An enzyme electrode for amperometric measurement of D-amino acid

A carbon paste enzyme electrode has been developed for measurement of D-amino acids that employs a fatty acid modified FAD to prevent leaching of this essential cofactor to the surrounding aqueous environment and which serves as an enzyme stabilizing agent. The lower limit of detection is at least 10(-4) M and the electrode has a linear range of 10(-4) to 3 x 10(-3) M and a response time of 180 s. Twenty L-amino acids were tested and none of them elicited responses when electrodes were exposed to 0.5 mM concentration increases over a baseline level. On the other hand, some response was observed when exposed to 18 of 20 D-amino acids varying from 2 to 200% of the corresponding D-alanine response. Electrodes were shown to have longevities of over 30 days while maintaining 85% of their original sensitivity. Electrodes showed activity over a pH of 6.2-11.7 with a maximum at 9.2 and over temperatures of 10-47 degrees C with a maximum at 37 degrees C.     

Modelling amperometric enzyme electrode with substrate cyclic conversion

A mathematical model of amperometric enzyme electrodes in which chemical amplification by cyclic substrate conversion takes place in a single enzyme membrane has been developed. The model is based on non-stationary diffusion equations containing a non-linear term related to Michaelis-Menten kinetic of the enzymatic reaction. The digital simulation was carried out using the finite difference technique. The influence of the substrate concentration, the maximal enzymatic rate as well as the membrane thickness on the biosensor response was investigated. The numerical experiments demonstrate significant (up to dozens of times) gain in biosensor sensitivity at low concentrations of substrate when the biosensor response is under diffusion control.     

Thin-film antimony-antimony-oxide enzyme electrode for penicillin determination

A potentiometric penicillinase electrode is reported in which the base pH transducer is a thin-film anti-mony-antimony-oxide electrode deposited by vacuum evaporation. Several enzyme immobilization procedures have been examined and a crosslinked protein film found to be the most appropriate to this type of sensor. The use of an adjacent antimony-antimony-oxide track as a pseudoreference electrode was successfully demonstrated. The overall response was shown to be independent of the stirring rate above 100 rpm, but the kinetics of the response were found to depend markedly on the stirring rate. The intrinsic linear response range was 3 x 10(-4)Mto 7 x 10(-3)M penicillin G. Linearizing transforms that extend the useful range were examined.     

A new amperometric nanostructured sensor for the analytical determination of hydrogen peroxide

A new amperometric, nanostructured sensor for the analytical determination of hydrogen peroxide is proposed. This sensor was constructed by immobilizing silver nanoparticles in a polyvinyl alcohol (PVA) film on a platinum electrode, which was performed by direct drop-casting silver nanoparticles that were capped in a PVA colloidal suspension. UV-vis spectroscopy, X-ray diffraction and transmission electron microscopy were used to give a complete characterization of the nanostructured film. Cyclic voltammetry experiments yielded evidence that silver nanoparticles facilitate hydrogen peroxide reduction, showing excellent catalytic activity. Moreover, the cronoamperometric response of modified sensors was dependent on nanoparticle lifetime. Experiments were performed, using freshly prepared solutions, after 4 and 8 days. Results concerning the quantitative analysis of hydrogen peroxide, in terms of detection limit, linear range, sensitivity and standard deviation (STD), are discussed for each tested sensor type. Utilization of two different linear ranges (40 microM to 6mM and 1.25 microM to 1.0mM) enabled the assessment of concentration intervals having up to three orders of magnitude. Moreover, the electrode made using a 4-day-old solution showed the maximal sensitivity of 128 nA microM(-1)(4090 nA microM(-1)cm(-2)), yielding a limit of detection of 1 microuM and STD of 2.5 microAmM(-1). All of these analytical parameters make the constructed sensors suitable for peroxide determination in aqueous solution.     

