Entrapped Horseradish Peroxidase Research Articles

Dual-Functional Peroxidase-Copper Phosphate Hybrid Nanoflowers for Sensitive Detection of Biological Thiols.

An effective strategy to detect biological thiols (biothiols), including glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), holds significant incentive since they play vital roles in many cellular processes and are closely related to many diseases. Here, we demonstrated that hybrid nanoflowers composed of crystalline copper phosphate and horseradish peroxidase (HRP) served as a functional unit exhibiting dual catalytic activities of biothiol oxidase and HRP, yielding a cascade reaction system for a sensitive one-pot fluorescent detection of biothiols. The nanoflowers were synthesized through the anisotropic growth of copper phosphate petals coordinated with the amine/amide moieties of HRP, by simply incubating HRP and copper(II) sulfate for three days at room temperature. Copper phosphates within the nanoflowers oxidized target biothiols to generate H2O2, which activated the entrapped HRP to oxidize the employed Amplex UltraRed substrate to produce intense fluorescence. Using this strategy, biothiols were selectively and sensitively detected by monitoring the respective fluorescence intensity. This nanoflower-based strategy was also successfully employed for reliable quantification of biothiols present in human serum, demonstrating its great potential for clinical diagnostics.

Study of the Interaction of a Novel Semi-Synthetic Peptide with Model Lipid Membranes.

Most linear peptides directly interact with membranes, but the mechanisms of interaction are far from being completely understood. Here, we present an investigation of the membrane interactions of a designed peptide containing a non-natural, synthetic amino acid. We selected a nonapeptide that is reported to interact with phospholipid membranes, ALYLAIRKR, abbreviated as ALY. We designed a modified peptide (azoALY) by substituting the tyrosine residue of ALY with an antimicrobial azobenzene-bearing amino acid. Both of the peptides were examined for their ability to interact with model membranes, assessing the penetration of phospholipid monolayers, and leakage across the bilayer of large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs). The latter was performed in a microfluidic device in order to study the kinetics of leakage of entrapped calcein from the vesicles at the single vesicle level. Both types of vesicles were prepared from a 9:1 (mol/mol) mixture of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1′-rac-glycerol). Calcein leakage from the vesicles was more pronounced at a low concentration in the case of azoALY than for ALY. Increased vesicle membrane disturbance in the presence of azoALY was also evident from an enzymatic assay with LUVs and entrapped horseradish peroxidase. Molecular dynamics simulations of ALY and azoALY in an anionic POPC/POPG model bilayer showed that ALY peptide only interacts with the lipid head groups. In contrast, azoALY penetrates the hydrophobic core of the bilayers causing a stronger membrane perturbation as compared to ALY, in qualitative agreement with the experimental results from the leakage assays.

Enzyme-loaded liposome with biocatalytic precipitation for potentiometric immunoassay of thyroid-stimulating hormone in thyroid carcinoma

A simple and feasible potentiometric immunosensing platform based on enzymatic biocatalytic precipitation technique was designed for the sensitive detection of thyroid-stimulating hormone (TSH; a typical kind of biomarkers for thyroid carcinoma), using horseradish peroxidase (HRP)-loaded liposome for the signal amplification. To construct such an assay system, a sandwich-type immunoreaction was readily carried out on monoclonal anti-TSH capture antibody-coated electrode by using polyclonal anti-TSH secondary antibody-conjugated HRP-loaded liposome. Accompanying the formation of sandwich-type immunocomplex, the carried liposome was lysed through the added Triton X-100 to release the entrapped HRP molecules, which catalyzed the oxidation of 4-chloro-1-naphthol to produce an insoluble and uncharged organic precipitation on the electrode surface, thereby causing the change of the local electrical potential. Two labeling protocols with and without the liposome were investigated for detection of target TSH, improved analytical features were achieved with HRP-entrapped liposome. Under optimal conditions, the potentiometric immunosensor had good responses for TSH detection within the linear range of 0.01–30 μIU/mL at a detection limit of 0.0067 μIU/mL. Good reproducibility, high specificity and long-time stability were acquired during the assay procedure. Importantly, a well-matched accuracy between the potentiometric immunosensor and commercial human TSH ELISA kit was gave for the analysis of human serum samples.

