Showing papers on "Amperometry published in 1994"

Separation-free sandwich enzyme immunoassays using microporous gold electrodes and self-assembled monolayer/immobilized capture antibodies

Abstract: A novel separation-free sandwich-type enzyme immunoassay for proteins is performed by designing an electrochemical detection system that enables preferential measurement of surface-bound enzyme-labeled antibody relative to the excess enzyme-labeled reagent in the bulk sample solution. In this initial model system, the assay is carried out using gold-coated microporous nylon membranes (pore size 0.2 micron) which are mounted between two chambers of a diffusion cell. The membrane serves as both a solid phase for the sandwich assay and the working electrode in the three-electrode amperometric detection system. The capture monoclonal antibody is immobilized covalently on the gold side of the membrane via a self-assembled monolayer of thioctic acid. In the separation-free sandwich assay, both model analyte protein (human chorionic gonadotropin; hCG) and alkaline phosphatase labeled anti-hCG (ALP-Ab) are incubated simultaneously with the immobilized capture anti-hCG antibody. Surface-bound ALP-Ab is spatially resolved from the excess conjugate in the bulk sample solution by introducing the enzyme substrate (4-aminophenyl phosphate) through the back side of the porous membrane. The substrate diffuses rapidly through the porous membrane where it first encounters bound ALP-Ab at the gold surface. The enzymatically generated product, aminophenol, is detected immediately by oxidation at the gold electrode (at +0.19 V vs Ag/AgCl), and the magnitude of current is directly proportional to the concentration of hCG in the sample. The response time after substrate addition is less than 1 min, although maximum response toward the analyte protein requires a sample/conjugate preincubation time of 30 min with the porous electrode.(ABSTRACT TRUNCATED AT 250 WORDS)

Design, Characterization, and One-Point in vivo Calibration of a Subcutaneously Implanted Glucose Electrode

Abstract: A 0.29-mm-diameter flexible electrode designed for subcutaneous in vivo amperometric monitoring of glucose is described. The electrode was designed to allow "one-point" in vivo calibration, i.e., to have zero output current at zero glucose concentration, even in the presence of other electroreactive species of serum or blood. A valid zero point, along with a measurement of the glucose concentration in a withdrawn sample of blood at which the current is known, defined the sensitivity in the linear response range. The electrode was four-layered, with the layers serially deposited within a 0.125-mm recess upon the tip of a polyimide-insulated 0.25-mm gold wire. The recessed structure reduced the sensitivity to movement and allowed, through control of the depth of the recess, control of the transport of glucose and thus the range of linearity. The recess contained the four polymeric layers, with a total mass less than 5 micrograms and no leachable components. The bottom glucose concentration-to-current transducing layer consisted of the enzyme "wiring" redox polymer poly[(vinylimidazole)Os(bipyridine)2Cl]+ , complexed with recombinant glucose oxidase and cross-linked with poly(ethylene glycol) diglycidyl ether, to form an electron-conducting hydrogel. The layer was overcoated with an electrically insulating layer of polyaziridine-cross-linked poly(allylamine), on which an immobilized interference-eliminating horseradish peroxidase based film was deposited. An outer biocompatible layer was formed by photo-cross-linking derivatized poly(ethylene oxide). The current output of a typical electrode at 10 mM glucose and at 37 degrees C was 35 nA, the apparent Km was 20 mM, and the 10-90% response time was approximately 1 min.(ABSTRACT TRUNCATED AT 250 WORDS) [on SciFinder (R)]

Electrical Communication between Electrodes and NAD(P)+-Dependent Enzymes Using Pyrroloquinolinequinone-Enzyme Electrodes in a Self-Assembled Monolayer Configuration: Design of a New Class of Amperometric Biosensors

Abstract: The development of an amperometric sensor utilizing the NAD(P) + -cofactor-dependent enzyme, malic enzyme, is described using a quinone-enzyme monolayer-modified electrode. Pyrroloquinolinequinone (PQQ, 1) was covalently linked to a self-assembled monlayer of cysteamine on an Au electrode. The resulting PQQ-monolayer electrode (PQQ surface coverage 1.98×10 -10 mol-cm -2 ) catalyzes the electrooxidation of NADPH and NADH. The developed anodic currents are controlled by NAD(P)H concentrations and provide an amperometric sensor for the cofactor

