Determination of glutamate pyruvate transaminase and pyruvate with an amperometric pyruvate oxidase sensor

Pyruvate oxidase (E.C. 1.2.3.3.) is immobilized by adsorption on a porous acetylcellulose membrane, and combined with an oxygen electrode to provide a sensor for pyruvate (0.1–0.8 mM). The response time is 2 min. Glutamate pyruvate transaminase (0.5–180 × 10^-3 I.U. ml^-1) is determined by its effect on pyruvate production by the alanine—α-ketoglutarate reaction. The sensor is stable for more than 10 days and 100 assays.     

Oxidase/peroxidase bilayer-modified electrodes as sensors for lactate,pyruvate, cholesterol and uric acid

Enzyme heterobilayer-modified electrodes were fabricated by successively covalently binding to the surface of a tin(IV) oxide plate horseradish peroxidase (HRP), then an oxidase (lactate, pyruvate or cholesterol oxidase or uricase), which liberates hydrogen peroxide by reaction with the respective substrate. The cooperative action of oxidase-HRP leads to an efficient amperometric sensor system with the minimum amount of enzyme immobilized on an electrode.     

Enzymatic immunoassay of α-fetoprotein,insulin and 17-α-hydroxyprogesterone base on chemiluminescence in a flow-injection system

Highly-sensitive enzymatic immunoassay procedures based on a chemiluminescent reaction are described. Glucose oxidase was used as the labelling enzyme conjugated with anti-α-fetoprotein IgG, insulin or 17-α-hydroxyprogesterone. Free and bound fractions present after the immune reaction were separated by an immobilized antibody or a second antibody. The enzyme activity was measured by the chemiluminescence produced by luminol and hydrogen peroxide, catalyzed by potassium hexacyanoferrate(III) after incubation with glucose. The chemiluminescence was measured in a flow-injection system, with a home-made luminescence detector equipped with a spiral flow cell. The detection limits for each substance were 10^?15–10^?17 mol. Recoveries of added α-fetoprotein from diluted serum were quantitative.     

Recycling of NAD+ using coimmobilized alcohol dehydrogenase andE. coli

The use of immobilized enzymes has opened the possibility of large scale utilization of NAD⁺-linked dehydrogenases, but the applications of this technique were limited by the necessity of providing the large amounts of NAD⁺ required by its stoichiometric consumption in the reaction. After immobilization of alcohol dehydrogenase and intactE. coli by glutaraldehyde in the presence of serum albumin, the respiratory chain was found to be capable of regenerating NAD⁺ from NADH. This NAD⁺ can be recycled at least 100 times, and thus the method is far more effective than any other, and, moreover, does not require NADH oxydase purification. The total NADH oxidase activity recovered was 10–30% of the initial activity. Although, NADH is unable to cross the cytoplasmic membrane, it was able to reach the active site of NADH dehydrogenase after immobilization. The best yield of NADH oxidase activity with immobilized bacteria was obtained without prior treatment of the bacteria to render them more permeable. The denaturation by heat of NADH oxidase in cells that are permeabilized was similar before and after immobilization. In contrast, the heat denaturation of soluble Β-galactosidase required either a higher temperature or a longer exposure after immobilization. The sensitivity of immobilized NADH oxidase to denaturation by methanol was decreased compared to permeabilized cells. As a result, it is clear that the system can function in the presence of methanol, which is necessary as a solvent for certain water insoluble substrates.     

