Immobilization of d-glucose oxidase onto a hydrogen peroxide permselective membrane and application for an enzyme electrode

A hydrogen peroxide permselective membrane with asymmetric structure was prepared and d-glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized d-glucose oxidase membrane was 0.34 units cm^?2 and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized d-glucose oxidase membrane was 1.6 × 10^?3 mol l^?1 and that of free enzyme was 4.8 × 10^?2 mol l^?1. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized d-glucose oxidase membrane. The enzyme electrode responded linearly to d-glucose over the concentration 0–1000 mg dl^?1 within 10 s. When the enzyme electrode was applied to the determination of d-glucose in human serum, within day precision (CV) was 1.29% for d-glucose concentration with a mean value of 106.8 mg dl^?1. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized d-glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of d-glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.     

Magnetic enzyme membranes as active elements of electrochemical sensors: specific amino acid enzyme elctrodes.

The basic principle of the described magnetic enzyme electrodes is a kinetic accumulation of CO2 at the active layer electrode interface. The local pCO2 level is linked to three simultaneous phenomena: substrate diffusion in, enzyme reaction CO2 diffusion out. After a transient state there is a stationary state between the quantity of CO2 produced by the enzyme reaction and the CO2 diffusing from the active membrane to the bulk solution. Continuous determination of free amino acids in biological media is useful in biological processing, fermentation, medicine, pharmaceutical industries and biological research. No methods are presently available for any specific continuous measurement of lysine which is of nutritional importance in protein industrial syntheses; of phenylalanine and tyrosine which have to be monitored in several inborn diseases (phenylketonuria being the most important of them); of arginine and histidine which play a still imperfectly understood part in neurochemistry. The use of decarboxylase bearing membranes as sensors in such measurements could offer several novel advantages: (a) a simple device made of a currently manufactured electrode slightly modified by the use of an enzyme membrane; (b) The absence of any enzymic consumption due to the immobilization and the negligible consumption of substrate during the measurements; (c) The sensitivity which can be sharpened by a systematic study of the membrane parameters; (d) the continuous response of the electrode as long as it is in contact with the substrate solution; (e) the further feasibility as a miniature sensor. The magnetic device introduced allows obviously a convenient use of the enzyme electrode, the active part can be removed and replaced without disturbance for the pCO2 electrode itself. The enzyme electrodes are not only useful at the applied point of view but also at the fundamental point of view by allowing a direct measurement of an intra membrane concentration. The influence of simple structures on enzyme kinetics was studied with enzyme electrodes by our group, in the case of memory and oscillations obtained with enzyme systems.     

Immobilization of -glucose oxidase onto a hydrogen peroxide permselective membrane and application for an enzyme electrode

A hydrogen peroxide permselective membrane with asymmetric structure was prepared and -glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized -glucose oxidase membrane was 0.34 units cm⁻² and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized -glucose oxidase membrane was 1.6 × 10⁻³ mol l⁻¹ and that of free enzyme was 4.8 × 10⁻² mol l⁻¹. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized -glucose oxidase membrane. The enzyme electrode responded linearly to -glucose over the concentration 0–1000 mg dl⁻¹ within 10 s. When the enzyme electrode was applied to the determination of -glucose in human serum, within day precision (CV) was 1.29% for -glucose concentration with a mean value of 106.8 mg dl⁻¹. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized -glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of -glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.     

Ultra-thin-polysiloxane-film-composite membranes for the optimisation of amperometric oxidase enzyme electrodes.

An outer ultra-thin-polydimethyldichlorosiloxane film composite membrane has been used as the outer covering barrier in an amperometric glucose oxidase enzyme electrode biosensor. The composite membrane was formed via the condensation polymerisation of dimethyldichlorosilane at the surface of a host porous alumina membrane. Homogeneous polydimethyldichlorosiloxane films of <100 nm thickness acted as effective substrate diffusional barriers and were supported by the underlying porous alumina surface. Glucose and oxygen permeability coefficients were determined using diffusion chamber apparatus. Polysiloxane composite membranes were found to offer some screening functionality towards anionic biological interferents such as ascorbate. On exposure to blood an approximate 25% signal drift was observed during the first 2 h exposure to blood; after this time responses remained almost stable. Whole blood glucose determinations showed a close correlation (r(2)=0.98) to analyses performed via standard hospital analyses.     

