Comparison of Oxalate Oxidase Enzyme Electrodes for Urinary Oxalate Determinations

Abstract

Enzyme electrodes for oxalate were constructed by immobilizing oxalate oxidase enzyme (E, C, 1,2,3,4) on both potentiometric carbon dioxide and amperometric hydrogen peroxide sensors. These systems were optimized, and the response characteristics of the two biosensors examined in a comparison study. Areas investigated include linear range, calibration curve slope, response time, limit of detection, and lifetime. Selectivity studies were carried out with the system and showed no measurable response to millimolar levels of various L-amino acids, metal ions or ascorbic acid.

This comparison was extended into the choice of an optimal sensor for urinary oxalate determinations with minimal sample pretreatment. The peroxide sensor performed best in urine samples, allowing for the 1:40 dilution necessary to minimize the effect of any interferents present. At this or greater dilution, the recovery of standard additions of oxalate averaged 95.5%, with an average relative standard deviation of 4.9%. The system retained its activity throughout two weeks of continued use.     

A New Amperometric Carbon Paste Enzyme Electrode for Ethanol Determination

Abstract

In this study, a new amperometric carbon paste enzyme electrode for determination of ethanol was developed. The carbon paste was prepared by mixing alcohol dehydrogenase, its coenzyme nicotinamide adenine dinucleotide (oxidized form, NAD⁺), poly(vinylferrocene) (PVF) that was used as a mediator, graphite powder and paraffin oil, then the paste was placed into cavity of a glass electrode body. Determination of ethanol was performed by oxidation of nicotinamide adenine dinucleotide (reduced form, NADH) generated enzymatically at +0.7 V. The effects of enzyme, coenzyme and PVF amounts; pH; buffer concentration and temperature were investigated. The linear working range of the enzyme electrode was 4.0×10^?4–4.5×10^?3 M, determination limit was 3.9×10^?4 M and response time was 50 s. The optimum pH, buffer concentration, temperature, and amounts of enzyme, NAD⁺ and PVF for enzyme electrode were found to be 8.5, 0.10 M, 37°C, 2.0, 6.0, and 12.0 mg, respectively. The storage stability of enzyme electrode at +4°C was 7 days. Enzyme electrode was used for determination of ethanol in two different wine samples and results were in good agreement with those obtained by gas chromatography.     

Liquid Chromatography with Electrochemical Detection of Phenol and NADH for Enzyme Immunoassay

Abstract

Enzyme immunoassays based on chromatographic separation and amperometric detection of an enzyme generated product have been investigated. These assays combine the selectivity of the antigen/antibody reaction with the high sensitivity of thin layer amperometry. The feasibility of utilizing LCEC as a detection scheme was demonstrated using the Syva EMIT® kit for phenytoin. NADH production by glucose-6-phosphate dehydrogenase was monitored following a homogeneous procedure. Heterogeneous assays were developed for alkaline phosphatase labeled species which were based upon LCEC determination of phenol. Assays were designed for a common serum glycoprotein (orosomucoid) and a clinically important drug (digoxin). Detection limits approach the pg/mL level and as such may prove fruitful in the quantitation of numerous antigens of clinical interest.     

Horseradish Peroxidase Enzyme Electrodes for Nadh and Nadph

Abstract

The aerobic oxidation of reduced pyridine phosphonucleotides catalyzed by horseradish peroxidase (E.C.1.11.1.7) (HRP) in combination with Mn²⁺-ions, phenolics and redox dyes was used to perform NADH and NADPH determinations with an amperometric HRP enzyme electrode. Optimal operational conditions have been elaborated for such a sensor and a linear relationship between derivative current (dI/dt) and NADH-concentration was obtained between 2.10^?5 and at least 2.10^?4 mol/l. The coefficient of variation was equal to or smaller than 4 %. The time for one measurement was less than 10 minutes including equilibration of the electrode.     

