Enzyme Sensor System Research Articles

Enzymatic biosensor for organophosphate pesticide detection using extract from the brain of Genyatremus luteus

This study developed a biosensor for the detection of pesticides, particularly organophosphates, employing an enzymatic inhibition technique using ECPP (Genyatremus luteus), a natural source of acetylcholinesterase. The stonefish is found from the Antilles to southern Brazil and is an important fishery resource for the states of Maranhão and Pará. This species is prominent in artisanal fisheries along the coast of Maranhão, standing out among several economically significant species due to its high abundance and popularity among the population of the state of Maranhão, where it ranks high in daily fish consumption preferences. High consumption consequently leads to waste generation. A byproduct of processing this species was utilized to construct the biosensor. Characterization of the crude extract, optimal temperature, pH, and kinetic studies with different substrate concentrations were conducted. To construct the biosensor, ECPP was incorporated into a carbon graphite paste with the mediator tetracyanoquinodimethane (TCNQ). Following sensor preparation, electrochemical characterization of the immobilized enzyme sensor system commenced. The results were chronocoulometrically recorded. The construction of this biosensor replaces the expensive and non-locally produced acetylcholinesterase from the commercially available electric eel marketed by Sigma, thus offering a cheaper alternative adapted to local conditions.

Amperometric determination of cadmium, lead, and mercury metal ions using a novel polymer immobilised horseradish peroxidase biosensor system

This work was undertaken to develop a novel Pt/PANI-co-PDTDA/HRP biosensor system for environmental applications to investigate the inhibition studies by specific heavy metals, to provide data suitable for kinetic studies and further application of the biosensor to environmental samples. The newly constructed biosensor was compared to the data of the well-researched Pt/PANI/HRP biosensor. Optimised experimental conditions, such as the working pH for the biosensor was evaluated. The functionality of the amperometric enzyme sensor system was demonstrated by measuring the oxidation current of hydrogen peroxide followed by the development of an assay for determination of metal concentration in the presence of selected metal ions of Cd2+, Pb2+ and Hg2+. The detection limits were found to be 8 × 10−4 μg L−1 for cadmium, 9.38 × 10−4 μg L−1 for lead and 7.89 × 10−4 μg L−1 for mercury. The World Health Organisation recommended that the maximum safety level of these metals should not exceed 0.005 mg L−1 of Cd2+, 0.01 mg L−1 of Pb2+ and 0.001 mg L−1 of Hg2+., respectively. The analytical and detection data for the metals investigated were observed to be lower than concentrations recommended by several bodies including World Health Organisation and Environmental Protection Agencies. Therefore the biosensors developed in this study can be used to screen the presence of these metals in water samples because of its low detection limit. The modes of inhibition of horseradish peroxidase by Pb2+, Cd2+ and Hg2+ as analysed using the double reciprocal plots of the Michaelis–Menten equation was found to be reversible and uncompetitive inhibition. Based on the Kmapp and Imax values for both biosensors the results have shown smaller values. These results also proved that the enzyme modified electrode is valuable and can be deployed for the determination or screening of heavy metals.

Chip-based amperometric enzyme sensor system for monitoring of bioprocesses by flow-injection analysis

A microfluidic chip integrating amperometric enzyme sensors for the detection of glucose, glutamate and glutamine in cell-culture fermentation processes has been developed. The enzymes glucose oxidase, glutamate oxidase and glutaminase were immobilized by means of cross-linking with glutaraldehyde on platinum thin-film electrodes integrated within a microfluidic channel. The biosensor chip was coupled to a flow-injection analysis system for electrochemical characterization of the sensors. The sensors have been characterized in terms of sensitivity, linear working range and detection limit. The sensitivity evaluated from the respective peak areas was 1.47, 3.68 and 0.28μAs/mM for the glucose, glutamate and glutamine sensor, respectively. The calibration curves were linear up to a concentration of 20mM glucose and glutamine and up to 10mM for glutamate. The lower detection limit amounted to be 0.05mM for the glucose and glutamate sensor, respectively, and 0.1mM for the glutamine sensor. Experiments in cell-culture medium have demonstrated a good correlation between the glutamate, glutamine and glucose concentrations measured with the chip-based biosensors in a differential-mode and the commercially available instrumentation. The obtained results demonstrate the feasibility of the realized microfluidic biosensor chip for monitoring of bioprocesses.

