Fabrication of herbicide biosensors based on the inhibition of enzyme activity that catalyzes the scavenging of hydrogen peroxide in a thylakoid membrane.

A novel herbicide biosensor with a thylakoid modified membrane electrode is presented. Thylakoid, isolated from spinach leaves, was entrapped in a membrane of poly (vinylalcohol) with the styrylpyridinium group (PVA-SbQ). The thylakoid membrane was fixed on the surface of a platinum electrode. It was found that the enzymes in thylakoid kept their activity for several months in the membrane. The oxidative current of hydrogen peroxide in a Tris-HCl buffer solution (pH 7.4) was detected at the modified electrode by a differential pulse voltammetric method. In the presence of herbicides, the oxidation current from the hydrogen peroxide decreased due to an inhibitor effect on the enzymes in thylakoid compared with that in the absence of the herbicides. The changes in the oxidation current at the electrode were proportional to the herbicide concentrations. The sensor could be used to detect herbicides in concentration ranges of 3 x 10(-9) - 1.5 x 10(-7) M for paraquat, 1 x 10(-8) - 3 x 10(-7) M for diuron, 4 x 10(-8) - 3 x 10(-6) M for prometryn, 5 x 10(-8) - 5 x 10(-6) M for atrazine and 1 x 10(-7) - 5 x 10(-6) M for ametryn, respectively. The enzyme activity on scavenging hydrogen peroxide in the modified PVA-SbQ membrane was examined.     

Homogenous electrogenerated chemiluminescence immunoassay for human immunoglobulin G using N-(aminobutyl)-N-ethylisoluminol as luminescence label at gold nanoparticles modified paraffin-impregnated graphite electrode

A homogeneous electrogenerated chemiluminescence (ECL) immunoassay for human immunoglobulin G (hIgG) has been developed using a N-(aminobutyl)-N-ethylisoluminol (ABEI) as luminescence label at gold nanoparticles modified paraffin-impregnated graphite electrode (PIGE). ECL emission was electrochemically generated from the ABEI-labeled anti-hIgG antibody and markedly increased in the presence of hIgG antigen due to forming a more rigid structure of the ABEI moiety. The concentration of hIgG antigen was determined by the increase of ECL intensity at a gold nanoparticles modified PIGE. It was found that the ECL intensity of ABEI in presence of hydrogen peroxide was dramatically enhanced at gold nanoparticles modified PIGE in neutral aqueous solution and the detection limit of ABEI was 2 x 10(-14)mol/L (S/N=3). The integral ECL intensity was linearly related to the concentration of hIgG antigen from 3.0 x 10(-11) to 1.0 x 10(-9)g/mL with a detection limit of 1 x 10(-11)g/mL (S/N=3). The relative standard deviation was 3.1% at 1.0 x 10(-10)g/mL (n=11). This work demonstrates that the enhancement of the sensitivity of ECL and ECL immunoassay at a nanoparticles modified electrode is a promising strategy.     

An amperometric hydrogen peroxide biosensor based on immobilizing horseradish peroxidase to a nano-Au monolayer supported by sol-gel derived carbon ceramic electrode

A novel strategy for fabricating horseradish peroxidase (HRP)-based H(2)O(2) sensor has been developed by combining the merits of carbon sol-gel supporting matrix and nano-scaled particulate gold (nano-Au) mediator. The thiol functional group-derived carbon ceramic electrode (CCE) was first constructed using (3-mercaptopropyl) trimethoxy silane as sol-gel monomer. Then, the stable nano-Au monolayer was obtained through covalent linkage between nano-Au and thiol group on the surface of CCE. The experimental results showed that nano-Au monolayer formed not only could steadily immobilize HRP but also efficiently retain its bioactivity. Hydrogen peroxide was detected with the aid of hydroquinone mediator to transfer electrons between the electrode and HRP. The process parameters for the fabrication of the enzyme electrode and various experimental variables such as the operating potential, mediator concentration and pH of background electrolyte were explored for optimum analytical performance of the enzyme electrode. The biosensor had a fast response of less than 8 s with linear range of 1.22 x 10(-5) to 1.10 x 10(-3)mol l(-1) and a detection limit of 6.1 x 10(-6)mol l(-1). The sensitivity of the sensor for H(2)O(2) was 0.29 A l mol(-1) cm(-2). The activation energy for enzyme reaction was calculated to be 10.1 kJ mol(-1). The enzyme electrode retained 75% of its initial activity after 5 weeks storage in phosphate buffer at pH 7.     