Glutathione Amperometric Enzyme Microsensor

Abstract

Glutathione micro-enzyme sensors were developed based on the immobilization of glutathione oxidase at the tip of a 25 μm Pt wire sealed in glass. An inner membrane constructed from cellulose acetate (CA) was deposited onto the tip of the Pt microelectrode prior to enzyme immobilization with glutaraldehyde. The final outer diameter of the microelectrodes is approximately 30 μm. The analytical characteristics of the microelectrodes, including calibration curves, apparent K_M′, pH response curves, stability and selectivity over enzymatic interferences were determined. The response was linear in the concentration range 1.0 × 10^?5 M - 1.9 × 10^?4 M glutathione, reaching 95% steady-state current in 15–20 seconds. The microelectrodes were useful for more than two months.     

Continuous-Flow Determination of Phenol With Chemically Immobilized Polyphenol Oxidase (Tyrosinase)

Abstract

The use of covalently bound mushroom polyphenol oxidase (tyrosinase, EC 1.10.3.1) for the determination of μg/mL and ng/mL concentrations of phenol in water samples with use of continuous-flow sample/reagent processing is described. Immobilization on controlled-pore glass, CPG, was accomplished via diazo coupling. Detection was effected with hexacyanoferrate(II) as a redox mediator and was either spectrophotometric or amperometric. the immobilized enzyme preparation was part of an open tubular reactor (CPG thermally embedded on Tygon tubing). the redox mediator was used either in solution or as part of a thin-layer cell and immobilized on poly(4-vinylpyridine) incorporated in a carbon paste electrode. Different spectrophotometric and amperometric strategies are compared and the method is applied to the determination of phenol in water samples and quality control standards.     

A Prospect for the Analysis of Species Acting as Enzyme Inhibitors as Illustrated by Heavy Metal Inhibition

Abstract

A flow system incorporating an amperometric glucose oxidase enzyme electrode has been used to study the inhibitory effects of 16 metal cations on glucose oxidase. Only copper(II), mercury(II) and silver(I) caused any significant inhibition. the enzyme electrode could be reactivated by EDTA, the reactivation being most effective for copper(II) and least so for silver(I). Other complexing agents were tried for reactivation but proved to be unsatisfactory.

The ability to reactivate the enzyme on the electrode following copper(II) inhibition, and the linear response of the system to the level of this inhibitor according to I/A = -9.49 × 10^?7 log([Cu]/M) + 4.84 × 10^?8; r = 0.994 between 2.5 × 10^?4M and 5 × 10^?3M [Cu]²⁺ indicates a prospect for the use of a flow system for determining enzyme inhibitors in samples.     

Applications of Amperometric Silica Sol-Gel Modified Enzyme Biosensors

ABSTRACT

The applications of amperometric sol-gel modified enzyme biosensors in numerous analyses of clinical, industrial and environmental importance have been demonstrated. Certain biosensors have been employed as the sensor in a FIA amperometric detector. The results obtained by the biosensors generally corroborate well with the classical or official method. In many cases, the recovery results obtained by the biosensor have been satisfactory.     

Amperometric Sensor and Immobilized Enzyme Electrode for the Determination of Enzymatic Activities

Abstract

A sensitive method for the rapid determination of activities of soluble or immobilized enzymes, based on the electrochemical detection of hydrogen peroxide is described. Kinetic studies (Vmax and K_M determinations) can be performed for all H₂O₂ generating enzymes (i.e. most of the oxidases) using an amperometric probe with a platinum anode at a fixed potential.

When associated with an immobilized glucose oxidase membrane, this sensor constitutes a glucose electrode and the activity of any hydrolase which releases glucose can be measured. There is no need for other auxiliary enzymes and no preincubation step is required. The possibility to carry out continuous analysis constitutes the main advantage of the described method.     

A Multistep Enzyme Sensor for Sucrose Based on S-Layer Microparticles As Immobilization Matrix

Abstract

In this paper we report on the construction principle and performance of an amperometric 3-enzyme sensor for sucrose based on crystalline bacterial cell surface layers (S-layers) as immobilization matrix for the biological components.