Use of an optical biosensor with a silicone-immobilized enzyme to determine plasma total cholesterol concentrations in fish

Decreased plasma total cholesterol concentrations are a useful indicator in fish of reduced resistance to bacterial infection. Taking advantage of the hydrophobic properties of silicone rubber, we developed an optical enzyme sensor system for measuring plasma cholesterol concentrations in fish. The system was constructed using an immobilized enzyme membrane, optical oxygen probe, flow system, and personal computer. The enzyme membrane was prepared from cholesterol esterase, cholesterol oxidase, and silicone rubber. The calibration curves were linear in the range of 55–85 mg dl−1 for yellowtail and 20–90 mg dl−1 for rainbow trout. Assays could be completed within 3 min, and the sensor response was stabilized by the hydrophobic properties of silicone rubber. Storage of immobilized enzyme membranes at 5°C permitted stable measurements for 58 days. The sensor system was applied to determine plasma total cholesterol concentrations in fish. Good correlations were obtained between results obtained using the sensor and results obtained using conventional methods (correlation coefficients: yellowtail 0.9751, rainbow trout 0.9947). Our method required much less time than conventional cholesterol assays and can be used economically for continuous determination of plasma total cholesterol in fish.

Wireless enzyme sensor system for real-time monitoring of blood glucose levels in fish

Periodic checks of fish health and the rapid detection of abnormalities are thus necessary at fish farms. Several studies indicate that blood glucose levels closely correlate to stress levels in fish and represent the state of respiratory or nutritional disturbance. We prepared a wireless enzyme sensor system to determine blood glucose levels in fish. It can be rapidly and conveniently monitored using the newly developed needle-type enzyme sensor, consisting of a Pt–Ir wire, Ag/AgCl paste, and glucose oxidase. To prevent the effects of interfering anionic species, such as uric acid and ascorbic acid, on the sensor response, the Pt–Ir electrode was coated with Nafion, and then glucose oxidase was immobilized on the coated electrode. The calibration curve of the glucose concentration was linear, from 0.18 to 144 mg/dl, and the detection limit was 0.18 mg/dl. The sensor was used to wirelessly monitor fish glucose levels. The sensor-calibrated glucose levels and actual blood glucose levels were in excellent agreement. The fluid of the inner sclera of the fish eyeball (EISF) was a suitable site for sensor implantation to obtain glucose sample. There was a close correlation between glucose concentrations in the EISF and those in the blood. Glucose concentrations in fish blood could be monitored in free-swimming fish in an aquarium for 3 days.

Optical enzyme sensor for determining l-lysine content using l-lysine oxidase from the rockfish Sebastes schlegeli

L-lysine (L-Lys) in living bodies is critical for metabolism; therefore, determination of its levels in food is important. Most enzymatic methods for L-Lys analysis are performed using L-lysine oxidase (LyOx), but commercially manufactured LyOx is generally not highly selective for L-Lys among amino acids. We previously isolated LyOx as an antibacterial protein secreted from the skin of the rockfish Sebastes schlegeli. In the present study, we developed an optical enzyme sensor system for rapid and continuous determination of L-Lys using this LyOx. The system comprised an immobilized LyOx membrane, an optical oxygen probe, a flow system, and a personal computer. The amount of L-Lys was detected as a decrease in the oxygen concentration due to the LyOx reaction. The specificity of the sensor was examined against various amino acids. The sensor response was specific for L-Lys. Good reproducibility was obtained in 58 assays. The response of the sensor using commercially prepared LyOx was unstable compared with the response using LyOx isolated in our laboratory. Our sensor system could be used for 5 weeks without our having to change the enzyme membrane. The calibration curve for a standard L-Lys solution was linear from 0.1 to 3.0 mmol L(-1). One assay could be completed within 2 min. The sensor was applied to determine the L-Lys content in food samples such as bonito cooking water and scallop hepatopancreas. The values obtained using the sensor and conventional high-performance liquid chromatography methods were well correlated.