Needle enzyme electrodes for biological studies

Needle enzyme electrodes have been produced for measurement of glucose and lactate. They comprise glutaraldehyde-crosslinked oxidases immobilised over < 1·1 mm od needle-type sensors for H₂O₂. To obtain selectivity in blood, an underlying polyethersulphone membrane was used which excluded electrochemical interferents from the working (Pt) electrode. Linearity for the systems was extended to cover the clinical range by the use of outer low permeability polyurethane membranes. This type of external membrane also reduced the stirring dependence of electrodes. The glucose needle electrode was used in unstirred whole blood samples and gave an acceptable correlation with the routine spectrophotometric method (y = 0·954× + 0·202, r = 0·991, n = 48).     

Immunosensors

The current trends and future aspects of the research and development of immunosensors are overviewed. A non-labelled immunosensor, whose selectivity depends on immunochemical affinity of an antigen for its corresponding antibody, has been developed as the basis for the potentiometric determination of an antigen, with an antibody-bound membrane or electrode. Non-labelled immunosensors for syphilis antibody, blood typing, human chorionic gonadotropin (HCG), and human serum albumin have been investigated. In contrast with non-labelled immunosensors, labelled immunosensors may be characterized by marked enhancement of sensitivity. Of these labelled immunosensors, enzyme immunosensors that use the chemical amplification of a labelling enzyme for sensitivity are promising. Enzyme immunosensors with an oxygen electrode have been developed to determine AFP, HCG, IgG and toxin. Bioaffinity sensors with a preformed metastable ligand-receptor complex, which are similar to the enzyme immunosensor have been found effective for the determination of thyroxine (T4), biotin, and insulin.     

Monoamine oxidase electrode in freshness testing of meat

Monoamine oxidase (monoamine: oxygen oxidoreductase, EC 1.4.3.4 from Aspergillus niger and beef plasma) was immobilized in a collagen membrane. An enzyme electrode consisting of a monoamine oxidase - collagen membrane (10 units) and an oxygen electrode was prepared for the determination of monoamines. Monoamines were oxidized to aldehydes by the immobilized enzyme and oxygen consumption was monitored amperometrically by the oxygen electrode. The response time of the electrode was 4 min. The optimum conditions for the enzyme electrode were pH 7.4 and 30°C. A linear relationship was observed between the amine (tyramine) concentration in the range 50–200 μm and the difference in current. No decrease in the output current was observed over an observation period of one week. The difference in current was reproducible with an average relative error of 8%. Monoamines in meat extracts were determined by the enzyme electrode.     

Determination of substrate concentrations by a computerized enzyme electrode

A numerical treatment of the signal produced by an electrode onto which an artificial enzyme membrane is mounted can give the concentration of the substrate (glucose, saccharose, lactose, amino acids, etc.) in solution. In the example of a glucose analyzer, in which glucose oxidase catalyzes the oxidation of glucose, the computer receives pO(2) level data from the electrode and calculates the glucose concentration. The transient electrode signal, measured as the enzyme membrane is exposed to a solution of glucose, is least-square approximated by a third-degree polynomial whose slope at inflection point is characteristic of the external glucose concentration. A calibration procedure provides a cubic spline approximation of glucose concentration as a function of slope, thus enabling automatic measurement of samples. The computer performs the calculations, and actuates valves for air rinsing, introduction of the sample, and water rinsing.     

Application of L-(+)-Lactate electrode for clinical analysis and monitoring of tissue culture medium

L-(+)-Lactate oxidase (EC 1.1.3.2) was immobilized onto the porous side of a cellulose acetate membrane with asymmetric structure which has selective permeability to hydrogen peroxide. The lactate electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized enzyme membrane. Properties of the enzyme membrane and characteristics of the lactate electrode were clarified for the determination of L-(+)-lactic acid. The lactate electrode responded linearly to L-(+)-lactic acid over the final concentration 0-0.25 mmol/L within 30 s. When the enzyme electrode was applied to the determination of L-(+)-lactic acid in control serum, within-day precision (CV), analytical recovery, and correlation coefficient between the electrode method and the colorimetric method were 1.4% with a mean value of 4.54 mmol/L, 98.0%, and 0.986, respectively. The lactate electrode was sufficiently stable to perform 1040 assays over 13 days operation for the determination of L-(+)-lactic acid. The dried immobilized enzyme membrane retained 84% of its initial activity after storage at 4 degrees C for 12 months. Moreover, the enzyme electrode was applied to the monitoring of culture medium for human melanoma cells. L-(+)-Lactate production and D-glucose consumption were closely related to cell numbers.     