Spectrophotometric Quantification of Peroxidase with p-Phenylene-diamine for Analyzing Peroxidase-Encapsulating Lipid Vesicles.

A spectrophotometric assay for the determination of horseradish peroxidase (HRP) in aqueous solution with p-phenylenediamine (PPD, benzene-1,4-diamine) as electron donor substrate and hydrogen peroxide (H2O2) as oxidant was developed. The oxidation of PPD by HRP/H2O2 leads to the formation of Bandrowski's base ((3E,6E)-3,6-bis[(4-aminophenyl)imino]cyclohexa-1,4-diene-1,4-diamine), which can be quantified by following the increase in absorbance at 500 nm. The assay was applied for monitoring the activity of HRP inside ≈180 nm-sized lipid vesicles (liposomes), prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and purified by size exclusion chromatography. Because of the high POPC bilayer permeability of PPD and H2O2, the HRP-catalyzed oxidation of PPD occurs inside the vesicles once PPD and H2O2 are added to the vesicle suspension. In contrast, if instead of PPD the bilayer-impermeable substrate ABTS2- (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)) is used, the oxidation of ABTS2- inside the vesicles does not occur. Therefore, using PPD and ABTS2- in separate assays allows distinguishing between vesicle-trapped HRP and HRP in the external bulk solution. In this way, the storage stability of HRP-containing POPC vesicles was investigated in terms of HRP leakage and activity of entrapped HRP. It was found that pH 7.0 suspensions of POPC vesicles (2.2 mM POPC) containing on average about 12 HRP molecules per vesicle are stable for at least 1 month without any significant HRP leakage, if stored at 4 °C. Such high stability is beneficial not only for bioanalytical applications but also for exploring the kinetic properties of vesicle-entrapped HRP through simple spectrophotometric absorption measurements with PPD as a sensitive and cheap substrate.

Thermo- and Sulfate-Controllable Bioelectrocatalysis of Glucose Based on Horseradish Peroxidase and Glucose Oxidase Embedded in Poly(N,N-diethylacrylamide) Hydrogel Films

Dual-responsive poly(N,N-diethylacrylamide) (PDEA) hydrogel films with entrapped horseradish peroxidase (HRP) and glucose oxidase (GOD) were successfully prepared on electrode surface with a simple one-step polymerization procedure under mild conditions, designated as PDEA-HRP-GOD. Cyclic voltammetric (CV) response of electroactive probe K3Fe(CN)6 at the film electrodes displayed reversible thermo- and sulfate-responsive switching behavior. For example, at 25°C, the K3Fe(CN)6 demonstrated a well-defined CV peak pair with large peak currents for the films, showing the on state, while at 40°C, the CV response was greatly suppressed and the system was at the off state. The influence of temperature and Na2SO4 concentration on the switching behavior of the film system was not independent or separated, but was synergetic. The responsive mechanism of the system was ascribed to the structure change of PDEA component in the films with temperature and sulfate concentration. This switching property of the PDEA-HRP-GOD films could be further used to realize dual-responsive catalytic oxidation of glucose sequentially by HRP and GOD entrapped in the films with Fe(CN)6 (3-) as the mediator through changing the surrounding temperature and Na2SO4 concentration. This system may establish a foundation for fabricating a new type of multi-switchable electrochemical biosensors based on bienzyme electrocatalysis.