A needle-type optical enzyme sensor system for determining glucose levels in fish blood

A needle-type biosensor system was developed for rapid and simple determination of glucose levels in fish blood. The sensor comprises a needle-type hollow container (18-gauge needle), immobilized enzyme membrane and optic fiber probe with ruthenium complex. The enzyme membrane was prepared from glucose oxidase, azide-unit pendant water-soluble photopolymer and an ultra-thin dialysis membrane. The optic fiber probe was inserted into the rolled enzyme membrane placed in the needle-type hollow container. The calibration curve was linear for glucose levels in fish plasma. One assay was completed within 3 min. A good reproducibility was observed for 60 times without exchange of the enzyme membrane. The sensor was inserted into the caudal vein of fish to measure blood glucose levels. The sensor responded immediately after insertion and glucose levels could be monitored. Good correlations were observed between values determined using the sensor and conventional methods in the range of 48–157 mg dl −l (correlation coefficient, 0.9474).

PH-metric detection of alkaline phosphatase activity as a novel biosensing platform

A detection of alkaline phosphatase (ALP, EC 3.1.3.1) activity by the monitoring of pH changes caused by the biocatalytic action of the enzyme has been experimentally examined. Enzymatically catalyzed hydrolysis of monofluorophosphate has been found to be the best basis for such measurements. Protolytic equilibria connected with the developed biosensing system were recognized and the optimal conditions for the assay have been found. Advantages and disadvantages of the developed (bio)sensing scheme have been discussed. The prototype of pH-ALP based enzyme electrode has been demonstrated. Potential utility of such substrate–enzyme–sensor system for the development of a new group of biosensors has been announced.

Simultaneous assay of glucose, lactate, L-glutamate and hypoxanthine levels in a rat striatum using enzyme electrodes based on neutral red-doped silica nanoparticles

An electrochemical method suitable for the simultaneous measurement of cerebral glucose, lactate, L-glutamate and hypoxanthine concentrations from in vivo microdialysis sampling has been successfully performed for the first time using a neutral red-doped silica (NRDS) nanoparticle-derived enzyme sensor system. These uniform NRDS nanoparticles (about 50 +/- 3 nm) were prepared by a water-in-oil (W/O) microemulsion method, and characterized by a TEM technique. The neutral red-doped interior maintained its high electron-activity, while the exterior nano-silica surface prevented the mediator from leaching out into the aqueous solution, and showed high biocompability. These nanoparticles were then mixing with the glucose oxidase (GOD), lactate oxidase (LOD), L-glutamate oxidase (L-GLOD) or xanthine oxidase (XOD), and immobilized on four glassy carbon electrodes, respectively. A thin Nafion film was coated on the enzyme layer to prevent interference from molecules such as ascorbic acid and uric acid in the dialysate. The high sensitivity of the NRDS modified enzyme electrode system enables the simultaneous monitoring of trace levels of glucose, L-glutamate, lactate and hypoxanthine in diluted dialysate samples from a rat striatum.

Enzyme sensor system for determination of total cholesterol in fish plasma

: An enzyme biosensor system was developed for rapid and continuous determination of total cholesterol in fish plasma. The system was constructed with an immobilized enzyme membrane, Clark-type oxygen electrode, flow system and personal computer. The enzyme membrane was prepared from cholesterol esterase, cholesterol oxidase, photosensitive polymer and an ultra-thin dialysis membrane. The optimum assay conditions of the sensor system were established as follows: pH 6.8, temperature 25°C and flow rate 0.2 mL/min. The calibration curves were linear in the range of 15–80 mg/dL for yellowtail, 17–42 mg/dL for amberjack and 20–70 mg/dL for rainbow trout. One assay was completed within 5 min and it was possible to determine the sample continuously. The sensor system was applied to the determination of total cholesterol in yellowtail plasma. A good correlation was observed between the values determined by the sensor method and the conventional method (correlation coefficient was 0.90).