Electrode and cuvette for rapid continuous CO2 tension and pH measurement in blood

An electrode and cuvette system has been developed for the continuous and rapid measurement of either blood CO2 tension or pH. The CO2 electrode consists of a 1.5-mm-diameter flat-tip glass pH electrode covered by a film of carbonic anhydrase solution, over which a 25-micron-thick dimethyl silicone membrane is attached. Porous ceramic filled with 20% polyacrylamide, equilibrated with a salt solution, serves as a salt bridge between a Ag-AgCl reference electrode and the pH electrode surface. The electrode is housed in a four-port cuvette assembly. Blood from a vessel of interest is delivered to the cuvette by means of an occlusive roller pump. The cuvette maintains the electrode and blood at a constant temperature and directs a continuous jet of blood against the electrode surface. The cuvette also allows for easy and frequent calibration of the electrode with either gas or liquid standards. The 90% response time of the CO2 electrode is 3.0 s for liquids and 1.3 s for gases. Removal of the dimethyl silicone membrane and carbonic anhydrase film yields a pH electrode that can continuously measure blood pH with a 90% response time of 1.6 s.     

Fabrication of butyrylcholinesterase sensor using polyurethane-based ion-selective membranes.

A simple method of enzyme immobilization was investigated which is useful for fabrication of enzyme sensors based on polymeric ion-selective membranes. The enzyme membrane was built by coating a thin hydrophilic polyurethane (HPU) film directly mixed with an enzyme over an underlying polyurethane (PU)-based ion-selective membrane. This highly simple method of enzyme immobilization was applied to the fabrication of a potentiometric butyrylcholinesterase-based biosensor for the determination of organophosphorus pesticides. The enzyme was well entrapped within the HPU film and the intrinsic potentiometric response of the underlying ion-selective PU membrane was not influenced significantly by the outer HPU/enzyme membrane. The enzyme electrode was optimized by changing systematically the composition of the enzyme membrane to evaluate the effect of the changes on sensor response. The sensor was successfully applied to the analysis of paraoxon, an organophosphorus pesticide.     

Development of potentiometric urea biosensor based on urease immobilized in PVA-PAA composite matrix for estimation of blood urea nitrogen (BUN)

A urea biosensor was developed using the urease entrapped in polyvinyl alcohol (PVA) and polyacrylamide (PAA) composite polymer membrane. The membrane was prepared on the cheesecloth support by gamma-irradiation induced free radical polymerization. The performance of the biosensor was monitored using a flow-through cell, where the membrane was kept in conjugation with the ammonia selective electrode and urea was added as substrate in phosphate buffer medium. The ammonia produced as a result of enzymatic reaction was monitored potentiometrically. The potential of the system was amplified using an electronic circuit incorporating operational amplifiers. Automated data acquisition was carried by connecting the output to a 12-bit analog to digital converter card. The sensor working range was 1–1000 mM urea with a response time of 120 s. The enzyme membranes could be reused 8 times with more than 90% accuracy. The biosensor was tested for blood urea nitrogen (BUN) estimation in clinical serum samples. The biosensor showed good correlation with commercial Infinity™ BUN reagent method using a clinical chemistry autoanalyzer. The membranes could be preserved in phosphate buffer containing dithiothreitol, β-mercaptoethanol and glycerol for a period of two months without significant loss of enzyme activity.     

Amperometric enzyme electrodes

Three different types of amperometric enzyme electrode are described. The first type uses a conducting organic-salt electrode to oxidize NADH. Results for sensors for ethanol and for bile acids are presented. In the second type of sensor, flavoenzymes are directly oxidized on the surface of the conducting organic-salt electrode. Results for five different enzymes are described. The mechanism of the enzyme oxidation is discussed and the reaction is shown to take place by heterogeneous redox catalysis and not by homogeneous mediation. The enzymes are strongly adsorbed on the electrode; microelectrodes for in vivo studies can be constructed without a membrane. Results for in vivo studies of changing glucose levels in the brain of a freely moving rat are presented. The third type of sensor is designed to measure low levels of toxic gases such as H2S and HCN. This is done by monitoring the inhibition by the toxic gas of the activity of the respiratory enzyme cytochrome oxidase.     

Reconstitution of functional electron transfer between membrane biological elements in a two-dimensional lipidic structure at the electrode interface

These studies develop a methodology to form supported phospholipid bilayers at an electrode/solution interface that models biological membrane systems. Two kinds of electrode were used, a planar gold electrode and a microporous aluminium oxide electrode on which octadecanethiol or octadecyltrichlorosilane was self-assembled. The supported lipidic structures were produced by transfer of a phospholipid monolayer by the Langmuir—Blodgett technique or by direct fusion of phospholipid vesicles. Ubiquinone was introduced into the lipidic structures during their formation; electrochemical measurements demonstrated the mobility of ubiquinone along the plane of the bilayer. A membrane enzyme, pyruvate oxidase from E. coli, was successfully incorporated into this artificial bilayer and was found to be able to exchange electrons with ubiquinone present in the bilayer.     