Ionic liquid modified carbon paste electrode and investigation of its electrocatalytic activity to hydrogen peroxide

. This paper reports on the preparation and advantages of novel amperometric biosensors in the presence of hydrophobic ionic liquid (IL), 1-methyl-3-butylimidazolium bromide ([MBIB]). Carbon paste biosensor has been constructed by entrapping horseradish peroxidase in graphite and IL mixed with paraffin oil as a binder. The resulting IL/graphite material brings new capabilities for electrochemical devices by combining the advantages of ILs composite electrodes. Amounts of H2O2 were amperometrically detected by monitoring current values at reduction potential (–0·15 V) of K3Fe(CN)6. Decrease in biosensor responses were linearly related to H2O2 concentrations between 10 and 100 μM with 2 s response time. Limit of detection of the biosensor were calculated to be 3·98 μM for H2O2. In the optimization studies of the biosensor some parameters such as optimum pH, optimum temperature, enzyme amount, interference effects of some substances on the biosensor response, reproducibility and storage stability were carried out. The promising results are ascribed to the use of an ionic liquid, which forms an excellent charge-transfer bridge and wide electrochemical windows in the bulk of carbon paste electrode.

Core–shell structured Ag@C for direct electrochemistry and hydrogen peroxide biosensor applications

Ag@C core–shell nano-composites have been prepared by a simple one-step hydrothermal method and are further explored for protein immobilization and bio-sensing. The electrochemical behavior of immobilized horseradish peroxidase (HRP) on Ag@C modified indium–tin–oxide (ITO) electrode and its application as H2O2 sensor are investigated. Electrochemical and UV–vis spectroscopic measurements demonstrated that Ag@C nano-composites provide excellent matrixes for the adsorption of HRP and the entrapped HRP retains its bioactivities. It is found that on the HRP–Ag@C/ITO electrode, HRP exhibited a fast electron transfer process and good electrocatalytic reduction toward H2O2. Under optimum experimental conditions the biosensor linearly responds to H2O2 concentration in the range of 5.0×10−7–1.4×10−4M with a detection limit of 2.0×10−7M (S/N=3). The apparent Michaelis–Menten constant (KappM) of the biosensor is calculated to be 3.75×10−5M, suggesting high enzymatic activity and affinity toward H2O2. In addition, the HRP–Ag@C/ITO bio-electrode shows good reproducibility and long-term stability. Thus, the core–shell structured Ag@C is an attractive material for application in the fabrication of biosensors due to its direct electrochemistry and functionalized surface for efficient immobilization of bio-molecules.

Novel biosensing platform based on self-assembled supramolecular hydrogel

The supramolecular hydrogel self-assembled from α-cyclodextrin (α-CD) and an amphiphilic triblock copolymer was used for the first time as a biosensing platform by the in-situ incorporation of horseradish peroxidase and polyaniline (PANI) nanoparticles. It was found that the used triblock copolymer could disperse well PANI nanoparticles in aqueous system and then interact with α-CD in the presence of horseradish peroxidase for the formation of supramolecular hydrogel composite. The content of PANI nanoparticles was found to affect the gelation time and gel strength. The circular dichroism analyses showed that the entrapped horseradish peroxidase could retain its native conformation. By electrochemical experiments, the incorporated PANI nanoparticles were confirmed to improve the current response and enzymatic activity, and the fabricated biosensor was found to provide a fast amperometric response to hydrogen peroxide.

Miniature Enzyme-Based Electrodes for Detection of Hydrogen Peroxide Release from Alcohol-Injured Hepatocytes

Alcohol insult to the liver sets off a complex sequence of inflammatory and fibrogenic responses. There is increasing evidence that hepatocytes play a key role in triggering these responses by producing inflammatory signals such as cytokines and reactive oxygen species (ROS). In the present study, we employed a cell culture/biosensor platform consisting of electrode arrays integrated with microfluidics to monitor extracellular H(2)O(2), one of the major ROS types, produced by primary rat hepatocytes during alcohol injury. The biosensor consisted of hydrogel microstructures with entrapped horseradish peroxidase (HRP) immobilized on an array of miniature gold electrodes. These arrays of sensing electrodes were integrated into microfluidic devices and modified with collagen (I) to promote hepatocyte adhesion. Once seeded into the microfluidic devices, hepatocytes were exposed to 100 mM ethanol and the signal at the working electrode was monitored by cyclic voltammetry (CV) over the course of 4 h. The CV experiments revealed that hepatocytes secreted up to 1.16 μM H(2)O(2) after 3 h of stimulation. Importantly, when hepatocytes were incubated with antioxidants or alcohol dehydrogenase inhibitor prior to alcohol exposure, the H(2)O(2) signal was decreased by ~5-fold. These experiments further confirmed that the biosensor was indeed monitoring oxidative stress generated by the hepatocytes and also pointed to one future use of this technology for screening hepatoprotective effects of antioxidants.