Isoporous, crystalline surface layers (S-layers) have been identified as outermost cell envelope layer in many bacteria. Since they are composed of identical protein or glycoprotein subunits with functional groups in well defined positions and orientations, they represent ideal matrices for the controlled and reproducible immobilization of functional macromolecules, as required for the development of biosensors. Apart from single enzyme sensors, which were described earlier, a strikingly simple method for the assembly and optimization of multistep systems was developed. For the fabrication of an amperometric sucrose sensor invertase, mutarotase and glucose oxidase were individually immobilized on S-layer fragments isolated from Clostridium thermohydrosulfuricum L111-69 via aspartic acid as spacer molecules. Subsequently, appropriate mixtures of enzyme loaded S-layer fragments were deposited on a microfiltration membrane and finally, the composite multifunctional sensing layer was sputtered with gold in order to establish a good metal contact. Amperometric sucrose measurements based on H₂O₂ oxidation revealed a high signal level (1 μA^?1/cm^2?mmol sucrose), 5 min response time and a linear range up to 30 mM sucrose as the main characteristics of the S-layer sucrose sensor.     

Amperometric Multienzyme Sensor for Determination of D-Glucono-δ-Lactone

Abstract

An amperometric enzyme sensor for the determination of gluconolactone in glucose-containing samples has been developed. The interfering glucose is eliminated by an outer anti-interference layer containing hexokinase, whilst the gluconolactone reaches a glucose de-hydrogenase-glucose oxidase layer, where it is converted into glucose (by glucose dehydrogenase) and then transformed by glucose oxidase, the associated oxygen consumption can be measured at the electrode. Gluconolactone is determined over the concentration range, 0.02–1 mmo1/1, with a toleration of glucose concentration up to 2 mmo1/1.     

Amperometric Enzyme Based Biosensor For the Detection of Xanthine Via Superoxide

Abstract

An amperometric enzyme modified carbon paste electrode for the detection of xanthinc by the xanthine oxidase catalyzed reaction is described. the product of the enzyme reaction monitored is the highly unstable radical superoxide, detected at O mV (vs SCE) poised potential. A dual working electrode configuration consisting of an enzyme modified carboa paste electrode and an unmodified glassy carbon electrode are utilized for this purpose.     

An Enzyme Sensor For Determination of Acetylcholine and Choline In Rat Brain Extracts

Abstract

A choline enzyme sensor, recently developed by the authors, was used for choline and acetylcholine determination in rat brain extracts, using choline oxidase immobilized on cellulose triacetate membranes, and acetylcholinesterase in homogeneous solution. the method proved useful for assay of the acetylcholine content in a commercial pharmaceutical formulation used in ophthalmology.     

Determination of Gentamicin with an Amperometric Enzyme Immunosensor

A new version of enzyme immunoassay with the use of an amperometric immunosensor was proposed for the determination of an aminoglycoside antibiotic gentamicin. The biosensing part of the enzyme immunosensor simultaneously incorporates immobilized enzyme cholinesterase and antibodies against gentamicin. The detection limit for gentamicin in this method is 1 × 10^–9 mg/mL. The time of determination is no longer than 20 min. The stability constants of the immunocomplex and the kinetic parameters of the enzymatic reaction in its presence were determined. Gentamicin was determined in samples of pharmaceutical preparations and foodstuffs (milk).     

Enzyme thermistors for process monitoring and control

In today’s biotechnology there is an increasing demand for appropriate analytical systems for process control. At present the most widely used control systems are based on measurements of pH, pO₂, and pCO₂. Such systems do not allow the direct measurement of substrates and products. To overcome this drawback sensors such as enzyme thermistors and enzyme electrodes have been designed and their development into industrial useful sensors for monitoring and controlling is the subject of active research.     

Gas Diffusion Electrodes for Use in an Amperometric Enzyme Biosensor

The preparation of gas diffusion electrodes and their use in an amperometric enzyme biosensor for the direct detection of a gaseous analyte is described. The gas diffusion electrodes are prepared by covering a PTFE membrane (thickness 250 μm, pore size 2 μm, porosity 35%) with gold, platinum, or a graphite/PTFE mixture. Gold and platinum are deposited by e‐beam sputtering, whereas the graphite/PTFE layer is prepared by vacuum filtration of a respective aqueous suspension. These gas diffusion electrodes are exemplarily implemented as working electrodes in an amperometric biosensor for gaseous formaldehyde containing NAD‐dependent formaldehyde dehydrogenase from P. putida [EC. 1.2.1.46] as enzyme and 1,2‐naphthoquinone‐4‐sulfonic acid as electrochemical mediator. The resulting sensors are compared with regard to background current, signal noise, linear range, sensitivity, and detection limit. In this respect, sensors with gold or graphite/PTFE covered membranes outclass ones with platinum for this particular analyte and sensor configuration.     

