Direct electrochemistry of horseradish peroxidase immobilized on a colloid/cysteamine-modified gold electrode

Direct electron transfer of immobilized horseradish peroxidase on gold colloid and its application as a biosensor were investigated by using electrochemical methods. The Au colloids were associated with a cysteamine monolayer on the gold electrode surface. A pair of redox peaks attributed to the direct redox reaction of horseradish peroxidase (HRP) were observed at the HRP/Au colloid/cysteamine-modified electrode in 0.1 M phosphate buffer (pH 7.0). The surface coverage of HRP immobilized on Au colloid was about 7.6 x 10(-10) mol/cm(2). The sensor displayed an excellent electrocatalytic response to the reduction of H(2)O(2) without the aid of an electron mediator. The calibration range of H(2)O(2) was 1. 4 microM to 9.2 mM with good linear relation from 1.4 microM to 2.8 mM. A detection limit of 0.58 microM was estimated at a signal-to-noise ratio of 3. The sensor showed good reproducibility for the determination of H(2)O(2). The variation coefficients were 3. 1 and 3.9% (n = 10) at 46 microM and 2.8 mM H(2)O(2), respectively. The response showed a Michaelis-Menten behavior at higher H(2)O(2) concentrations. The K(app)(M) value for the H(2)O(2) sensor was found to be 2.3 mM.     

Lipid membrane immobilized horseradish peroxidase biosensor for amperometric determination of hydrogen peroxide

Stable films of didodecyldimethylammonium bromide (DDAB, a synthetic lipid) and horseradish peroxidase (HRP) were made by casting the mixture of the aqueous vesicle of DDAB and HRP onto the glassy carbon (GC) electrode. The direct electron transfer between electrode and HRP immobilized in lipid film has been demonstrated. The lipid films were used to supply a biological environment resembling biomembrane on the surface of the electrode. A pair of redox peaks attributed to the direct redox reaction of HRP were observed in the phosphate buffer solution (pH 5.5). The cathodic peak current increased dramatically while anodic peak decreased by addition of small amount H(2)O(2). The pH effect on amperometric response to H(2)O(2) was studied. The biosensor also exhibited fast response (5 s), good stability and reproducibility.     

Fabrication of hydrogen peroxide biosensor based on Ni doped SnO2 nanoparticles

Ni doped SnO(2) nanoparticles (0-5 wt%) have been prepared by a simple microwave irradiation (2.45 GHz) method. Powder X-ray diffraction (XRD) and transmission electron microscopy (TEM) studies confirmed the formation of rutile structure with space group (P(42)/mnm) and nanocrystalline nature of the products with spherical morphology. Direct electrochemistry of horseradish peroxidase (HRP)/nano-SnO(2) composite has been studied. The immobilized enzyme retained its bioactivity, exhibited a surface confined, reversible one-proton and one-electron transfer reaction, and had good stability, activity and a fast heterogeneous electron transfer rate. A significant enzyme loading (3.374×10(-10) mol cm(-2)) has been obtained on nano-Ni doped SnO(2) as compared to the bare glassy carbon (GC) and nano-SnO(2) modified surfaces. This HRP/nano-Ni-SnO(2) film has been used for sensitive detection of H(2)O(2) by differential pulse voltammetry (DPV), which exhibited a wider linearity range from 1.0×10(-7) to 3.0×10(-4)M (R=0.9897) with a detection limit of 43 nM. The apparent Michaelis-Menten constant (K(M)(app)) of HRP on the nano-Ni-SnO(2) was estimated as 0.221 mM. This excellent performance of the fabricated biosensor is attributed to large surface-to-volume ratio and Ni doping into SnO(2) which facilitate the direct electron transfer between the redox enzyme and the surface of electrode.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase based on clay-chitosan-gold nanoparticle nanocomposite