Amperometric detection of nitrate via a nitrate reductase immobilized and electrically wired at the electrode surface

Abstract: The electropolymerization of a nitrate reductase-amphiphilic pyrrole viologen mixture preadsorbed on the electrode surface provide the immobilization of the enzyme in a N-substituted viologen polypymole film. Furthermore, the electrogenerated redox polymer simultaneously entraps the enzyme and connects it electrically with the electrode. The immobilized enzyme catalyzes the reduction of nitrate to nitrite mediated by the viologen redox couple (V 2+/.+ ). This enzyme immobilization has been accomplished on electrode surface previously coated by a polypyrrole-viologen film, leading to a bilayer electrode configuration. The sensitivity and the detection light of this biosensor are 13.8 mA M -1 cm -2 and 4×10 - M, respectively

Voltammetric and amperometric studies of thiocholine at a screen-printed carbon electrode chemically modified with cobalt phthalocyanine: studies towards a pesticide sensor

Abstract: Cyclic voltammetry was used to investigate the electrochemical behaviour of thiocholine at screen-printed carbon electrodes chemically modified with cobalt phthalocyanine. Cyclic voltammograms exhibited one anodic peak (Ia) and two cathodic peaks (Ic and IIc) in the potential range –0.8–+0.3 V between pH 5.0 and 12.0. Peak Ia is considered to result from the electrocatalytic oxidation of the thiol moiety to produce the corresponding disulfide; peak Ic is likely to be the electrocatalytic reduction of the disulfide back to the original thiol species. Peak IIc is probably due to a reduction process occurring in the macrocyclic phthalocyanine molecule. Hydrodynamic voltammetry was performed in 0.05 mol dm–3 phosphate buffer (pH 8.0) containing thiocholine; one well-defined anodic wave was obtained. From this, the optimum potential for amperometric studies was found to be +0.1 V. Calibration studies were performed using amperometry in stirred solution; a linear response was obtained over the range 5.0 × 10–7–4.8 × 10–5 mol dm–3. It should be possible to use these screen-printed electrodes as the basis of a pesticide sensor.

Amperometric Glucose Sensors Based on Immobilized Glucose Oxidase-Polyquinone System

Abstract: Non-cross-linked and also cross-linked poly(ether amine quinone)s were prepared and tested for their efficiency as electron-transfer relay systems in amperometric glucose biosensors. Cyclic voltammetry and constant applied potential measurements showed that poly(ether amine quinone) relay systems efficiently mediated electron transfer from reduced glucose oxidase to a conventional carbon paste electrode. Sensors containing these relay systems respond rapidly to low (< 0.1 mM) glucose concentrations and reach steady-state current responses in less than 1 min. Electrodes constructed with cross-linked polymer and glucose oxidase were stable, indicating that the glucose oxidase was trapped in the polymer matrix and did not freely diffuse away from the electrode surface into aqueous solutions. The cross-linked polymer is a large molecular system which facilitates a flow of electrons from enzyme to the electrode, acting as an electron-transfer relay system and not as a diffusional mediator.

Novel, reagentless, amperometric biosensor for uric acid based on a chemically modified screen-printed carbon electrode coated with cellulose acetate and uricase

Abstract: Amperometry in stirred solution has been used for the systematic evaluation of modified screen-printed carbon electrodes (SPCEs) with a view to developing a reagentless biosensor for uric acid. The developed system consists of a base cobalt phthalocyanine (CoPC) electrode tailored to the electrocatalytic oxidation of H2O2 by means of a cellulose acetate (CA)–uricase bilayer. Uricase was immobilized by drop-coating the enzyme onto the CA membrane covering the CoPC-SPCE. The device exploits the near-universal H2O2-generating propensity of oxidases, the permselectivity of the CA film towards H2O2 and the electrocatalytic oxidation of this product at the CoPC-SPCE. The electrochemical oxidation of the resulting Co+ species was used as the analytical signal, facilitating the application of a greatly reduced operating potential when compared with that required for direct oxidation of H2O2 at unmodified electrodes. The time required to achieve 95% of the steady-state current (t95iSS) was 44 s [relative standard deviation = 7.5%(n= 10)]. Amperometric calibrations were linear over the range from 13 × 10–6 to 1 × 10–3 mol dm–3, with the former representing the limit of detection. The CA membrane extended the linear range of the biosensor by over two orders of magnitude, when apparent Michaelis–Menten constants (Km′) of immobilized and free enzymes are compared. This suggests that the process is diffusion-controlled and not governed by the kinetics of the enzyme. The precision of electrode fabrication was determined by cyclic voltammetry to be 4.9%(n= 6). Successive amperometric calibrations (n= 7) over 7 d using a single sensor revealed only a 14.0% diminution in sensitivity from the original response. Sensor stability and the dependence of the steady-state current on the pH, ionic strength and temperature of the supporting electrolyte were studied and the results are presented. The functioning of the biosensor is indifferent to a wide range of potential interferences studied in a synthetic sample and results correlate favourably with those of a standard hospital method.