Bioactivity of horseradish peroxidase entrapped in silica nanospheres

Interest in the fabrication of micro/nanoreactors for evaluation of the function of biomolecules in biological processes, enzymatic reaction kinetics occurring inside the nanospace is rapidly increasing. With a simple reverse-micelle microemulsion method, horseradish peroxidase (HRP), a model biomolecule, was herein skillfully confined in silica nanoshells (HRP@SiO2) and its biocatalytical behaviors were investigated in detail. Spectroscopic measurements showed that the entrapped HRP molecules retained their native structure and had high enzymatic activity toward 3,3′,5,5′-tetramethylbenzidine (TMB) with Michaelis constant (Km) of 3.02×10−5molL−1. The entrapped HRP displayed a good direct electron transfer behavior and sensitive electrocatalytic response toward the reduction of H2O2, which could be enhanced using thionine and o-phenylenediamine (o-PD) as electron mediators. When using thionine as mediator, the mass transport between the substrates in electrolyte and HRP confined in silica nanospheres through the mesoporous tunnels was slower than that of o-PD, which slowed down the electron transfer between heme in HRP in the confined nanospace and the electrode, and resulted in low sensitivity to H2O2 with thionine as mediator when compared to o-PD.

PH-, Sugar-, and Temperature-Sensitive Electrochemical Switch Amplified by Enzymatic Reaction and Controlled by Logic Gates Based on Semi-Interpenetrating Polymer Networks

Phenylboronic acid (PBA) moieties are grafted onto the backbone of poly(acrylic acid) (PAA), forming the PAA-PBA polyelectrolyte. The semi-interpenetrating polymer network (semi-IPN) films composed of PAA-PBA and poly(N,N-diethylacrylamide) (PDEA) were then synthesized on electrode surface with entrapped horseradish peroxidase (HRP), designated as PDEA-(PAA-PBA)-HRP. The films demonstrated reversible pH-, fructose-, and thermo-responsive on-off behavior toward electroactive probe K(3)Fe(CN)(6) in its cyclic voltammetric (CV) response. This multiswitchable CV behavior of the system could be further employed to control and modulate the electrochemical reduction of H(2)O(2) catalyzed by HRP immobilized in the films with K(3)Fe(CN)(6) as the mediator in solution. The responsive mechanism of the system was also explored and discussed. The pH-sensitive property was attributed to the electrostatic interaction between the PAA component of the films and the probe at different pH; the thermo-responsive behavior originated from the structure change of PDEA hydrogel component of the films with temperature; the fructose-sensitive property was ascribed to the structure change of the films induced by the complexation between the PBA constituent and the sugar. This smart system could be used as a 3-input logic network composed of enabled OR (EnOR) gates in chemical or biomolecular computing by combining the multiresponsive property of the films and the amplification effect of bioelectrocatalysis and demonstrated the potential perspective for fabricating novel multiswitchable electrochemical biosensors and bioelectronic devices.