Urate oxidase electrode based on dissolved oxygen probe for urine uric acid determination

A biosensor for the specific determination of uric acid in urine was developed using urate oxidase (EC 1.7.3.3) in combination with a dissolved oxygen probe. Urate oxidase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated teflon membrane to serve as enzyme electrode. The electrode response was maximum when 50 mM glycine buffer was used at pH 9.2 and 35 degrees C. The enzyme electrode response depends linearly on uric acid concentration between 5-40 microM with a response time of 5 min. The enzyme electrode is stable for more than 2 weeks and during this period over 35 assays were performed.     

Enzyme electrode composed of the pyruvate oxidase from pediococcus species coupled to an oxygen electrode for measurements of pyruvate in biological media

Pyruvate oxidase from Pediococcus species was immobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde. The film was fixed onto the tip of an oxygen electrode. The enzyme electrode was specific for pyruvate measurements. This electrode was sensitive to 0.1 mM and could be used up to a final pyruvate concentration of 2 mM.At each step of the enzymatic film preparation and assay 0.7 mM thiamine pyrophosphate, 10 μM flavin adenine dinucleotide, 5mM Mg²⁺ and 10 mM phosphate buffer were necessary.A computerized probe allowed successive measurements every 3 min for more than 20 h with the same enzymatic film. The reproducibillty for the same pyruvate concentration was 2% during 400 assays without special optimization.This enzyme electrode has many applications in basic (metabolism, enzymology) and applied (blood, yoghurt) research. Results obtained from assays carried out in yoghurt are presented.     

Miniaturized multi-enzyme biosensors integrated with pH sensors on flexible polymer carriers for in vivo applications

An electrochemical glucose sensor has been integrated, together with a pH sensor, on a flexible polyimide substrate for in vivo applications. The glucose sensor is based on the measurement of H₂O₂ produced by the membrane-entrapped enzyme glucose oxidase (GOD). To minimize electrochemical interference, an electrode configuration was designed to perform differential measurements. The solid-state pH sensor employs a PVC-based neutral carrier membrane. The enzymes GOD and catalase were immobilized into two layers of photolithographically patterned hydrogels. The intended use of this device is the short-term monitoring of glucose and pH in intensive care units and operating theatres, especially for neurosurgical applications. The developed immobilization technique can also be used to create integrated multi-sensor chips for clinical analysers. The glucose and pH sensor exhibited excellent performance during tests in buffer solutions, serum and whole blood.     

肌苷酶电极生物传感器

为了构建肌苷酶电极生物传感器,以固定化核苷磷酸化酶(EC 2.4.2.1)、黄嘌呤氧化酶(EC 1.2.3.2)与过氧化氢电极组成电流型酶电极生物传感器,用于检测肌苷片中的肌苷,其输出电流可达500nA.结果发现,肌苷测定的线性范围为1-268 mg/L,精度:RSD小于0.14%,响应时间:60 s,使用寿命大于25 d,实际测定肌苷片中肌苷含量回收率:100.8%.由此表明:采用双酶电极法测定肌苷片中的肌苷含量,由于酶促反应专一性高、样品不需分离直接进样分析、处理条件温和、反应时间短暂因而结果较为可靠.     

Biocompatible glucose sensor prepared by modifying protein and vinylferrocene monomer composite membrane

This paper proposes a very simple procedure for preparing a biocompatible sensor based on a protein (bovine serum albumin, BSA), enzyme and vinylferrocene (VF) composite membrane modified electrode. The membrane was prepared simply by first casting vinylferrocene and then coating it with BSA and glucose oxidase immobilised with glutaraldehyde. The sensor response was independent of dissolved oxygen concentration from 3 to 10 ppm and showed good stability for serum sample measurement, unlike the commonly used BSA/enzyme modified electrode. The sensor response was almost unchanged over the measurement time (>10 h) whereas the responses of a BSA and glucose oxidase modified platinum electrode and an osmium-polyvinylpyridine wired horseradish peroxidase modified electrode (Ohara et al., 1993) fell to 68% of their initial value in a serum sample containing 10mM glucose.     

醋酸纤维素膜为基础的葡萄糖生物传感器的研制

用共价法将酶固定在醋酸纤维素膜上,方法简便易行,制造的酶膜稳定,比活力高。同时采用该方法制备了葡萄糖氧化酶酶膜,与氧电极组装成测定葡萄糖的生物传感器,线性范围为50~800mg／dl,仪器工作的最适pH为6．0,最适温度为40℃。将该膜与过氧化氢电极组装得到的传感器具有以下特性：线性范围为10~200mg／dl,最适pH为6．0,测定结果与酶试制盒有良好相关性。