An Unmediated Hydrogen Peroxide Sensor Based on a Hemoglobin-sds Film Modified Electrode

ABSTRACT

An unmediated hydrogen peroxide sensor is designed in this paper by employing a hemoglobin-SDS film modified electrode. Hemoglobin exhibits direct (unmediated) electrochemistry at the modified electrode. The protein also shows elegant catalytic activity towards the electrochemical reduction of hydrogen peroxide. Consequently, a prototype hydrogen peroxide sensor is prepared. Under optimum conditions, this sensor provides a linear response over the hydrogen peroxide concentrations in the range of 1×10^-5～1×10^-4 mol/L. The detection limit was 2×10^-6 mol/L The relative standard deviation was 4.2% for 6 successive determinations of the hydrogen peroxide at 1×10^-5 mol/L. This configuration is shown to be sensitive, stable and easily fabricated. It might be useful in the biological and industrial fields.     

Electrocatalytic Reduction of H2O2 and Oxygen on the Surface of Thionin Incorporated onto MWCNTs Modified Glassy Carbon Electrode: Application to Glucose Detection

A new H₂O₂ enzymeless sensor has been fabricated by incorporation of thionin onto multiwall carbon nanotubes (MWCNTs) modified glassy carbon electrode. First 50 μL of acetone solution containing dispersed MWCNTs was pipetted onto the surface of GC electrode, then, after solvent evaporations, the MWCNTs modified GC electrode was immersed into an aqueous solution of thionin (electroless deposition) for a short period of time <5–50 s. The adsorbed thin film of thionin was found to facilitate the reduction of hydrogen peroxide in the absence of peroxidase enzyme. Also the modified electrode shows excellent catalytic activity for oxygen reduction at reduced overpotential. The rotating modified electrode shows excellent analytical performance for amperometric determination of hydrogen peroxide, at reduced overpotentials. Typical calibration at ?0.3 V vs. reference electrode, Ag/AgCl/3 M KCl, shows a detection limit of 0.38 μM, a sensitivity of 11.5 nA/μM and a liner range from 20 μM to 3.0 mM of hydrogen peroxide. The glucose biosensor was fabricated by covering a thin film of sol–gel composite containing glucose oxides on the surface of thionin/MWCNTs modified GC electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The detection limit, sensitivity and liner calibration rang were 1 μM, 18.3 μA/mM and 10 μM–6.0 mM, respectively. In addition biosensor can reach 90% of steady currents in about 3.0 s and interference effect of the electroactive existing species (ascorbic acid–uric acid and acetaminophen) is eliminated. The usefulness of biosensor for direct glucose quantification in human blood serum matrix is also discussed. This sensor can be used as an amperometric detector for monitoring oxidase based biosensors.     

Diffusion coefficient measurements in microfluidic devices

A glassy carbon electrode (GCE) modified with Pd/IrO(2) provides excellent electrocatalytic oxidation of hydrogen peroxide. Glucose oxidase (GOD) and xanthine oxidase (XOD) were co-immobilized on the modified electrode with a thin film Nafion coated on the enzyme layer to form a glucose (Glu)/hypoxanthine (Hx) sensor, without interference from electroactive species such as ascorbic acid (AA) and uric acid (UA). Its response was evaluated with respect to the enzyme amount on the electrode, pH and temperature of the electrolyte. The prepared bienzymic biosensor, used as the detector of HPLC gave a detection limit of 1.0x10(-6) mol l(-1) Glu and 2.0x10(-7) mol l(-1) Hx (Hx) with a linear concentration range of 5.0x10(-6)-2.5x10(-3) mol l(-1) and 1.0x10(-6)-5.0x10(-4) mol l(-1), respectively. Coupled with microdialysis, it was used to monitor the concentrations of Glu and Hx in rat brain.     

Development of a diamine biosensor

An amperometric diamine sensor is developed for clinical applications in diagnosis of bacterial vaginosis (BV). The sensor is based on crosslinked putrescine oxidase (PUO) which catalyzes the conversion of diamines (mainly putrescine and cadaverine) to products including hydrogen peroxide. The hydrogen peroxide is detected anodically at platinum electrode polarized at 0.5 V versus Ag/AgCl. Platinum-plated gold electrodes used as a substrate for the sensor construction, are batch-fabricated on a flexible polyimide foil (Kapton(R), DuPont). A three-electrode cell configuration is used in all amperometric measurements. The sensor construction is based on three layers: an inner layer to reject the interference effect of oxidizable molecules, an outer diffusion controlling layer, and in addition, an enzyme middle layer. The enzyme layer was immobilized by crosslinking PUO with bovine serum albumin (BSA) using glutaraldehyde (GA). An optimization study of the enzyme solution composition was carried out. With the optimized enzyme layer, the biosensor showed a very high sensitivity and fast response time of ca. 20 s. The sensor has a linear dynamic range from (0.5-300 muM) for putrescine that covers the expected biological levels of the analyte. Details on sensor fabrication and characterization are given in the present work.     