Gold nanoparticles stabilized by chitosan (AuCS) were hybridized with exfoliated clay nanoplates through electrostatic interaction. The resulting clay-chitosan-gold nanoparticle nanocomposite (Clay/AuCS) was used to modify glassy carbon electrode (GCE). HRP, a model peroxidase, was entrapped between the Clay/AuCS film and another clay layer. UV-vis spectrum suggested HRP retained its native conformation in the modified film. Basal plane spacing of clay obtained by X-ray diffraction (XRD) indicated that there was an intercalation-exfoliation-restacking process among HRP, AuCS and clay during the modified film drying. The immobilized HRP showed a pair of quasi-reversible redox peaks at -0.195 V (vs. saturated Ag/AgCl electrode) in 0.1M PBS (pH 7.0), and the biosensor displayed a fast amperometric response to H(2)O(2) with a wide linear range of 39 microM to 3.1 mM. The detection limit was 9.0 microM based on the signal to noise ratio of 3. The kinetic parameters such as alpha (charge transfer coefficient), k(s) (electron transfer rate constant) and K(m) (Michaelis-Menten constant) were evaluated to be 0.53, 2.95+/-0.20s(-1) and 23.15 mM, respectively.     

The potential use of hydrazine as an alternative to peroxidase in a biosensor: comparison between hydrazine and HRP-based glucose sensors

The potential use of hydrazine sulfate was examined for the catalytic reduction of enzymatically generated H2O2 in a biosensor system. The performance of the hydrazine-based sensor was compared with an HRP-based glucose sensor as a model of a biosensor. Hydrazine and HRP were covalently immobilized onto a conducting polymer layer with glucose oxidase. The direct electron transfer reactions of the immobilized hydrazine and HRP onto the poly-5,2':5,2'-terthiophene-3'-carboxylic acid (poly-TTCA) layer were investigated by using cyclic voltammetric method and the electron transfer rate constants were determined. The glucose oxidase- and hydrazine-immobilized sensor efficiently reduced the enzymatically generated H2O2 at -0.15 V versus Ag/AgCl. The surface of this GOx/hydrazine/poly-TTCA-based glucose sensor was characterized by QCM, SEM, and ESCA. Glucose-sensing properties were studied using cyclic voltammetric and chronoamperometric techniques. Various experimental parameters were optimized according to the amount of hydrazine, pH, the temperature, and the applied potential. A linear calibration plot was obtained in the concentration range between 0.1 and 15.0 mM, and the detection limit was determined to be 40.0+/-7.0 microM. Interferences from other biological compounds were studied. The long-term stability of the GOx/hydrazine sensor was better than that of the one based on a GOx/HRP biosensor. The proposed glucose sensor was successfully applied to human whole blood and urine samples for the detection of glucose.     

A third-generation hydrogen peroxide biosensor based on horseradish peroxidase immobilized in a tetrathiafulvalene-tetracyanoquinodimethane/multiwalled carbon nanotubes film

A new third-generation biosensor for H(2)O(2) assay was developed on the basis of the immobilization of horseradish peroxidase (HRP) in a nanocomposite film of tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ)/multiwalled carbon nanotubes (MWCNTs) modified gold electrode. The prepared HRP/TTF-TCNQ/MWCNTs/Au electrode was used for the bioelectrocatalytic reduction of H(2)O(2), with a linear range from 0.005 to 1.05mM and a detection limit of 0.5muM for amperometric sensing of H(2)O(2). In addition, a novel method on the basis of electrochemical quartz crystal microbalance (EQCM) measurements was proposed to determine the effective enzymatic specific activity (ESA) of the immobilized HRP for the first time, and the ESA was found to be greater at the TTF-TCNQ/MWCNTs/Au electrode than that at the MWCNTs/Au or TTF-TCNQ/Au electrode, indicating that the TTF-TCNQ/MWCNTs film is a good HRP-immobilization matrix to achieve the direct electron transfer between the enzyme and the electrode.     