Enzyme microcapsules with substrate selective permeability constructed via layer-by-layer polyelectrolyte self-assembly

Polyelectrolyte multilayer microcapsules with entrapped horseradish peroxidase (HRP) have been prepared via a layer-by-layer (LbL) self-assembly strategy of polycation and polyanion on CaCO3 microparticles as templates. Preparation conditions have been studied and optimized. Within the investigated ranges, use of buffer solutions with lower pH value or lower ionic strength in the buffer solution for polyelectrolyte self-assembly has resulted in thinner polyelectrolyte film and higher permeability of the microcapsules. For dissolving the CaCO3 templates, use of weakly acidic (pH 4.0) buffer solution in place of routinely used EDTA reagent has improved the catalytic activity of microcapsules. The HRP-containing microcapsules have exhibited catalytic activity to pyrogallol as substrate, while the catalytic activity to 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) was severely suppressed. No less than 90% of the maximum enzymatic activity to pyrogallol has remained after 30days. Promising prospects in various biocatalysis applications have been expected for the enzyme microcapsules, due to their selective permeability, stability and reusability.

Triply Responsive Films in Bioelectrocatalysis with a Binary Architecture: Combined Layer-by-Layer Assembly and Hydrogel Polymerization

In this work, triply responsive films with a specific binary architecture combining layer-by-layer assembly (LbL) and hydrogel polymerization were successfully prepared. First, concanavalin A (Con A) and dextran (Dex) were assembled into {Con A/Dex}(5) LbL layers on electrode surface by the lectin-sugar biospecific interaction between them. The poly(N,N-diethylacrylamide) (PDEA) hydrogels with entrapped horseradish peroxidase (HRP) were then synthesized by polymerization on the surface of LbL inner layers, forming {Con A/Dex}(5)-(PDEA-HRP) films. The films demonstrated reversible pH-, thermo-, and salt-responsive on-off behavior toward electroactive probe Fe(CN)(6)(3-) in its cyclic voltammetric responses. This multiple stimuli-responsive films could be further used to realize triply switchable electrochemical reduction of H(2)O(2) catalyzed by HRP immobilized in the films and mediated by Fe(CN)(6)(3-) in solution. The responsive mechanism of the films was explored and discussed. The pH-sensitive property of the system was attributed to the electrostatic interaction between the {Con A/Dex}(5) inner layers and the probe at different pH, and the thermo- and salt-responsive behaviors should be ascribed to the structure change of PDEA hydrogels for the PDEA-HRP outermost layers under different conditions. The concept of binary architecture was also used to fabricate {Con A/Dex}(5)-(PDEA-GOD) films on electrodes, where GOD = glucose oxidase, which was applied to realize the triply switchable bioelectrocatalysis of glucose by GOD in the films with ferrocenedicarboxylic acid as the mediator in solution. This film system with the unique binary architecture may establish a foundation for fabricating a novel type of multicontrollable biosensors based on bioelectrocatalysis with immobilized enzymes.

Determination of sphingomyelinase-D activity of Loxosceles venoms in sphingomyelin/cholesterol liposomes containing horseradish peroxidase

Based on degradation of sphingomyelin/cholesterol liposomes containing entrapped horseradish peroxidase, we evaluated the Sphingomyelinase-D (SMase-D) activity of scorpion, spider and snake venoms by monitoring spectrophotometrically the product of oxidation of HRP released. The results indicate that Loxosceles crude venoms (Loxosceles intermedia, Loxosceles laeta, Loxosceles gaucho and Loxosceles similis) displayed SMase-D activity in a concentration-dependent manner. Furthermore, this activity was blocked by the anti-loxoscelic antivenom. However, Tityus serrulatus scorpion venom, Phoneutria nigriventer spider venom and Bothrops jararaca, Crotalus durissus, Lachesis muta and Micrurus frontalis snake venoms did not show measurable SMase-D activity.