Pulsed amperometric detection of thaumatin using antibody-containing poly(pyrrole) electrodes

Abstract: An electrochemical sensor produced by direct incorporation of anti-thaumatin into a polypyrrole film was developed. The use of flow injection analysis and pulsed amperometric detection permitted the development of a sensitive, reversible and rapid electrochemical immunoassay for thaumatin.

Pharmaceutical and biomedical applications of capillary electrophoresis/electrochemistry

Abstract: The use of capillary electrophoresis/electrochemistry (CEEC) for the analysis of microdialysis samples obtained for pharmacokinetic and neurochemical studies is described, as well as the development of new types of electrodes and waveforms which increase the selectivity of this technique for specific classes of analytes. CEEC with a carbon fiber electrode was employed for the analysis of microdialysis samples. Microdialysis is an in vivo sampling technique that yields very small samples for analysis (less than 1 microL). Therefore, capillary electrophoresis, with its small volume requirements, is an excellent choice for the analytical method. CEEC was used to study the pharmacokinetics of L-dopa and the release of aspartate and glutamate following a high K+ infusion in the brain. Several modified electrodes which increase the applicability of CEEC in pharmaceutical and biomedical analysis are described. One of these is a gold/mercury electrode which is highly selective for thiols and was used for the determination of glutathione in a rat brain. An alternative method for the detection of thiols employed a chemically modified electrode containing cobalt phthalocyanine. In this case, an electrocatalyst reduces the overpotential of thiols at the carbon electrode and makes it possible to detect them at a much lower and more selective oxidation potential. This electrode was used for the detection of cysteine in urine. The development of pulsed amperometric detection for capillary electrophoresis is also described and is demonstrated by the detection of glucose in blood. Lastly, a method for the detection of peptides based on the formation of a copper complex and detection at a carbon fiber electrode is discussed.

Model for an immobilized oxidase enzyme electrode in the presence of two oxidants

Abstract: In mediated oxidase electrodes, the artificial electron acceptor competes with oxygen for the reoxidation of the enzyme. A steady-state model for a three-substrate electrode is employed to investigate the influence of oxygen on the current response of an amperometric enzyme electrode and concentration profiles predicted in the enzyme layer for the substrates. The model predicts the electrode response for different O 2 levels, and the influence of model parameters on the electrode performance is tested and discussed with the view of improved sensor design

Activated carbon paste electrodes for biosensors

Abstract: A reagentless amperometric glucose biosensor was constructed using glucose oxidase and hydroquinone (H2Q) coimmobilized in carbon paste. The sensor response was studied by amperometry and cyclic voltammetry in quiescent solutions, as well as in a flow injection apparatus. Studies were conducted as a function of surface activation and sensor working conditions such as glucose concentration, storage, aging, and reusability. Surface activation proved to be useful to improve the electro-chemical reversibility of the mediator and the analytical characteristics of the sensor. The harsh conditions of the surface pretreatment did not deactivate the enzyme. Results from the assay of a clinical sample gave a glucose concentration value of 76 +/- 6 mg/dL, which compared favorably with the expected range of 72-88 mg/dL.