Electropolymerized Diphenylamine on Functionalized Multiwalled Carbon Nanotube Composite Film and Its Application to Develop a Multifunctional Biosensor

Diphenylamine (DPA) monomers have been electropolymerized on the amino‐functionalized multiwalled carbon nanotube (AFCNT) composite film modified glassy carbon electrode (GCE) by cyclic voltammetry (CV). The surface morphology of PDPA‐AFCNT was studied using field‐emission scanning electron microscopy (FE‐SEM). The interfacial electron transfer phenomenon at the modified electrode was studied using electrochemical impedance spectroscopy (EIS). The PDPA‐AFCNT/GCE represented a multifunctional sensor and showed good electrocatalytic behavior towards the oxidation of catechol and the reduction of hydrogen peroxide. Rotating‐disk electrode technique was applied to detect catechol with a sensitivity of 1360 µA mM^?1 cm^?2 and a detection limit of 0.01 mM. Amperometric determination of hydrogen peroxide at the PDPA‐AFCNT film modified electrode results in a linear range from 10 to 800 µM, a sensitivity of 487.1 µA mM^?1 cm^?2 and detection limit of 1 µM. These results show that the nano‐composite film modified electrode can be utilized to develop a multifunctional sensor.     

An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a beta-type zeolite modified electrode

A new biosensor for the amperometric detection of hydrogen peroxide was developed based on the coimmobilization of horseradish peroxidase (HRP) and methylene blue on a beta-type zeolite modified glassy carbon electrode without the commonly used bovine serum albumin-glutaraldehyde. The intermolecular interaction between enzyme and zeolite matrix was investigated using FT-IR. The cyclic voltammetry and amperometric measurement demonstrated that methylene blue co-immobilized with HRP in this way displayed good stability and could efficiently transfer electrons between immobilized HRP and the electrode. The sensor responded rapidly to H2O2 in the linear range from 2.5 x 10(-6) to 4.0 x 10(-3) M with a detection limit of 0.3 microM. The sensor was stable in continuous operation.     

Enhanced electrochemiluminescence employed for the selective detection of methyl parathion based on a zirconia nanoparticle film modified electrode

A simple, rapid and sensitive electrochemiluminescence (ECL) sensor was proposed for direct measurements of methyl parathion (MP) based on the strong affinity of a nano zirconia particles (ZrO(2) NPs) modified film on the electrode to the phosphoric group. ZrO(2) NPs, which could provide a larger absorption area to immobilize organophosphorus, was firstly modified on the glassy carbon electrode surface to prepare the proposed ECL sensor (ZrO(2)/GC). Subsequently, the ZrO(2)/GC electrode was scanned from -0.8 to +0.6 V to obtain the background signal at 0.44 V in a luminol/KCl solution. Then, a certain concentration of MP was added to an aqueous solution for 240 s, which was absorbed onto the ZrO(2)/GC electrode surface. Moreover, the MP absorbed on the surface of the ZrO(2)/GC electrode enhanced the ECL signal of luminol in the luminol/KCl solution, which increased with the concentration of MP. As a result, a novel ECL sensor was obtained in a luminol/KCl solution. The MP was determined in the range of from 3.8 × 10(-11) to 3.8 × 10(-6) mol L(-1), with a low detection limit of 1.27 × 10(-11) mol L(-1) (S/N = 3). The proposed ECL sensor performance for MP detection will open a new field in the application of rapid and screen detection of ultra-trace amounts of organ phosphorus pesticides (OPs) of vegetables used in farm markets.     

Electrocatalytic Oxidation of Hydrogen Peroxide on Poly(m‐toluidine)‐Nickel Modified Carbon Paste Electrode in Alkaline Medium