An amperometric xanthine oxidase enzyme electrode based on hydrogen peroxide electroreduction

A xanthine oxidase enzyme electrode (xanthine oxidase immobilized on electrochemically modified graphite and conveniently coated with gelatine electrode working surface) for quantitative analysis of xanthine is proposed. The detection of thus developed electrochemical system is based on the electroreduction of hydrogen peroxide generated in enzyme layer and offered L-ascorbic and uric acid reducing interference effect on the substrate determination. At a working potential -50 mV (vs. Ag/AgCl) the detection limit of 4.5 microM and the linearity of the amperometric signal up to substrate concentration of about 40 microM were found. At that working potential, the electrode is practically inert towards L-ascorbic- and uric acid present. The response time did not exceed 2 min.     

Polyvinylferrocenium modified Pt electrode for the design of amperometric choline and acetylcholine enzyme electrodes

A simple method of enzyme immobilization was investigated, which is useful for development of enzyme electrodes based on polyvinylferrocenium perchlorate coated Pt electrode surface. Enzymes were incorporated into the polymer matrix via ion exchange process by immersing polyvinylferrocenium perchlorate coated Pt electrode in enzyme solution for several times. Choline and acetylcholine enzyme electrodes were developed by co-immobilizing choline oxidase and acetylcholinesterase in polyvinylferrocenium perchlorate matrix coated on a Pt electrode surface. The amperometric responses of the enzyme electrodes were measured at +0.70 V versus SCE, which was due to the electrooxidation of enzymatically produced H2O2. The effects of the thickness of the polymeric film, pH, temperature, substrate and enzyme concentrations on the response of the enzyme electrode were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. The steady-state current of these enzyme electrodes were reproducible within +/-5.0% of the relative error. Response time was found to be 30-50s and upper limit of the linear working portions was found to be 1.2mM choline and acetylcholine concentrations in which produced detectable currents were 1.0 x 10(-6)M substrate concentrations. The apparent Michaelis-Menten constant and the activation energy of this immobilized enzyme system were found to be 1.74 mM acetylcholine and 14.9 kJ mol(-1), respectively. The effects of interferents and stability of the enzyme electrodes were also investigated.     

A new type of enzyme electrode: The ascorbic acid eliminator electrode

A new type of enzyme electrode has been developed for $\text{in}$$\text{vivo}$ electrochemical measurements which allows discrimination between ascorbic acid and catecholamines and their metabolites. The electrode employs the enzyme ascorbic acid oxidase held between the voltammetric electrode and solution by a dialysis membrane or immobilized on the outer surface of serous membrane from rat small intestine. The electrode gives linear calibration curves for all catecholamines and metabolites independent of any ascorbic acid concentrations significant in physiological measurements. The electrode has been tested in brain slice measurements and shown to respond to releases of catecholamines initiated by potassium ion stimulation.     

An enzyme electrode for specific determination of L-lysine: A real-time control sensor

Lysine decarboxylase (L-lysine carboxylyase, E.C.4.1.1.18) is immobolized on a carbon dioxide gas-sensing electrode, by copolymerization with gelatin using the bifuncitional agent glutaraldehyde. The enzyme electrodes thus prepared are used in a continuous flow system to measure the concentration of L-lysine in a mixture of amino acids. The measuring time for each sample is about 3 min, including response and rinsing times. The electrode response is linear between 0.01-1 g/L and has a high specificity for L-lysine. The enzyme electrode response to lysine at concentrations below 0.5 g/L is stable on repeated use for at least 500 assays.     

A novel amperometric bienzymatic biosensor based on alcohol oxidase coupled PVC reaction cell and nanomaterials modified working electrode for rapid quantification of alcohol

Abstract

A new amperometric sensor has been fabricated for sensitive and rapid quantification of ethanol. The biosensor assembly was prepared by covalently immobilizing alcohol oxidase (AOX) from Pichia pastoris onto chemically modified surface of polyvinylchloride (PVC) beaker with glutaraldehyde as a coupling agent followed by immobilization of horseradish peroxidase (HRP), silver nanoparticles (AgNPs), chitosan (CHIT), carboxylated multi-walled carbon nanotubes (c-MWCNTs) and nafion (Nf) nanocomposite onto the surface of Au electrode (working electrode). Owing to properties such as chemical inertness, light weight, weather resistance, corrosion resistance, toughness and cost-effectiveness, PVC membrane has attracted a growing interest as a support for enzyme immobilization in the development of biosensors. The amperometric biosensor displayed optimum response within 8?s at pH 7.5 and 35°C temperature. A linear response to alcohol in the range of 0.01mM–50?mM and 0.0001?µM as a minimum limit of detection was displayed by the proposed biosensor with excellent storage stability (190?days) at 4°C. The sensitivity of the sensor was found to be 155?µA mM^?1?cm^?2. A good correlation (R²?=?0.99) was found between alcohol level in commercial samples as evaluated by standard ethanol assay kit and the current biosensor which validates its performance.     