Development of a flow-injection analysis (FIA) enzyme sensor for fructosyl amine monitoring.

An enzyme-sensor system with flow-injection analysis (FIA) has been developed for the detection of fructosyl amine compounds; the sensor utilizes fructosyl amine oxidase isolated from the marine yeast Pichia sp. N1-1 strain. With this FIA system 0.2 to 10 mmol L(-1) fructosyl valine can be determined. The sensor is approximately five times more sensitive to fructosyl valine, a model compound for glycated hemoglobin HbA1c, than to N(epsilon)-fructosyl lysine, a model compound for glycated albumin. This FIA system can also be used to detect fructosyl dipeptides. The operational stability of the sensor enabled more than 120 consecutive sample injections over a period of approximately 20 h.

フェリ磁性膜を用いた熱型酵素センサの基礎特性

The introduction of biochemistry into electronics technology is advancing and various biosensors have been developed. Ferri-magnetic film, FMF, with 1-μm thickness can be utilized as a highly sensitive temperature sensor. The FMF is prepared using an annealing treatment after RF sputtering of temperature-sensitive ferrite as a target. FMF with a marked temperature dependence has the ability to detect minute temperature variations of as little as 10-3 °C, so application of the FMF to an enzyme sensor was possible. Up to now, enzyme sensors utilizing the electrical characteristics of a thermistor have been used, but a magnetic enzyme sensor has not yet been proposed. The paper describes construction of a thermal enzyme sensor system using the FMF's magnetic characteristics for detecting minute heat variations caused by chemical reactions. We also discuss a technique for discrimination of chemical substances and metabolites.

Rapid determination of glucose and sucrose by an amperometric glucose-sensing electrode combined with an invertase/mutarotase-attached measuring cell

Glucose and sucrose are simultaneously determined by the use of an enzyme sensor system consisting of a glucose-sensing electrode based on a lipid-modified glucose oxidase and a measuring cell that contains an invertase/mutarotase-coimmobilized layer. From the current response of the enzyme electrode after the addition of a glucose/sucrose mixture, the concentrations of the two kinds of sugars can be separately determined: the concentration of glucose (0·2 μM–3 mM) is determined from the steady-state current increase obtained from 2 to 6 s after the addition of the mixture, and that of sucrose (10 μM–6 mM), from the rate of current increase from 8 to 20 s after the addition. The relative standard deviations are 1·7% for glucose and 3·1% for sucrose ( n=10). The system can be applied to the rapid determination of glucose and sucrose in food samples.

Preparation and Application to Enzyme Sensor of Composite Metal Oxide Semiconductor Thin Film

The metal oxide devices such as a thermistor and a varistor in the form of bulk or thick film which are applied for instruments, control and electronic circuits, have been developed. However, preparation of metal oxide thin film is difficult because of a high melting point of oxide material and irregular components including impurities. To solve this problem, the authors perceived that a low melting point metal becomes a metal oxide with a high melting point by oxidation, then devised the new method which after deposited according to order of Mn and Fe powder with high melting point from Zn with low melting point in vacuum, its multilayered metal can be converted into a metal oxide via profile of high temperature annealing in air. Through this process, the metal film with low melting point than annealing temperature does not disperse, so that a composite metal oxide semiconductor thin-film (COT) with thickness of 0.7-1.5μm is prepared. A markedly temperature dependent COT is suitable for a high sensitive microsensor responding to minute temperature change. The paper describes preparation of COT and construction of an enzyme sensor system based on chemical reaction heat for detecting nitrogen metabolite such as urea.

Kinetic analysis of arginase-urease coupled reaction with a surface acoustic wave enzyme sensor system

An extended set of equations which describe coupled arginase-urease reactions is presented. The mathematical treatment leads to the development of new equations which relate the lag-time required for the concentration of urea to reach a defined fraction of its steady-state concentration to the kinetic parameters of the enzymes, when the steady-state concentration of urea is small compared to its Michaelis constant value, the coupled arginase-urease reaction was monitored with a surface acoustic wave (SAW) enzyme sensor system, to couple the biochemical selectivity of enzymes with the sensitivity of SAW sensors. The proposed theoretical expressions were verified experimentally. The kinetic parameters of urease and arginase (extracted directly from bovine liver) were examined under theoretical guidance. Dependence of the lag-time of the coupled enzyme reaction on the concentrations of urease and arginase is also described.