Immobilization of glucose oxidase on electrodeposited nickel oxide nanoparticles: direct electron transfer and electrocatalytic activity

For the first time glucose oxidase (GOx) was successfully co-deposited on nickel-oxide (NiO) nanoparticles at a glassy carbon electrode. In this paper we present a simple fabrication method of biosensor which can be easily operated without using any specific reagents. Cyclic voltammetry was used for electrodeposition of NiO nanoparticle and GOx immobilization. The direct electron transfer of immobilized GOx displays a pair of well defined and nearly reversible redox peaks with a formal potential (E(0')) of -0.420 V in pH 7 phosphate buffer solution and the response shows a surface controlled electrode process. The surface coverage and heterogeneous electron transfer rate constant (k(s)) of GOx immobilized on NiO film glassy carbon electrode are 9.45 x 10(-13)mol cm(-2) and 25.2+/-0.5s(-1), indicating the high enzyme loading ability of the NiO nanoparticles and great facilitation of the electron transfer between GOx and NiO nanoparticles. The biosensor shows excellent electrocatalytical response to the oxidation of glucose when ferrocenmethanol was used as an artificial redox mediator. Furthermore, the apparent Michaelis-Menten constant 2.7 mM, of GOx on the nickel oxide nanoparticles exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. In addition, this glucose biosensor shows fast amperometric response (3s) with the sensitivity of 446.2nA/mM, detection limit of 24 microM and wide concentration range of 30 microM to 5mM. This biosensor also exhibits good stability, reproducibility and long life time.     

Amperometric biosensor based on coentrapment of enzyme and mediator by gold nanoparticles on indium-tin oxide electrode

A disposable pseudo-mediatorless amperometric biosensor has been fabricated for the determination of hydrogen peroxide (H2O2). In the current study, an indium-tin oxide (ITO) electrode was modified with thiol functional group by (3-mercaptopropyl)trimethoxysilane. The stable nano-Au-SH monolayer (AuS) was then prepared through covalent linking of gold nanoparticles and thiol groups on the surface of the ITO. The horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) were finally coentrapped by the colloidal gold nanoparticles. The immobilized TMB was used as an electron transfer mediator that displayed a surface-controlled electrode process at a scan rate of less than 50mV/s. The biosensor was characterized by photometric and electrochemical measurements. The results showed that the prepared AuS monolayer not only could steadily immobilize HRP but also could efficiently retain HRP bioactivity. Parameters affecting the performance of the biosensor, including the concentrations of the immobilized TMB and HRP, the pH value, and the reaction temperature, were optimized. Under the optimized experimental conditions, H(2)O(2) could be determined in a linear calibration range from 0.005 to 1.5mM with a correlation coefficient of 0.998 (n=14) and a detection limit of 1microM at a signal/noise ratio of 3. The proposed method provides a new alternative to develop low-cost biosensors by using ITO film electrodes from industrial mass production.     

Multiwalled carbon nanotubes dispersed in carminic acid for the development of catalase based biosensor for selective amperometric determination of H(2)O(2) and iodate

We report the preparation of stable dispersion of multiwalled carbon nanotubes (MWCNTs) using carminic acid (CA) as a dispersing agent. The transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) results confirmed that MWCNT is well dispersed in CA aqueous solution and CA has been well adsorbed at MWCNT walls. Fourier transform infrared (FTIR) and UV-vis absorption spectra results also confirmed the adsorption of CA at MWCNT. To develop a highly selective amperometric biosensor for H(2)O(2) and iodate, the model enzyme catalase (CAT) was immobilized at CACNT modified glassy carbon electrode surface. The immobilized CAT exhibits well defined quasi reversible redox peaks at a formal potential (E°') of -0.559V in 0.05M pH 7 phosphate buffer solution (PBS). The proposed CAT/CACNT biosensor exhibits excellent amperometric response towards H(2)O(2) and iodate in the linear concentration range between 10μM to 3.2mM and 0.01-2.16mM. The sensitivity values are 287.98μAmM(-1)cm(-2) and 0.253mAmM(-1)cm(-2), respectively. Moreover, the developed CAT biosensor exhibits high affinity for H(2)O(2) and iodate with good selectivity.     