A dual enzyme amperometric biosensor for monitoring organophosphorous pesticides

An amperometric biosensor consisting of two enzyme membranes, one a potato layer rich in acid phosphatase and the other immobilized glucose oxidase membrane, when operated in conjunction with a Clark type dissolved O 2 elec-trode, detected the pesticide, Paraoxon, at 1 g/ml. The advantage of this biosensor is that the inhibition of acid phosphatase by the pesticide is reversible and thereby eliminates the problem of enzyme inactivation and the necessity for its reactivation which is not efficient.     

An enzyme electrode for on-line determination of ethanol and methanol

Since a stable alcohol oxidase with a high specific activity is not commercially available, we propose to produce and purify this enzyme from a strain of the yeast Hansenula polymorpha. This alcohol oxidase was immobilized into a gelatin matrix and its activity was estimated by a pO(2) sensor. The enzyme electrode obtained was then used in a continuous flow system to measure methanol or ethanol concentrations. The sample oxygen content dependence of the signal was minimized by the support properties. Measuring time for each sample were less than two minutes including response data treatment and rinsing step. The enzyme electrode response was set for ethanol from 0.5mM to 15mM and for methanol from 10mM to 300mM. On repeated use, the electrode signal for 10mM of ethanol was stable for at least 500 assays. Analysis have been performed in different beverages such as wine and beer, and the results compared to those obtained with classical methods of analysis.     

Amperometric enzyme electrode for determination of theophylline in serum.

This paper describes an amperometric enzyme electrode for the rapid determination of theophylline in serum. The method is based on the catalysed oxidation of theophylline by the haem-containing enzyme theophylline oxidase. Results are presented for two approaches. First, ferrocene monocarboxylic acid was used as a mediator. The second-order rate constant was 1.1 x 10(3) 1 mol-1 s-1. Secondly, the organic conducting salt NMP.TCNQ was used to construct enzyme electrodes. These electrodes were employed for the rapid (60 s) measurement of theophylline in serum at a working potential of +100 mV versus Ag/AgCl. Linear calibration curves were obtained over the clinically relevant range (y = 0.13x + 0.22, n = 8). Caffeine, theobromine and 3-methylxanthine at levels up to 100 mg l-1 do not interfere and 1-methylxanthine shows cross-reactivity at concentrations greater than 50 mg l-1.     

A nylon membrane based amperometric biosensor for polyphenol determination

A nylon membrane based amperometric biosensor employing banana fruit polyphenol oxidase (PPO) is presented for polyphenol detection. Nylon membrane was first activated and then coupled with chitosan. PPO was covalently attached to this membrane through glutaraldehyde coupling. The membrane bioconjugate was characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) study and then mounted onto Au electrode using parafilm to construct a working electrode. Once assembled along with Ag/AgCl as reference and Pt as auxiliary electrode, the biosensor gave optimum response within 15 s at pH 7.5 and 30 °C, when polarized at +0.4 V. The response (in mA) was directly proportional to polyphenol concentration in the range 0.2–400 μM. The lower detection limit of the biosensor was 0.2 μM. The biosensor was employed for determination of polyphenols in tea, beverages and water samples. The enzyme electrode showed 25% decrease in initial activity after 150 reuses over 6 months, when stored at 4 °C.     