Direct electrochemistry and bioelectrocatalysis of horseradish peroxidase based on gold nano-seeds dotted TiO 2 nanocomposite

Gold nano-seeds (GNSs) ( Ф = 2–5 nm) were dotted in TiO 2 colloids and the horseradish peroxidase (HRP) was successfully immobilized on the as-made GNSs–TiO 2 nanocomposite by a convenient and effective method. The matrix integrates the merits of both GNSs and TiO 2, which provides a favorable microenvironment for the immobilization of HRP. The cyclic votammetric results demonstrated that the entrapped HRP achieves direct electron transfer at glassy carbon electrode (GCE). A pair of stable and quasi-reversible redox peaks with a small peak-to-peak separation of 43 mV was observed in phosphate buffer solution. The GNSs stabilized by TiO 2 colloids acted sufficiently as the conducting tunnel to promote the electron transfer. As a result, the electrochemical behaviors were improved in virtue of the synergic effect of TiO 2 and GNSs. The Nafion/HRP–GNSs–TiO 2/GCE displayed an excellent and rapid electrocatalytic response to the reduction of H 2O 2. The proposed biosensor exhibited a good linear response in the range from 4.1 × 10 −5 to 6.3 × 10 −4 mol L −1, with a detection limit of 5.9 × 10 −6 mol L −1 (at the ration of signal to noise, S/N = 3). The apparent Michaelis–Menten constant was estimated to be 0.63 mmol L −1. Furthermore, the biosensor possesses satisfactory stability and good reproducibility.

Zymographic assay of plant diamine oxidase on entrapped peroxidase polyacrylamide gel electrophoresis. A study of stability to proteolysis

A zymographic assay of diamine oxidase (DAO, histaminase, EC 1.4.3.6), based on a coupled peroxidase reaction, and its behavior at proteolysis in simulated gastric and intestinal conditions, are described. The DAO activity from a vegetal extract of Lathyrus sativus seedlings was directly determined on sodium dodecyl sulfate polyacrylamide electrophoretic gels containing entrapped horseradish peroxidase, with putrescine as substrate of histaminase and ortho-phenylenediamine as co-substrate of peroxidase. The accumulation of azo-aniline, as peroxidase-catalyzed oxidation product, led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of DAO. After image analysis of gels, a linear dependency of DAO content (Coomassie-stained protein bands) and of its enzymatic activity (zymographic bands) with the concentration of the vegetal extract was obtained. In simulated gastric conditions (pH 1.2, 37 °C), the DAO from the vegetal extract lost its enzymatic activity before 15 min of incubation, either in the presence or absence of pepsin. The protein pattern (Coomassie-stained) revealed that the DAO content from the vegetal extract was kept almost constant in the simulated intestinal fluid (containing pancreatin or not), with a slight diminution in the presence of pancreatic proteases. After 10 h of incubation at 37 °C, the DAO enzymatic activity from the vegetal extract was 44.7% in media without pancreatin and 13.6% in the presence of pancreatin, whereas the purified DAO retained only 4.65% of its initial enzymatic activity in the presence of pancreatin. FigureFigure Zymographic assay of DAO with peroxidase included in polyacrylamide gels

Using flowerlike polymer–copper nanostructure composite and novel organic–inorganic hybrid material to construct an amperometric biosensor for hydrogen peroxide

A new type of amperometric hydrogen peroxide biosensor was fabricated by entrapping horseradish peroxidase (HRP) in the organic–inorganic hybrid material composed of zirconia–chitosan sol–gel and Au nanoparticles (ZrO 2–CS–AuNPs). The sensitivity of the biosensor was enhanced by a flowerlike polymer–copper nanostructure composite (pPA–FCu) which was prepared from co-electrodeposition of CuSO 4 solution and 2,6-pyridinediamine solution. Several techniques, including UV–vis absorption spectroscopy, scanning electron microscopy, cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy were employed to characterize the assembly process and performance of the biosensor. The results showed that this pPA–FCu nanostructure not only had excellent redox electrochemical activity, but also had good catalytic efficiency for hydrogen peroxide. Also the ZrO 2–CS–AuNPs had good film forming ability, high stability and good retention of bioactivity of the immobilized enzyme. The resulting biosensors showed a linear range from 7.80 × 10 −7 to 3.7 × 10 −3 mol L −1, with a detection limit of 3.2 × 10 −7 mol L −1 (S/N = 3) under optimized experimental conditions. The apparent Michaelis–Menten constant was determined to be 0.32 mM, showing good affinity. In addition, the biosensor which exhibits good analytical performance, acceptable stability and good selectivity, has potential for practical applications.