A novel hydrogen peroxide sensor based on the direct electron transfer of horseradish peroxidase immobilized on silica-hydroxyapatite hybrid film

The direct electron transfer of immobilized horseradish peroxidase (HRP) on silica-hydroxyapatite (HAp) hybrid film-modified glassy carbon electrode (GCE) and its application as H(2)O(2) biosensors were investigated. On silica/HRP-HAp/GCE, HRP displayed a fast electron transfer process accompanied with one proton participate in. This sensor exhibited an excellent electrocatalytic response to the reduction of H(2)O(2) without the aid of an electron mediator. The proposed biosensor showed good reproducibility and high sensitivity to H(2)O(2) with the detection limit of 0.35 microM. In the range of 1.0-100 microM, the catalytic reduction current of H(2)O(2) was proportional to H(2)O(2) concentration. The apparent Michaelis-Menten constant (k(m)(app)) of the biosensor was calculated to be 21.8 microM, exhibiting a high enzymatic activity and affinity for H(2)O(2).     

Hydrogen peroxide detection at a horseradish peroxidase biosensor with a Au nanoparticle-dotted titanate nanotube|hydrophobic ionic liquid scaffold

In this work, a novel sensing scaffold, consisting Au nanoparticle (GNP)-dotted TiO(2) nanotubes (TNTs) as the rigid material and the hydrophobic ionic liquid (HIL), 1-decyl-3-methylimidazolium tetrafluoroborate, as the entrapping agent, was applied to facilitate the electron transfer of horseradish peroxidase (HRP) on a glassy carbon electrode. GNPs were immobilised on the TNTs in our work using a one-step reduction of HAuCl(4)·3H(2)O by sodium borohydride in the presence of sodium citrate as a stabilising reagent. The morphology and composition of the as-synthesised composite materials were characterised by transmission electron microscopy, scanning electron microscopy, X-ray diffraction and Fourier-transform infrared spectroscopy. Cyclic voltammetry of HRP at the modified electrode presented a pair of reproducible, quasi-reversible redox peaks with a peak-to-peak separation of 69 mV, indicating electron transfer between HRP and composite electrode. The GNP-TNT|HIL|HRP electrode was then applied to the detection of H(2)O(2) in a pH 7.0 phosphate buffer using chronoamperometry. The biosensor exhibited a linear response in the 15-750 μM range, and a limit of detection of 2.2 μM. The biosensor also exhibited stability with 90% of the detection signal retained over a two-week duration.     

Highly sensitive and selective hydrogen peroxide biosensor based on hemoglobin immobilized at multiwalled carbon nanotubes-zinc oxide composite electrode

A highly sensitive and selective amperometric hydrogen peroxide (H(2)O(2)) biosensor based on immobilization of hemoglobin (Hb) at multiwalled carbon nanotubes-zinc oxide (MWCNT/ZnO) composite modified glassy carbon electrode (GCE) is reported. ZnO microsponges were electrochemically grown on MWCNT surface by the simple, cost-effective, green, electrochemical method at room temperature. The MWCNT/ZnO/Hb composite film showed a pair of well-defined, quasi-reversible redox peaks with a formal potential (E°') of -0.336V, characteristic features of heme redox couple of Hb. The electron transfer rate constant (k(s)) of immobilized Hb was 1.26s(-1). The developed biosensor showed a very fast response (>2s) toward H(2)O(2) with good sensitivity, wide linear range, and low detection limit of 0.02μM. The fabricated biosensor showed interesting features, including high selectivity, acceptable stability, good reproducibility, and repeatability along with excellent conductivity, facile electron mobility of MWCNT, and good biocompatibility of ZnO. The fabrication method of this biosensor is simple and effective for determination of H(2)O(2) in real samples with quick response, good sensitivity, high selectivity, and acceptable recovery.     

Electrochemically deposited chitosan hydrogel for horseradish peroxidase immobilization through gold nanoparticles self-assembly

A new strategy for immobilization of horseradish peroxidase (HRP) has been presented by self-assembling gold nanoparticles on chitosan hydrogel modified Au electrode. From a mildly acidic chitosan solution, a chitosan film is electrochemically deposited on Au electrode surface via a negative voltage bias. This process is accompanied by the hydrogen evolution reaction, and the released hydrogen gas made the deposited chitosan film with porous structure, which facilitates the assembly of gold nanoparticles and HRP. The resulting substrates were characterized by atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). The immobilized HRP displayed an excellent catalytic property to the reduction of H2O2 in the presence of methylene blue mediator. The resulting biosensor (HRP-modified electrode) showed a wide dynamic range of 8.0 microM-15 mM H2O2, and the linear ranges were 8.0 microM-0.12 mM and 0.50-12 mM, with a detection limit of 2.4 microM estimated at a signal-to-noise ratio of 3. Moreover, the biosensor remained about 85% of its original sensitivity after four weeks' storage.     