A new multi-wavelength model-based method for determination of enzyme kinetic parameters

Lineweaver-Burk plot analysis is the most widely used method to determine enzyme kinetic parameters. In the spectrophotometric determination of enzyme activity using the Lineweaver-Burk plot, it is necessary to find a wavelength at which only the substrate or the product has absorbance without any spectroscopic interference of the other reaction components. Moreover, in this method, different initial concentrations of the substrate should be used to obtain the initial velocities required for Lineweaver-Burk plot analysis. In the present work, a multi-wavelength model-based method has been developed and validated to determine Michaelis-Menten constants for some enzyme reactions. In this method, a selective wavelength region and several experiments with different initial concentrations of the substrate are not required. The absorbance data of the kinetic assays are fitted by non-linear regression coupled to the numeric integration of the related differential equation. To indicate the applicability of the proposed method, the Michaelis-Menten constants for the oxidation of phenanthridine, 6-deoxypenciclovir and xanthine by molybdenum hydroxylases were determined using only a single initial concentration of the substrate, regardless of any spectral overlap.     

Electrochemically platinized carbon paste enzyme electrodes: A new design of amperometric glucose biosensors

A platinized carbon paste prepared via electrodeposition had a preferential electrocatalytic action toward H₂O₂. Therefore, we have developed a new amperometric glucose biosensor based on the immobilization of glucose oxidase on to the electrochemically platinized carbon paste. The proposed biosensor is free of potential interferences due to its cathodic detection of glucose at the potential of 0.0 V (vs. Ag/AgCl). It also shows acceptable analytical performance in terms of linearity (6 × 10⁻⁵ to 1.2 × 10⁻² M, r = 0.998), detection limit (2 × 10⁻⁵ M), response time (20–30 s), reproducibility (RSD = 4.4%), and storage life (t _0.80 = 45 days). All these advantages of the biosensor raise potential possibilities for its medical or other biotechnical applications.     

Implantable enzyme amperometric biosensors

The implantable enzyme amperometric biosensor continues as the dominant in vivo format for the detection, monitoring and reporting of biochemical analytes related to a wide range of pathologies. Widely used in animal studies, there is increasing emphasis on their use in diabetes care and management, the management of trauma-associated hemorrhage and in critical care monitoring by intensivists in the ICU. These frontier opportunities demand continuous indwelling performance for up to several years, well in excess of the currently approved seven days. This review outlines the many challenges to successful deployment of chronically implantable amperometric enzyme biosensors and emphasizes the emerging technological approaches in their continued development. The foreign body response plays a prominent role in implantable biotransducer failure. Topics considering the approaches to mitigate the inflammatory response, use of biomimetic chemistries, nanostructured topographies, drug eluting constructs, and tissue-to-device interface modulus matching are reviewed. Similarly, factors that influence biotransducer performance such as enzyme stability, substrate interference, mediator selection and calibration are reviewed. For the biosensor system, the opportunities and challenges of integration, guided by footprint requirements, the limitations of mixed signal electronics, and power requirements, has produced three systems approaches. The potential is great. However, integration along the multiple length scales needed to address fundamental issues and integration across the diverse disciplines needed to achieve success of these highly integrated systems, continues to be a challenge in the development and deployment of implantable amperometric enzyme biosensor systems.     

A method for determination of xanthine in meat by amperometric biosensor based on silver nanoparticles/cysteine modified Au electrode

A method is described for construction of an amperometric xanthine biosensor based on covalent Immobilization of xanthine oxidase (XOD) onto citrate capped silver nanoparticles deposited on Au electrode surface through cysteine self assembled monolayers (SAM). The biosensor showed optimum response within 5 s at pH 7.0 and 35 °C, when polarized at 0.5 V vs. Ag/AgCl. The linear working range of biosensor for xanthine was from 2 to 16 μM, with a detection limit of 0.15 μM and sensitivity of 0.17 mA/μM/cm². The mean analytical recovery of exogenously added xanthine in fish meat extract (5 g/l and 10 g/l) was 96.2 ± 2.3% and 95.2 ± 3.4%, respectively. Within and between batches coefficients of variation were <2.6% and <3.4%, respectively. The biosensor measured xanthine in fish, chicken, pork, and beef meat. The enzyme electrode lost 20% of its initial activity after its regular 180 uses over a period of 60 days, when stored at 4 °C in dry state.