Determination of lipase activity in porcine pancreas and clinical analysis of lipase in human serum with surface acoustic wave enzyme sensor system

A new kind of surface acoustic wave (SAW) enzyme sensor system has been applied to the assay of extracted lipase activity based on triacetin solution as substrate. A linear relationship between the frequency change and the enzyme activity up to 500 U/L has been obtained, and the detection limit has been evaluated to 0.3 U/L. Kinetic parameters of the extracted lipase as well as that of the standard enzyme have been estimated. The effects of temperature, pH value and surfactants have been investigated. A comparison between SAW sensor system and the conductometric method has been carried out. The technique has been applied to the determination of lipase activity in serum samples.

Study on the urease activity in different plant seeds and clinical analysis of urea in human body fluids by a surface acoustic wave enzyme sensor system

Urease was extracted from several kinds of plant seeds and the kinetic frequency response, the urease activity, Michaelis constants and other kinetic parameters were measured by means of a new device, i.e. a surface acoustic wave (SAW) enzyme sensor system, which was composed of a 61 MHz SAW resonator and a pair of parallel platinum electrodes. Influences such as extracting conditions, pH and temperature and the specificity were also investigated. The method has been successfully applied in the clinical assay of urea in human urine and blood. The results are in agreement with the reported values and also support the clinical diagnosis.

Study on a modified algorithm—iterative Kalman filter and its application in enzymic kinetics

A modified algorithm, the iterative Kalman filter, for the analysis of enzymic kinetic data was investigated and successfully applied to the determination of urea in human blood by a urease catalytic reaction. This reaction was monitored with a novel sensor, the surface acoustic wave enzyme sensor system recently proposed by our laboratory. The results were compared with those provided by a calibration graph and classical colorimetry methods. In contrast to these conventional methods, the iterative extended-Kalman filter is shown to be an excellent data analysis method for enzyme kinetic analysis. The urease kinetic parameters were also estimated according to the iterative Kalman filtering results.

Determination of dehydroascorbic acid by an enzyme sensor system with a parallel flow cell

An enzyme sensor system was developed for the determination of dehydroascorbic acid and L-ascorbic acid. The system was constructed from a parallel flow cell separated by a dialysis membrane, an immobilized ascorbate oxidase membrane, coupled to a Clark-type oxygen electrode, and L-cysteine, which was present as a reductant. The method is based on the reduction of dehydroascorbic acid to L-ascorbic acid with L-cysteine in a parallel flow cell. One assay for dehydroascrobic acid could be completed within 8 min. Linear calibration curves for dehydroascorbic acid and L-ascorbic acid were observed in the range of 20 mM–100 mM and 2 mM–20 mM, respectively.

Development of a biosensor for assaying postmortem nucleotide degradation in fish tissues

An enzyme sensor system has been developed to assess the freshness level in fish tissue. The system was designed to measure the K value, the concentration ratio of [Hx + HxR] and [Hx + HxR + IMP], where Hx, HxR, and IMP are hypoxanthine, inosine and inosine-5'-monophosphate, respectively. The [Hx + HxR] concentration in tissue extract was measured by nucleoside phosphorylase and xanthine oxidase immobilized on a preactivated nylon membrane and attached to the tip of a polarographic electrode. The electrode amperometrically detected the products of degradation, hydrogen peroxide and uric acid. For determination of [IMP + HxR + Hx], IMP was first converted to HxR by nucleotidase immobilized on the wall of a polystyrene tube. The enzyme electrode consisting of nucleoside phosphorylase and xanthine oxidase provided excellent reproducible results for at least 40 repeated assays and immobilized nucleotidase was good for at least 40 assays as well. The K value for each sample could be determined in ca. 10 min. When applied to K value measurements in several fish meats, the results obtained agreed well with those obtained by the conventional enzymatic method.