Direct electrochemistry of horseradish peroxidase on TiO(2) nanotube arrays via seeded-growth synthesis

Horseradish peroxidase (HRP) was successfully immobilized on vertically oriented TiO(2) nanotube arrays (NTAs), which was prepared by a seeded-growth mechanism. The nanotubular structure of TiO(2) was characterized by scanning electron microscope (SEM). After encapsulated HRP on TiO(2) nanotube arrays, the direct electron transfer of HRP was observed. Owing to the redox reaction of electroactive center of HRP, the HRP/TiO(2) NTAs modified electrode exhibited a pair of quasi-reversible peaks with the peak-to-peak separation of 70mV and the formal potential of -0.122V (vs. SCE) in 0.2molL(-1) phosphate buffer solution (PBS, pH 7.0). The number of transference electron was 0.84 and the direct electron transfer (ET) constant (k(s)) was 3.82s(-1). The HRP/TiO(2) NTAs modified electrode displayed an excellent electrocatalytic performance for H(2)O(2) and the formal Michaelis-Menten constant (K(m)(app)) was 1.9mmolL(-1). The response currents had a good linear relation with the concentration of H(2)O(2) from 5.0x10(-7)molL(-1) to 1.0x10(-5)molL(-1) and 5.0x10(-5)molL(-1) to 1.0x10(-3)molL(-1), respectively.     

Direct electrochemistry and electrocatalysis based on a film of horseradish peroxidase intercalated into Ni-Al layered double hydroxide nanosheets

Positively charged Ni-Al layered double hydroxide nanosheets (Ni-Al LDHNS) have been used for the first time as matrices for immobilization of horseradish peroxidase (HRP) in order to fabricate enzyme electrodes for the purpose of studying direct electron transfer between the redox centers of proteins and underlying electrodes. X-ray diffraction (XRD) and high-resolution transmission electron microscopy (HRTEM) revealed that the HRP-Ni-Al LDHNS film had an ordered structure and that HRP was intercalated into Ni-Al LDHNS with a monolayer arrangement. Field emission scanning electron microscopy (FESEM) showed that the HRP-Ni-Al LDHNS film had a uniform, porous morphology. UV-vis spectroscopy indicated that the intercalated HRP retained its native structure after incorporation in the Ni-Al LDHNS film. The immobilized HRP in Ni-Al LDHNS on the surface of a glassy carbon electrode (GCE) exhibited good direct electrochemical and electrocatalytic responses to the reduction of hydrogen peroxide (H(2)O(2)) and trichloroacetic acid (TCA). The resulting H(2)O(2) biosensor showed a wide linear range from 6.00x10(-7)M to 1.92x10(-4)M, low detection limit (4.00x10(-7)M) and good stability. The results show that Ni-Al LDHNS provide a novel and efficient platform for the immobilization of enzymes and realizing direct electrochemistry and that the materials have potential applications in the fabrication of third-generation biosensors.     

Electrochemical studies on horseradish peroxidase covalently coupled with redox dyes

The present study aims at investigating the use of redox dyes as non-diffusional electron mediators in hydrogen peroxide biosensors using horseradish peroxidase (HRP). We observe that the two redox dyes Safranine O and Neutral Red covalently bound to HRP, efficiently mediate electron transfer from the active site of the enzyme to the electrode surface. Dyes bound to the enzyme using a spacer arm diaminohexane further enhance the electron transfer. The enzyme electrodes show a linear response to the concentration of H2O2 up to 500 microM concentration and with a detection limit of around 50 microM. The dyes can be used as coupled mediators to develop a successful electro-optical biosensor.     

