay. The enzyme-functionalized MSN were fabricated by simultaneous coimmobilization of HRP and the carcinoembryonic antigen antibody (anti-CEA) onto the surface of MSN using 3-aminopropyltriethoxysilane (APTES) as the linkage. Because the large surface area of MSN carriers increased the amount of HRP bound per sandwiched immunoreaction, the conjugates provided a much higher signal and increased sensitivity. This is an improvement over the traditional sandwich immunoassay which often has one or two enzyme molecules per antibody. This approach was successfully demonstrated as a simple, cost-effective, specific, and potent method to detect CEA in practical samples. The analysis showed a linear response within the range of 0.1–40ng/mL (r =0.9912). The relative standard deviation (RSD) for 11 parallel measurements of 20ng/mL CEA was 3.9%. The sensitivity of the immunosensor using MSN–HRP–Ab2 as labels was about 10-fold higher than that of traditional labels. These labels for immunosensors may provide many potential applications for detection of different biomolecules. [Copyright &y& Elsevier]

Abstract: Horseradish peroxidase (HRP) was chemically modified using cyanuric chloride (CC) as a linking agent onto a carbon felt (CF), which is a microelectrode ensemble of micro carbon fiber (>7μm, diameter) with a random three-dimensional structure. The resulting HRP-modified CF (HRP-ccCF) exhibited well-defined redox waves based on the HRP heme FeIII/FeII redox couple at −0.23V vs. Ag/AgCl (at pH 7.0), while the HRP-adsorbed CF (HRP-CF) showed no apparent redox couple in the same potential range, indicating that the chemical modification of HRP via CC facilitated the direct electron transfer (DET) between HRP and CF. The apparent heterogeneous electron transfer rate constant k s was estimated to be 35s−1. Cyclic voltammetry and electrochemical impedance spectroscopy revealed that the interfacial properties (i.e., structure, morphology of enzyme-layer) of covalently modified HRP (HRP-ccCF) and physically adsorbed HRP (HRP-CF) are different, resulting in the difference in the electron transfer properties. The HRP-ccCF was successfully used as a working electrode unit in bioelectrocatalytic flow-through detector for highly sensitive amperometric determination of H2O2. Under the optimized conditions (i.e., applied potential, 0V vs. Ag/AgCl; carrier flow rate, 3.25ml/min; and carrier pH 7.0), the cathodic peak current of H2O2 linearly increased up to 3μM (sensitivity, 1.94μA/μM; the detection limit, 0.08μM [S/N=3]) with sample through-put of ca. 90samples/h. [Copyright &y& Elsevier]

Abstract: A biosensor for detection of hydrogen peroxide was designed based on immobilization of anthraquinone 2-carboxylic acid modified horseradish peroxidase. Direct electrochemistry of the modified enzyme exhibited a quasi-reversible voltammogram with a formal potential of −459mV. The surface charge transfer rate constant, k s, and the charge transfer coefficient, α c, for electron transfer between the glassy carbon electrode and the modified enzyme were estimated to be 3.165s−1 and 0.455, respectively. The modified electrode exhibited a remarkable electrocatalytic activity towards hydrogen peroxide. Under the optimized experimental conditions, H2O2 could be detected in a linear concentration range from 2.65×10−9 to 1.08×10−7 M and a detection limit of 1.44×10−9 M at S/N=3. The response time was 6s and when the electrode was stored at 4°C, the biosensor was used repeatedly for 17 days with 10% deterioration in response. [Copyright &y& Elsevier]

Abstract: Direct electrochemistry and bioelectrocatalytic ability toward the reduction of hydrogen peroxide of horseradish peroxidase (HRP) were found at the multiwalled carbon nanotube/alumina-coated silica (MWCNT/ACS) nanocomposite modified glassy carbon electrode. The surface morphology of the HRP/MWCNT/ACS nanobiocomposite modified glassy carbon electrode was characterized by scanning electron microscopy. In cyclic voltammetric response, the immobilized HRP at the MWCNT/ACS nanocomposite modified glassy carbon electrode displayed a pair of well-defined and quasi-reversible redox peaks with a formal potential of −0.328V versus Ag/AgCl at a scan rate of 200mVs−1. The formal potential of HRP varied linearly in the range of solution pH values from 6.0 to 8.0 with a slope of −56mVpH−1, which is close to the theoretical value of −59mVpH−1 for a reversible one-electron coupled with one-proton electrochemical reaction process. Several important experimental variables were optimized. The optimized hydrogen peroxide biosensor exhibited a sensitivity of 0.157AM−1 cm−2, a detection limit of 0.63μM, a linear range of 1–500μM, and an apparent Michaelis–Menten constant of 0.39mM. The performance of the prepared biosensor was evaluated with respect to four possible interferences. [Copyright &y& Elsevier]