A Novel Enzyme Membrane Electrode for Oxalate Determination in Foodstuffs

Abstract

An enzyme membrane electrode usable for the assay of oxalate in foodstuffs is described. A commercially available preactivated polyamide membrane was used for the immobilization of oxalate oxidase. The bioactive disk thus obtained was associated with an amperometric transducer. The resulting self-contained enzyme electrode wich allows oxalate determination in various materials with minimal pretreatment exhibits a linear calibration ranging from 10–7 M and 10–4 M in the cell. The response-time was comprised between 20 seconds and 1 minute, depending on the oxalate content in the sample. The electrode-response was very stable for at least 4 months, a period during which more than 150 assays were performed.

The results obtained with several food materials were in good agreement with those obtained with the conventional spectrophotometric method. Assays were also performed with a microprocessor-based analyzer normally used for glucose measurements with a glucose oxidase electrode When the analyzer is equipped with an oxalate oxidase membrane, without further setting, oxalate can be determined in the range 5 10^?3 M-10^?1 M in the sample.     

Polyurethane Enzyme Membranes for Chip Biosensors

Abstract

The coverage of electrochemical chip sensors based on silicon technology with a polyurethane enzyme membrane is described. After crosslinking of the surface by polyfunctional isocyanates the enzyme membrane shows good adhesion, complete retention of the enzyme molecules, and low diffusional resistance to both analytes and products. Using thin film noble metal electrodes and ion sensitive field effect transistors, glucose and urea sensors with good long term stability and short response time have been prepared.     

An Electrochemical Determination of Uric Acid in Sera by a Flow-Through Equipment with and Without Uricase

Abstract

A comparison of an enzymeless direct electrochemical oxidation procedure at a platinum electrode for the determination of uric acid, and an enzyme sensor with immobilized urate: oxygen oxidoreductase (uricase), was performed in flow stream systems. The uricase enzyme electrode is based on the H₂O₂ oxidation current. Both amperometric methods were related to the wall-known photometric uricase-catalase-procedure (UCM) as a reference method. The measured values of both methods are of the first derivatives of current change (dI/dt) due to the electrochemical or electrochemical enzymatic reaction, respectively. The analytical quality of the measurements is characterized by: precision s% within run < 2% day to day < 5% accuracy acceptable (control materials) correlation to reference method r >0.93 analysis rate 80 samples/hr     

Glucose Biosensor Based on Oxygen Electrode. Part II: Long-Term Operational Stability of the Rechargeable Glucose Biosensor

Abstract

A potentially implantable glucose biosensor for measuring blood or tissue glucose levels in diabetic patients has been developed. The glucose biosensor is based on an amperometric oxygen electrode and immobilized glucose oxidase enzyme, in which the immobilized enzyme can be replaced (the sensor recharged) without surgical removal of the sensor from the patient. Recharging of the sensor is achieved by injecting fresh immobilized enzyme into the sensor using a septum. A special technique for immobilization of the enzyme on Ultra-Low Temperature Isotropic (ULTI) carbon powder held in a liquid suspension has been developed.

In vitro studies of the sensors show stable performance during several recharge cycles over a period of 3 months of continuous operation.

Diffusion membranes which ensure linear dependence of the sensor response on glucose concentration have been developed. These membranes comprise silastic latex-rubber coatings over a microporous polycarbonate membrane. Calibration curves of the amperometric signal show linearity over a wide range of glucose concentrations (up to 16 mM), covering hypoglycemic, normoglycemic and hyperglycemic conditions.

The experimental results confirm the suitability of the sensors for in vitro measurements in undiluted human sera.