A facile approach to immobilize protein for biosensor: self-assembled supported bilayer lipid membranes on glassy carbon electrode

A kind of solid substrate, glassy carbon (GC) electrode, was selected to support self-assembled lipid layer membranes. On the surface of GC electrode, we made layers of dimyristoylphosphatidylcholine (DMPG, a kind of lipid). From electrochemical impedance experiments, we demonstrated that the lipid layers on the GC electrode were bilayer lipid membranes. We immobilized horseradish peroxidase (HRP) into the supported bilayer lipid membranes (s-BLM) to develop a kind of mediator-free biosensor for H2O2. The biosensor exhibited fine electrochemical response, stability and reproducibility due to the presence of the s-BLM. As a model of biological membrane, s-BLM could supply a biological environment for enzyme and maintain its activity. So s-BLM is an ideal choice to immobilize enzyme for constructing the mediator-free biosensor based on GC electrode.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in sol-gel-derived ceramic-carbon nanotube nanocomposite film

The sol-gel-derived ceramic-carbon nanotube (SGCCN) nanocomposite film fabricated by doping multiwall carbon nanotubes (MWNTs) into a silicate gel matrix was used to immobilize protein. The SGCCN film can provide a favorable microenvironment for horseradish peroxidase (HRP) to perform direct electron transfer (DET) at glassy carbon electrode. The HRP immobilized in the SGCCN film shows a pair of well-defined redox waves and retains its bioelectrocatalytic activity to the reduction of O2 and H2O2, which is superior to that immobilized in silica sol-gel film.     

A novel approach to construct a horseradish peroxidase|hydrophilic ionic liquids|Au nanoparticles dotted titanate nanotubes biosensor for amperometric sensing of hydrogen peroxide

The direct electrochemistry of horseradish peroxidase (HRP) on a novel sensing platform modified glassy carbon electrode (GCE) has been achieved. This sensing platform consists of Nafion, hydrophilic room-temperature ionic liquid (RTIL) and Au nanoparticles dotted titanate nanotubes (GNPs-TNTs). The composite of RTIL and GNPs-TNTs was immobilized on the electrode surface through the gelation of a small amount of HRP aqueous solution. The composite was characterized by transmission electron microscopy (TEM), powder X-ray diffraction (XRD) and infrared spectroscopy (IR). UV-Vis and IR spectroscopy demonstrated that HRP in the composite could retain its native secondary structure and biochemical activity. The HRP-immobilized electrode was investigated by cyclic voltammetry and chronoamperometry. The results from both techniques showed that the direct electron transfer between the nanocomposite modified electrodes and heme in HRP could be realized. The biosensor responded to H(2)O(2) in the linear range from 5×10(-6) to 1×10(-3) mol L(-1) with a detection limit of 2.1×10(-6) mol L(-1) (based on the S/N=3).     

Direct electrochemistry of glucose oxidase and biosensing for glucose based on boron-doped carbon nanotubes modified electrode

Due to their unique physicochemical properties, doped carbon nanotubes are now extremely attractive and important nanomaterials in bioanalytical applications. In this work, selecting glucose oxidase (GOD) as a model enzyme, we investigated the direct electrochemistry of GOD based on the B-doped carbon nanotubes/glassy carbon (BCNTs/GC) electrode with cyclic voltammetry. A pair of well-defined, quasi-reversible redox peaks of the immobilized GOD was observed at the BCNTs based enzyme electrode in 0.1M phosphate buffer solution (pH 6.98) by direct electron transfer between the protein and the electrode. As a new platform in glucose analysis, the new glucose biosensor based on the BCNTs/GC electrode has a sensitivity of 111.57 microA mM(-1)cm(-2), a linear range from 0.05 to 0.3mM and a detection limit of 0.01mM (S/N=3). Furthermore, the BCNTs modified electrode exhibits good stability and excellent anti-interferent ability to the commonly co-existed uric acid and ascorbic acid. These indicate that boron-doped carbon nanotubes are the good candidate material for the direct electrochemistry of the redox-active enzyme and the construction of the related enzyme biosensors.