Abstract: We developed a new flowerlike ZnO–gold nanoparticles (GNPs)–Nafion nanocomposite which can promote the direct electron transfer of horseradish peroxidase (HRP) immobilized in the film effectively. The cyclic voltammetric results show that HRP entrapped in the composite could undergo a direct quasi-reversible electrochemical reaction with a formal potential (E 0′) of −0.32V (versus Ag/AgCl) in phosphate buffer solution (PBS). The flowerlike ZnO and GNPs show synergistic effect and the ZnO–GNPs–Nafion–HRP modified GC electrode gives an enhanced electrocatalytic activity towards the reduction of hydrogen peroxide (H2O2). The calculated apparent Michaelis–Menten constant () is 1.76mM, which is much lower than that reported, indicating a high catalytic activity of HRP. The catalysis currents increase linearly to the H2O2 concentration in a wide range of 1.5×10−5 to 1.1×10−3 M, with a correlation coefficient of 0.998. The detection limit is 9.0×10−6 M (at the ratio of signal to noise, S/N=3), demonstrating that the formed film provides a favorable microenvironment for the enzyme to retain its activity. Moreover, the modified electrode displays rapid response to H2O2 and possesses good stability and reproducibility. [Copyright &y& Elsevier]

Abstract: A novel biocompatible polyquaternium (QY)-manganese oxide nanosheet (MNS) nanocomposite has been prepared and shown to be a promising matrix for horseradish peroxidase (HRP) immobilization. The resulting HRP-QY-MNS film was characterized by Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy, which indicated that HRP retained its native structure in the nanocomposite film. An HRP-QY-MNS film-modified glassy carbon electrode exhibited a pair of well-defined and quasi-reversible cyclic voltammetric peaks centered at −0.272V (vs. Ag/AgCl) in pH 7.0 phosphate buffer solution. The direct electrochemical behavior of HRP was greatly enhanced in the QY-MNS nanocomposite film compared with that in single-component QY or MNS films. The immobilized HRP showed excellent electrocatalysis in the reduction of hydrogen peroxide (H2O2), which was exploited in the construction of an H2O2 biosensor. The linear range of the biosensor for H2O2 was found to be from 1.0×10−7 to 3.2×10−5 M with a correlation coefficient of 0.998. The detection limit was 7.8×10−8 M at a signal-to-noise ratio of 3. The biosensor exhibited rapid response and good long-term stability. [Copyright &y& Elsevier]

Abstract: Methylene blue (MB) has been intercalated into layered manganese oxide (birnessite) by a delamination/reassembly process and the resulting MB-intercalated birnessite (MB-Bir) material characterized by X-ray powder diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and electrochemical measurements. A novel reagentless hydrogen peroxide biosensor was fabricated by using the MB-Bir as an electron mediator. Cyclic voltammetry and amperometric measurements demonstrated that methylene blue coimmobilized with horseradish peroxidase (HRP) displayed good stability and that electrons were efficiently shuttled between HRP and the electrode. The biosensor showed a rapid and linear response to H2O2 over the range from 1.5×10−6 M to 8.1×10−3 M, with a detection limit of 1.3μM (S/N=3/1). Moreover, the biosensor retained about 85% of its original response to H2O2 after storage for 2 months. Addition of glucose, sucrose, citric acid, acetic acid, or l-ascorbate did not interfere with the H2O2 response. [Copyright &y& Elsevier]

Abstract: Horseradish peroxidase was immobilized on a glass carbon electrode by poly (N-isopropylacyamide-co-3-methacryloxy-propyl-trimethoxysilane) (PNM). Horseradish peroxidase entrapped in the PNM film exhibited a pair of well-defined, quasi-reversible cyclic voltammetric peaks at about −0.36V versus a saturated calomel electrode (SCE) in pH 7.0 buffer solution, corresponding to hemeFeIII +e→hemeFeII. Some electrochemical parameters were calculated by performing nonlinear regression analysis of square wave voltammetry (SWV) experimental data. Fourier transform infrared (FTIR) spectra suggested that horseradish peroxidase entrapped in the PNM film retained the secondary structure. Hydrogen peroxide could be reduced by the catalysis of the entrapped horseradish peroxidase without any mediator. The reagentless hydrogen peroxide sensor had a fast response of less than 2.5s with linear range of 0.19–1.35μM with a detection of 4.75×10−8 molL−1. The sensitivity of the sensor for H2O2 was 0.62Amol−1 cm−2. The activation energy for enzyme reaction was calculated to be 14.84kJmol−1. [Copyright &y& Elsevier]