Electrocatalytic activity of horseradish peroxidase/chitosan/carbon microsphere microbiocomposites to hydrogen peroxide

Colloidal carbon microspheres (CMS) are dispersed in chitosan (CHIT) solution to form an organic–inorganic hybrid with excellent micro-environment for the immobilization of biomolecules. A novel amperometric biosensor for the determination of hydrogen peroxide (H 2O 2) has been constructed by entrapping horseradish peroxidase (HRP) in as-synthesized CMS/CHIT hybrid. The modification of glassy carbon electrode is made by a simple solution-evaporation method. The electrochemical properties of the biosensor are characterized in electrochemical methods. The proposed biosensor shows high sensitive determination and fast response to H 2O 2 at −0.15 V. The constructed HRP/CHIT/CMS/GC electrode also exhibits a fine linear correlation with H 2O 2 concentration. The calculated value of the apparent Michaelis–Menten constant, 2.33 mM, suggests that the HRP in CMS/CHIT hybrid keeps its native bioactivity and has high affinity for H 2O 2.

Immobilization of horseradish peroxidase on chitosan/silica sol–gel hybrid membranes for the preparation of hydrogen peroxide biosensor

A simple and effective strategy for fabrication of hydrogen peroxide (H 2O 2) biosensor has been developed by entrapping horseradish peroxidase (HRP) in chitosan/silica sol–gel hybrid membranes (CSHMs) doped with potassium ferricyanide (K 3Fe(CN) 6) and gold nanoparticles (GNPs) on platinum electrode surface. The hybrid membranes are prepared by cross-linking chitosan (CS) with 3-aminopropyltriethoxysilane (APTES), while the presence of GNPs improved the conductivity of CSHMs, and the Fe(CN) 6 3−/4− was used as a mediator to transfer electrons between the electrode and HRP due to its excellent electrochemistry activity. UV–Vis absorption spectroscopy was employed to characterize the different components in the CSHMs and their interaction. The parameters influencing the performance of the resulting biosensor were optimized and the characteristic of the resulting biosensor was characterized by cyclic voltammetry and chronoamperometry. Linear calibration for hydrogen peroxide was obtained in the range of 3.5 × 10 − 6 to 1.4 × 10 − 3 M under the optimized conditions with the detection limit (S/N = 3) of 8.0 × 10 − 7 M. The apparent Michaelis–Menten constant of the enzyme electrode was 0.93 mM. The enzyme electrode retained about 78% of its response sensitivity after 30 days. The system was applied for the determination of the samples, and the results obtained were satisfactory.

A bienzyme channeling glucose sensor with a wide concentration range based on co-entrapment of enzymes in SBA-15 mesopores

A novel bienzyme-channeling sensor was constructed by entrapping glucose oxidase (GOD) and horseradish peroxidase (HRP) in the mesopores of well-ordered hexagonal mesoporous silica structures (SBA-15). The SBA-15 mesoporous materials accelerated the electron transfer between the entrapped HRP and electrode. Both HRP and GOD retained their catalytic activities in the bienzyme-entrapped SBA-15 film. In presence of glucose the enzymatic reaction of GOD-glucose-dissolved oxygen system generated hydrogen peroxide in the bienzyme-entrapped mesopores, which was immediately reduced at −0.40 V by an electrocatalytic reaction with the HRP entrapped in the same mesopore to lead to a sensitive and fast amperometric response. Thus the bienzyme channeling could be used for the detection of glucose with excellent performance without the addition of any mediator. Optimization of the experimental parameters was performed with regard to pH, operating potential and temperature. The detection limit was down to 2.7 × 10 −7 M with a very wide linear range from 3.0 × 10 −6 to 3.4 × 10 −2 M. The constructed bienzyme channeling provided a strategy for amperometric detection of oxidase substrates by co-entrapping the corresponding oxidase and HRP in the mesoporous materials.