The poly(m‐toluidine) film was prepared by using the repeated potential cycling technique in an acidic solution at the surface of carbon paste electrode. Then transition metal ions of Ni(II) were incorporated to the polymer by immersion of the modified electrode in a 0.2 M NiSO₄, also the electrochemical characterization of this modified electrode exhibits stable redox behavior of the Ni(III)/Ni(II) couple. The electrocatalytic ability of Ni(II)/poly(m‐toluidine)/modified carbon paste electrode (Ni/PMT/MCPE) was demonstrated by electrocatalytic oxidation of hydrogen peroxide with cyclic voltammetry and chronoamperometry methods in the alkaline solution. The effects of scan rate and hydrogen peroxide concentration on the anodic peak height of hydrogen peroxide oxidation were also investigated. The catalytic oxidation peak current showed two linear ranges with different slopes dependent on the hydrogen peroxide concentration and the lower detection limit was 6.5 μM (S/N=3). The catalytic reaction rate constant, (k_h), was calculated 5.5×10² M^?1 s^?1 by the data of chronoamperometry. This modified electrode has many advantages such as simple preparation procedure, good reproducibility and high catalytic activity toward the hydrogen peroxide oxidation. This method was also applied as a simple method for routine control and can be employed directly without any pretreatment or separation for analysis cosmetics products.     

An electrochemiluminescent biosensor for glucose based on the electrochemiluminescence of luminol on the nafion/glucose oxidase/poly(nickel(II)tetrasulfophthalocyanine)/multi-walled carbon nanotubes modified electrode

A poly(nickel(II) tetrasulfophthalocyanine)/multi-walled carbon nanotubes composite modified electrode (polyNiTSPc/MWNTs) was fabricated by electropolymerization of NiTSPc on MWNTs-modified glassy carbon electrode (GCE). The modified electrode was found to be able to greatly improve the emission of luminol electrochemiluminescence (ECL) in a solution containing hydrogen peroxide. Glucose oxidase (GOD) was immobilized on the surface of polyNiTSPc/MWNTs modified GC electrode by Nafion to establish an ECL glucose sensor. Under the optimum conditions, the linear response range of glucose was 1.0 × 10⁻⁶ to 1.0 × 10⁻⁴ mol L⁻¹ with a detection limit of 8.0 × 10⁻⁸ mol L⁻¹ (defined as the concentration that could be detected at the signal-to-noise ratio of 3). The ECL sensor showed an outstanding well reproducibility and long-term stability. The established method has been applied to determine the glucose concentrations in real serum samples with satisfactory results.     

A novel biosensor for specific determination of hydrogen peroxide: catalase enzyme electrode based on dissolved oxygen probe

A biosensor for the specific determination of hydrogen peroxide was developed using catalase (EC 1.11.1.6) in combination with a dissolved oxygen probe. Catalase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated teflon membrane served as enzyme electrode. The electrode response was maximum when 50 mM phosphate buffer was used at pH 7.0 and at 35 degrees C. The biosensor response depends linearly on hydrogen peroxide concentration between 1.0x10(-5) and 3.0x10(-3) M with a response time of 30 s. The sensor is stable for >3 months so in this period >400 assays can be performed.     

A glass capillary microelectrode based on capillarity and its application to the detection of L-glutamate release from mouse brain slices.

A new glass capillary microelectrode for L-glutamate is described using pulled glass capillaries (tip size, approximately 12.5 microm) with a very small volume (approximately 2 microl) of inner solution containing glutamate oxidase (GluOx) and ascorbate oxidase. The operation of the electrode is based on capillary action that samples L-glutamate into the inner solution. The enzyme reaction by GluOx generates hydrogen peroxide that is detected at an Os-gel-HRP polymer modified Pt electrode in a three-electrode configuration. The amperometric response behavior of the electrode was characterized in terms of the capillarity, response time, sensitivity and selectivity for measurements of L-glutamate. The currents at 0 V vs. Ag/AgCl increased linearly with the L-glutamate concentration from 10 to 150 microM for in vitro and in situ calibrations. The response was highly selective to L-glutamate over ascorbate, dopamine, serotonin and other amino acids. The detection of L-glutamate in the extracellular fluids of different regions of mouse hippocampal slices under stimulation of KCl was demonstrated.     

Amplified Electrochemical DNA Sensor Based on Polyaniline Film and Gold Nanoparticles

In this work, an electrochemical DNA biosensor, based on a dual signal amplified strategy by employing a polyaniline film and gold nanoparticles as a sensor platform and enzyme‐linked as a label, for sensitive detection is presented. Firstly, polyaniline film and gold nanoparticles were progressively grown on graphite screen‐printed electrode surface via electropolymerization and electrochemical deposition, respectively. The sensor was characterized by scanning electron microscopy (SEM), cyclic voltammetry and impedance measurements. The polyaniline‐gold nanocomposite modified electrodes were firstly modified with a mixed monolayer of a 17‐mer thiol‐tethered DNA probe and a spacer thiol, 6‐mercapto‐1‐hexanol (MCH). An enzyme‐amplified detection scheme, based on the coupling of a streptavidin‐alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalyzed the hydrolysis of the electroinactive α‐naphthyl phosphate to α‐naphthol; this product is electroactive and has been detected by means of differential pulse voltammetry. In this way, the sensor coupled the unique electrical properties of polyaniline and gold nanoparticles (high surface area, fast heterogeneous electron transfer, chemical stability, and ease of miniaturisation) and enzymatic amplification. A linear response was obtained over a concentration range (0.2–10 nM). A detection limit of 0.1 nM was achieved.     