Abstract: Direct electron transfer process of immobilized horseradish peroxidase (HRP) on a gold colloid-cysteine-nafion membrane, and its application as a biosensor were investigated by using electrochemical methods. The electrochemical characteristics of the biosensor were studied by cyclic voltammetry, linear sweep voltammetry and chronoamperometry. The modified process was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The sensor displayed an excellent electrocatalytic response to the reduction of H2O2 without the aid of an electron transfer mediator. Analytical parameters such as pH and temperature were also studied. Linear calibration for H2O2 was obtained in the range 3.50×10−7 to 5.87×10−3 M under the optimized conditions. The sensor was highly sensitive to H2O2 with a detection limit of 1.05×10−7 M (S/N =3), and the sensor achieved 95% of the steady-state current within 10s. The sensor exhibited high sensitivity, selectivity and stability. [Copyright &y& Elsevier]

Abstract: A highly stable and sensitive amperometric ethanol bi-enzyme biosensor was developed using alcohol oxidase isolated from thermotolerant methylotrophic yeast Hansenula polymorpha and horseradish peroxidase as biorecognition elements. Enzyme immobilization was performed by means of electrodeposition paints (EDP) with a first layer integrating horseradish peroxidase within an Os-complex modified EDP in order to assure fast electron transfer between the enzyme and the electrode surface. On top of this layer alcohol oxidase was entrapped within an EDP layer thus assuring fast substrate diffusion within the hydrogel layer concomitantly with a stabilization of the enzyme. A variety of sensor architectures were investigated aiming on the optimization of the electrochemical communication between the immobilized enzymes and the electrode surface. The immobilized enzymes activities were highly dependent on the applied potential during the electrochemically induced EDP precipitation and on the chemical composition of the used EDP. Bioanalytical properties of an optimized alcohol biosensor such as response time, dynamic range for different analytes (ethanol, methanol, n-propanol, n-butanol, formaldehyde), operational and storage stability were investigated. Moreover, the developed biosensor was applied for the determination of ethanol in wine samples. [Copyright &y& Elsevier]

Abstract: Horseradish peroxidase (HRP) to a nano-Au monolayer, which was supported by PAMAM dendrimer/cystamine modified gold electrode. The high affinity of PAMAM dendrimer for nano-Au with its amine groups was used to realize the role of nano-Au as an intermediator to retain the high bioactivity of HRP. A hydrogen peroxide biosensor was fabricated based on the effective immobilization of the high bioactivity of HRP. In the presence of the hydroquinone mediator in the solution, the immobilized HRP exhibited excellent electrocatalytical response to the reduction of H2O2. The effects of various experimental variables were optimized. The resulting biosensor showed a linear response to H2O2 over a concentration range from 1 × 10-5 to 2.5 × 10-3molL-1 with a sensitivity of 0.53ALmol-1cm-2 and a detection limit of 2.0μmolL-1 based on a signal-to-noise ratio of 3. The apparent Michaelis-Menten constant () of the biosensor was evaluated to be 0.52mmolL-1. The biosensor exhibited high sensitivity and good stability and reproducibility. Performance of the biosensor was evaluated with respect to possible interferences and the application to real sample analysis. [Copyright &y& Elsevier]

An amperometric enzyme electrode employing horseradish peroxidase (HRP) immobilized on a ferrocene containing polymer electrochemically deposited onto a Pt electrode is used for the determination of hydrogen peroxide and organic peroxides. The polymer consists on a siloxane-based homopolymer with pendant dendritic wedges possessing electronically communicated ferrocenyl moieties. The steady-state amperometric response of the sensors is investigated as a function of the applied potential, substrate concentration and polymer film thickness. The effects of interferences and storage on the response were also studied. The new biosensors respond to substrates at a work potential value of -50 mV (versus SCE) and show a good linear response region and protection from interferences. Moreover, their sensitivity and detection limits are competitive with other peroxides based electrodes reported. [Copyright &y& Elsevier]