Development of amperometric sensors for uric acid based on chemically modified graphite-epoxy resin and screen-printed electrodes containing cobalt phthalocyanine

Abstract: Cyclic voltammetry, hydrodynamic voltammetry (HDV) and amperometry were used to investigate the behaviour of uric acid at carbon(graphite)–epoxy resin electrodes modified with the following electrocatalysts: ferrocenes, phthalocyanines, phenoxazines, phenazines and hexacyanoferrate species. As no direct electrocatalytic oxidation of uric acid was apparent, enzyme-coupled mediation was studied using uricase with electrodes modified with ferrocene, cobalt phthalocyanine (CoPC) and Meldola's Blue, in the presence and absence of oxygen. Again, no electrocatalysis was observed so we exploited the specificity of the enzyme uricase, which is known to produce hydrogen peroxide from uric acid. This product has been shown to undergo electrocatalysis at modified electrodes, and preferentially traverses certain membranes, so this rationale was adopted for the ensuing studies. Cyclic voltammetry and amperometry were used to study the electrochemical behaviour of H2O2 in 0.05 mol dm–3phosphate buffer (pH 9.3). Hydrogen peroxide was found to be electroactive at substantially lower potentials (<600 mV) at CoPC-modified electrodes than at unmodified electrodes. The precision of surface–surface reproducibility of epoxy resin electrodes was calculated by cyclic voltammetry to be 7.3%(n= 5). Further studies were performed with and without cellulose acetate (CA) membranes using amperometry in stirred solution. Epoxy resin electrodes were simply dropcoated with CA. The membrane functioned as an effective barrier against a range of potential interferences, but allowed the selective detection of enzymically generated H2O2 from urate. This methodology was then translated to screen-printed electrodes (SPEs). Cyclic voltammetry was first used to establish the precision of electrode manufacture; this was 4.9%(n= 5). The SPEs were then modified by placing each inside a tube, one end of which was covered with a solvent cast (CA) membrance. This electrode–polymer configuration furnished similar permselective properties as the drop-coated devices. The solvent cast membranes were treated with various concentrations of uricase; when the membrane was loaded with 0.48 U of enzyme a measurable H2O2 signal was obtained at the CoPC–SPE for a solution containing 0.18 mmol dm–3 uric acid.

Method for measuring sample by enzyme electrodes

Abstract: To reduce measurement time, methods of operating an amperometric, enzyme electrode (14) before steady state is established are described. In a first method current is monitored for a short time after injection of buffer solution into a cell (1) containing the electrode and the values used to establish a background current via time extrapolution using a least squares technique. Sample is then injected into the cell, and the current again measured. The analyte (e.g. glucose, lactic acid, uric acid) concentration is determined by subtracting the background current extrapolated to the measurement time (t₃) from the measured current.

Development of a disposable amperometric sensor for reduced nicotinamide adenine dinucleotide based on a chemically modified screen-printed carbon electrode

Abstract: The evaluation of novel, disposable, screen-printed electrodes by means of cyclic voltammetry with potassium hexacyanoferrate (II), phenazine methosulfate, and Meldola's Blue is described. Meldola's Blue was incorporated into selected electrodes, and the electrocatalytic oxidation of reduced nicotinamide ademine dinucleotide (NADH) was investigated using cyclic voltammetry and hydrodynamic voltammetry. The modified electrodes were used for quantitative measurements of NADH by means of amperometry in stirred solutions at applied potentials of –0.05 V (versus a saturated calomel electrode). The limit of detection was found to be 2.5 × 10–6 mol dm–3; the response was linear up to 2.5 × 10–4 mol dm–3.

Detection of Galactose and Lactose by a Poly(Amphiphilic Pyrrole)-Galactose Oxidase Electrode

Abstract: The entrapment of galactose oxidase (GAO) on an electrode surface by coadsorption with a cationic amphiphilic pyrrole and electropolymerization of this pyrrole monomer is described. This simple and rapid procedure for biosensor construction provides very fast responsive and sensitive GAO-based sensors to galactose and lactose. The electrode response is based on the electrochemical detection of enzymically generated hydrogen peroxide. The stability, optimum pH and selectivity of the bioelectrode as well as the characteristics of the immobilized galactose oxidase have been determined. Poly(amphiphilic pyrrole) films have been electrogenerated on the surface of the bioelectrode and the effect of such additional coatings on the biosensor selectivity have also been examined.

Electrocatalytic oxidation and amperometric determination of sulfhydryl compounds at a copper hexacyanoferrate film glassy carbon electrode in liquid chromatography

Abstract: Electrocatalytic oxidation of sulfhydryl compounds was effective on a copper hexacyanoferrate (CuHCF) film glassy carbon electrode, at a significantly reduced overpotential (0.55 to 0.65 V) and for a broader pH range (2.0 to 7.0). The electrocatalysis was