Development of a bienzyme reactor sensor system for the determination of ornithine

A bienzyme reactor sensor system with amperometric detection was developed for the determination of ornithine. The system based on the immobilized enzymes (ornithine carbamyl transferase and pyruvate oxidase) consisted of a buffer tank, a peristaltic pump, an enzyme reactor, an oxygen electrode and a recorder. Then, 0.1 M MOPS buffer, containing pyruvic acid (0.5 mM) and carbamyl phosphate (0.5 mM), was continuously transferred into the system at 35 °C. Phosphate ion was formed enzymatically by transformation of ornithine in the presence of carbamyl phosphate. Pyruvate oxidase is activated by the presence of phosphate. Therefore, ornithine was determined from the oxygen consumed upon oxidation of pyruvic acid catalyzed by pyruvate oxidase in the presence of phosphate ion. The limit of detection was 0.05 mM and the response was linear to 3 mM (R²=0.9905). The variation coefficients were 4.9 (n=15) and 3.9% (n=15) for 1.1 and 3.0 mM standard ornithine, respectively. Good comparative results (R²=0.9238) were observed between ornithine contents in prawn muscle determined by the proposed system and by the HPLC. One assay was completed within 4 min. The immobilized enzymes were stable for 2 months at 4 °C and more than 150 samples could be continually determined using this enzyme reactor.     

Determination of glucose in blood by flow injection analysis and an immobilized glucose oxidase column

A flow-injection system for glucose determination is described. Glucose oxidase is immobilized on controlled porosity glass (CPG) and used in a glass column (2.5 mm diameter × 2.5 cm). The hydrogen peroxide produced by the enzymatic reaction (? 1 × 10^?6 M) is detected by the current produced in a flow-through cell, with two platinum electrodes having a potential difference of 0.6 V. Glucose (0–20 mmol l^?1) can be determined in blood plasma either with a dialyser in the system or, better, by incorporating a column of copper(II) diethyldithiocarbamate on CPG before the enzyme column. The results compared well with those obtained by a conventional analyser system. The glucose oxidase column showed little change in activity over a 10-month period.     

Oxygen electrode-based enzyme immunoassay for the amperometric determination of hepatitis B surface antigen

Specific antibodies labelled with glucose oxidase are immobilized onto a gelatin membrane, which is fixed over an oxygen electrode. The sensor is immersed in a standard glucose solution and a signal is obtained by measuring the consumption of oxygen by the enzyme catalyzed reaction. The response increases linearly with increasing antigen concentration over the range 0.1–100 μg 1^?1. A microcomputer is used for data acquisition and processing.     

Flow injection analysis for L-lactate with immobilized lactate dehydrogenase

Immobilized lactate dehydrogenase (LDH) is used for determination of L-lactate in a continuous flow system. The LDH is immobilized by reaction with glutaraldehyde onto the surface of alkylamino-bonded silica gel and packed into a column in the flow system. The reduction of NAD⁺ occurs simultaneously, and the NADH formed is detected amperometrically. The peak current is linearly related to the L-lactate concentration in the range 1–80 × 10^-6 M; 30 samples h^-1 can be analyzed with a r.s.d. of 0.5–1.5%. The immobilized LDH retains over 90% of its initial activity after repetitive use for 3 months.     

Flow-injection determination of sulphite and assay of sulphite oxidase

Sulphite (1–80 × 10^?5 M) in formaldehyde-stabilized solutions is determined by injection into a flowing stream of pH 8.5 phosphate buffer, passing through a mini-column of sulphite oxidase immobilized on controlled-pore glass, with amperometric detection of the hydrogen peroxide produced. Sulphite oxidase (5–100 U l^?) is determined by injection into a flowing stream of formaldehyde-stabilized 2 × 10^?3 M sodium sulphite in pH 8.0 phosphate buffer; hydrogen peroxide is again monitored.     

A sensitive xanthine oxidase electrode with silk net for estimating fish freshness

An enzyme biosensor was constructed using a plate platinum electrode and immobilized xanthine oxidase (XOD).Only a very small quantity of enzyme was chemically immobilized on a special silk net.Hydrogen peroxide released during the enzymatic reaction was detected by the electrode at+0.65 V (vs.Ag/AgCl).The electrode was very sensitive to hypoxanthine and its detection limit was 1×10-7 mol/L.When it was applied to the determination of fish freshness,the results agreed well with those obtained by traditional methods-determination of total volatile basic nitrogen (TVB-N) and microbial count.A range for estimating the freshness of river fish was suggested.     