A comparative physical study of two different hydrophilic synthetic latex matrices for the construction of a glucose biosensor

Two different biodegradable latex polymers functionalised by hydroxy (1) or gluconamide (2) groups proved to be good immobilisation matrixes for glucose oxidase. The responses of these biosensors to glucose additions were measured by potentiostating the modified electrodes at 0.6 V/SCE in order to oxidise the hydrogen peroxide generated by the enzymatic oxidation of glucose in the presence of oxygen. The response of such electrodes was evaluated as a function of film thickness, pH and temperature. Rotating disk electrode experiments showed the influence of the enzyme on the structure of both latex films, namely a marked improvement in matrix permeability. The high permeability of the latex 1 based enzyme sensor (bilayer, P(m)=8.10x10(-4) cm s(-1)) resulted in a high dynamic range. Furthermore, the activation energy for a latex 1 sensor was determined to be 44.55 and 18.03 kJ mol(-1), respectively depending on the conformation of the enzyme.     

原位氧化复合法制备聚苯胺/金纳米纤维及过氧化氢生物传感器的构建

采用原位氧化复合法制备了聚苯胺/金纳米纤维,并将其固载在玻碳电极上构建了一种新型的过氧化氢生物传感器。该传感器对过氧化氢的还原具有良好的催化活性,在优化的实验条件下,该传感器对过氧化氢测定的线性范围为1.010-6～1.0510-4mol/L,检出限为5.7510-7mol/L(S/N=3)。     

Sol-gel-derived amperometric biosensor for hydrogen peroxide based on methylene green incorporated in Nafion film

A hydrogen peroxide biosensor was fabricated by coating a sol-gel-peroxidase layer onto a Nafion-methylene green modified electrode. Immobilization of methylene green (MG) was attributed to the electrostatic force between MG(+) and the negatively charged sulfonic acid groups in Nafion polymer, whereas immobilization of horseradish peroxidase was attributed to the encapsulation function of the silica sol-gel network. Cyclic voltammetry and chronoamperometry were employed to demonstrate the feasibility of electron transfer between sol-gel-immobilized peroxidase and a glassy carbon electrode. Performance of the sensor was evaluated with respect to response time, sensitivity as well as operational stability. The enzyme electrode has a sensitivity of 13.5 muA mM(-1) with a detection limit of 1.0x10(-7) M H(2)O(2), and the sensor achieved 95% of the steady-state current within 20 s.     

Liposome Immunoassay for Determination of Phenytoin

Inthispaper,arapid,sensitive,andsimpleliposomeimmunoassay(LIA)lbrdetectingphenytoinhasbeenestablished.Phenytoinisuscdtocurecpilepsy,butinclinicalpractice,theefficientconcentrationofphenytoininpatient'sseraisverynarrow,about1O~2Opg/ml.Iftheconcentrationofphenytoininserumislessthantheefficientconcentration,thereisnoeffectofmedicine.Butiftheconcentrationrangewerc2O~4Ollg/ml,themedicinecouldbringsideeffect.Ifthcconcentrationbecomesgreater,thenitcancausepoisoning.Becausetherearemanyfactorswhich…     

Stability Improvement of Prussian Blue by a Protective Cellulose Acetate Membrane for Hydrogen Peroxide Sensing in Neutral Media

The cellulose acetate covered Prussian blue modified glassy carbon electrode (GCE/PB/CA) was fabricated as a novel hydrogen peroxide sensor. It was shown by scanning electron microscope (SEM) and atomic force microscope (AFM) that Prussian blue was covered and protected by cellulose acetate perfectly. The modified electrode showed a good electrocatalytic activity for H₂O₂ reduction in neutral aqueous solution. H₂O₂ was detected amperometrically in 0.05 mol/L phosphate buffer solutions (pH 7.0, containing 0.1 mol/L KCl as supporting electrolyte) at an applied potential of ?0.2 V (vs. SCE). The response current was proportional to the concentration of H₂O₂ in the range of 1.0×10^?5 mol/L to 2.5×10^?4 mol/L with the detection limit of 2.2×10^?6 mol/L at a signal to noise ratio 3.