Amperometric determination of choline and acetylcholine with enzymes immobilized in a photocross-linkable polymer

Choline and acetylcholine sensors were prepared by using choline oxidase and acetylcholinesterase, entrapped in photocross- linkable poly(vinyl alcohol) bearing styrylpyridinium (PVA-SbQ). The measurements were based on the detection of hydrogen peroxide liberated by an enzyme reaction (choline oxidase) or two sequential enzyme reactions (acetylcholine esterase and choline oxidase). The determination range for choline was 2.5-2-150 αmol 1^-1 and for acetylcholine 20-2-750 αmol 1^-1. The response times were 2-2-4 min. The immobilized enzyme membranes stored in a dry state were very stable and no loss of activity was observed after storage for 60 days.     

Bioelectrochemical response of the polyaniline galactose oxidase electrode

Galactose oxidase has been immobilized in a polyaniline film. The response current of the galactose oxidase electrode is a function of the applied potential and increases as the pH increases from 5.61 to 7.25. The optimum pH of the immobilized galactose oxidase is 7.25. The activation energy of the enzyme-catalysed reaction is 41.8 kJ mol⁻¹. The response current of the enzyme electrode shows good reproducibility at temperatures below the optimum temperature of 30.4°C and increases as the galactose concentration increases from 0.2 to 6 mmol dm⁻³. Thus the polyaniline galactose oxidase electrode can be used to determine galactose concentration.     

An enzyme reactor electrode for determination of amino acids

L-Leucine can be determined with an enzyme reactor electrode containing L-amino acid oxidase immobilized with glutaraldehyde to glass. The reactor also contains immobilized catalase which splits the hydrogen peroxide formed. Oxygen for the reaction is also supplied by adding hydrogen peroxide to the samples. The electrode is an ammonia gas sensor. The calibration curve is strictly linear with Nernstian slope between 3·10^-5 and 10^-3 M leucine.     

Novel Multiplexed Biosensor System for the Determination of Lactate and Pyruvate in Blood Serum

Multiplexing is one of the main current trends in biosensors, especially important for clinical diagnosis. However, simultaneous determination of several substances in one sample is often difficult due to different performance and working conditions of separate biosensors. This work was aimed at the development of a multiplexed biosensor system for the determination of lactate and pyruvate concentrations in liquid samples (i. e., blood serum). The system consisted of two amperometric biosensors based on lactate oxidase and pyruvate oxidase, which worked simultaneously in a single measuring cell. Conditions for the biosensor system work were selected. Linear range of lactate determination was 5–1000 μM, pyruvate – 10–5000 μM. Steady‐state response time was 30 s and 50 s for the lactate and pyruvate biosensors, respectively. After 2 weeks of storage biosensor responses decreased to 95 % (lactate biosensor) or 82 % (pyruvate biosensor) of the initial value. A scheme of analysis of the concentrations of lactate and pyruvate in human blood serum was proposed. The lactate and pyruvate concentrations as well as their ratio in human blood serum samples were determined and compared with the control method. The proposed biosensor system is suitable for the rapid detection of lactate, pyruvate and their ratio and can be used for clinical diagnosis, e. g., evaluation of the reasons of lactic acidosis and prognosis of patient's recovery.     

Micropatterning of proteins on the surface of three-dimensional poly(ethylene glycol) hydrogel microstructures

This paper describes micropatterning of proteins on the surface of three-dimensional hydrogel microstructures. Poly(ethylene glycol) (PEG)-based hydrogel microstructures were fabricated on a glass substrate using a poly(dimethylsiloxane) (PDMS) replica as a molding insert and photolithography. The lateral dimension and height of the hydrogel microstructures were easily controlled by the feature size of the photomask and depth of the PDMS replica, respectively. Bovine serum albumin (BSA), a model protein, was covalently immobilized to the surface of the hydrogel microstructure via a 5-azidonitrobenzoyloxy N-hydroxysuccinimide bifunctional linker at a surface density of 1.48 mg cm⁻². The immobilization of BSA on the PEG hydrogel surface was demonstrated with XPS by confirming the formation of a new nitrogen peak, and the selective immobilization of fluorescent-labeled BSA on the outer region of the three-dimensional hydrogel micropattern was demonstrated by fluorescence. A hydrogel microstructure could immobilize two different enzymes separately, and sequential bienzymatic reaction was demonstrated by reacting glucose and Amplex Red with a hydrogel microstructure where glucose oxidase was immobilized on the surface and peroxidase was encapsulated. Activity of immobilized glucose oxidase was 16.5 U mg⁻¹, and different glucose concentration ranged from 0.1 to 20 mM could be successfully detected.     

Gas-diffusion dilution flow-injection method for the determination of ethanol in beverages without sample pretreatment

The combination of immobilized alcohol oxidase (AOD) with a gas-diffusion membrane in a flow-injection system greatly enhanced the specificity of ethanol determination. This gas-diffusion flow-injection system with amperometric detection of hydrogen peroxide had a linear range of analysis from 0.0006 up to 60% (v/v) ethanol. Silicone-modified polypropylene membranes of different thickness were used. Undiluted samples of beer, wine, spirits and medicine could be investigated with excellent correlation with standard methods (r > 0.997). The frequency of analysis was 120–180 samples h^?1 and the operational half-life of the immobilized enzyme was 8000 injections within 44 h.     

Determination of heavy metal ions by an amperometric biosensor based on glucose oxidase immobilized onto single-walled carbon nanotubes/Nile blue nanocomposite

An enzymatic electrochemical biosensor was prepared based on glucose oxidase (GOD) immobilized on single-walled carbon nanotubes/Nile blue (Nb–SWCNTs) nanocomposite cross-linked with glutaraldehyde on graphite electrode. The function of glucose biosensor is based on the electrocatalytic reduction of H₂O₂ generated by the reaction of the enzyme with glucose molecules. The biosensor was characterized by Fourier transform infrared (FTIR) spectroscopy, emission scanning (FESEM), UV–Vis spectrometry, and voltammetric and amperometric methods. Parameters influencing the performance of the biosensor, namely pH and applied potential, were optimized. Inhibition was carried out for the detection of Hg²⁺ and Pb²⁺ species under optimized condition. Inhibition investigations showed that the type of inhibition for both the abov- mentioned heavy metal ions were reversible and competitive.     

Amperometric determination of cholinesterase with use of an immobilized enzyme electrode

A choline-sensitive electrode consisting of an immobilized choline oxidase layer and an oxygen electrode is described. Cholinesterase (0.5–60 I.U. l^-1) is measured by addition of acetylcholine, and detection of the choline produced. The precision is 3%, and the electrode is stable for more than 2 weeks (140 assays).     

Production of α-keto acids Part I. Immobilized cells ofTrigonopsis variabilis containing D-amino acid oxidase

Whole cells ofTrigonopsis variabilis were immobilized by entrapment in Ca²⁺-alginate and used for the production of α-keto acids from the corresponding D-amino acids. The D-amino acid oxidase within the immobilized cells has a broad substrate specificity. Hydrogen peroxide formed in the enzymatic reaction was efficiently hydrolyzed by manganese oxide co-immobilized with the cells. The amino acid oxidase activity was assayed with a new method based on reversed-phase HPLC. Oxygen requirements, bead size, concentration of cells in the beads, flow rate, and other factors were investigated in a “ trickle-bed ” reactor.     

Amperometric assay of glucose and lactic acid by flow injection analysis

A computer-controlled flow-injection system is described for the assay of D-glucose and L-lactic acid in undiluted plasma. Glucose or lactate is quantified by coupling an immobilized glucose oxidase or lactate oxidase membrane with an amperometric sensor; the hydrogen peroxide generated is directly related to the concentration of glucose or lactate. The linear range is 0–40 mM and 0–10 mM for glucose and lactic acid, respectively. The sample frequency is 60 h^?1 with a standard deviation of less than 1.5%. Correlation with the results for blood plasma obtained by routine clinical analyzers was good for both glucose and lactic acid.