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).     

Amperometric determination of sulfite with sulfite oxidase immobilized at a platinum electrode surface

Sulfite oxidase is immobilized on collagen membrane at the surface of a platinum electrode and catalyzes the oxidation of sulfite to sulfate with stoichiometric production of hydrogen peroxide. The hydrogen peroxide is detected amperometically at the platinum electrode at an applied potential of 700 mV. The system responds linearly to sulfite in the range 1–150 μM, with a detection limit of 0.2 μM. The enzyme retains over 95% of its activity for three weeks if stored at ?20° C when the probe is not in use.     

Selective oxoammonium salt oxidations of alcohols to aldehydes and aldehydes to carboxylic acids

The oxidation of alcohols to aldehydes using stoichiometric 4-acetamido-2,2,6,6-tetramethylpiperidine-1-oxoammonium tetrafluoroborate (1) in CH(2)Cl(2) at room temperature is a highly selective process favoring reaction at the carbinol center best able to accommodate a positive charge. The oxidation of aldehydes to carboxylic acids by 1 in wet acetonitrile is also selective; the rate of the process correlates with the concentration of aldehyde hydrate. A convenient and high yield method for oxidation of alcohols directly to carboxylic acids has been developed.     

The use of immobilized alcohol oxidase in the continuous flow determination of ethanol with an oxygen electrode

Immobilized alcohol oxidase was used in the determination of blood alcohol. The alcohol oxidase catalyzed the aerobic oxidation of ethanol and the oxygen concentration was monitored with an oxygen membrane electrode in a flow cell. The enzyme was immobilized either by covalent attachment via glutaraldehyde to the inside walls of nylon tubing, or by adsorption onto three separate controlled-pore glass support materials: TiO₂, SiO₂, or AL₂O₃. The supports were packed into 10 cm lengths of 3 mm i.d. glass tubing or 30 cm lengths of 5 mm i.d. nylon tubing. The five methods of immobilization were compared for stability and activity toward ethanol. Immobilization on silanized glass beads results in the highest activity and greatest stability of the reactor.     

Protease determination using an optimized alcohol enzyme electrode

A new method for the determination of protease activities is described. In this large family, trypsin is used as a protease model that cleaves the enthyl or methyl ester of artificial substrates producing ethanol or methanol. Alcohol is detected using an alcohol oxidase enzyme electrode. The H₂O₂ production that occurs is measured amperometrically. At 30°C, in a 0.1M phosphate buffer, pH 7.5, the enzyme electrode response for ethanol was calibrated at 3.10^?6–3.10^?3 M and for methanol from 3.10^?7 to 4.10^?4 M in the cell measurement. Trypsin levels as determined by the proposed method and by a conventional spectrophotometric method are in good agreement when using the same measurement conditions. A detection limit of 10 U·L^?1 and a linear calibration curve of 10–100,000 U·L^?1 in the sample were obtained. Measuring time for the required trypsin solution concentration was from 4 min (for the most dilute samples) to 1 min (for the most concentrate samples). In a typical experiment, protease measurements did not inactivate the alcohol oxidase on the probe, nor did a more classical use for alcohol detection. The procedure developed could permit any protease estimation on the condition that they hydrolyze ester bonds from synthetic substrate.     

Amperometric detection of uric acid and hypoxanthine with Xanthine oxidase immobilized and carbon based screen-printed electrode. Application for fish freshness determination

Carbon-based screen-printed electrodes are suitable for uric acid detection. Xanthine oxidase (XO) was immobilized either directly on the surface of the electrode or in a reactor with CPG aminopropylsilane in a FIA assembly. Higher reproducibility and lifetime was obtained with the reactor. Optimum conditions were found for the determination of Hypoxanthine (Hx), Inosine (HxR) and Inosine monophosphate (IMP). Calibration curves for IMP, HxR and Hx are linear up to 50 muM with detection limit of 1 muM for 50 mul injection. One assay is completed within 30 s. The reproducibility of 20 muM of Hx was obtained with CV 2%.     

Amperometric determination of choline with enzyme immobilized in a hybrid mesoporous membrane

Choline sensor is successfully prepared by using immobilized enzyme, i.e., choline oxidase (ChOx) within a hybrid mesoporous membrane with 12 nm pore diameter (F127M). The measurement was based on the detection of hydrogen peroxide, which is the co-product of the enzymatic choline oxidation. The determination range and the response time are 5.0-800 μM and approximately 2 min, respectively. The sensor is very stable compared to the native enzyme sensor and 85% of the initial response was maintained even after storage for 80 days. These results indicate that ChOx is successfully immobilized and well stabilized, and at the same time, enzyme reaction proceeds efficiently. Such ability of hybrid mesoporous membrane F127M suggests great promise for effective immobilization of enzyme useful for electrochemical biosensors.     

Amperometric determination of glucose with a ferrocene-mediated glucose oxidase/polyacrylamide gel electrode

Enzyme electrodes were constructed by immobilization of glucose oxidase and ferrocene into cross-linked polyacrylamide gels. Electrogenerated ferrocinium ion acts as a direct electron mediator between glucose oxidase and a reticulated vitreous carbon (RVC)/graphite support bed. The electrode is easily constructed, gives a current response proportional to glucose concentrations up to 30 mM, and has good chemical stability in water and air.     

Flow-injection analysis for l-glutamate using immobilized l-glutamate oxidase: comparison of an enzyme reactor and enzyme electrode

An enzyme electrode and enzyme based on immobilized l-glutamate oxidase are used for the determination of l-glutamate in a flow-injection system. The hydrogen peroxide produced is monitored amperometrically. The enzyme reactor system surpasses the enzyme electrode system with regard to sensitivity and analytical speed. For both systems, the peak current is linearly related to the l-glutamate concentration in the range 5 × 10^?6-1 × 10^?3 M. l-Glutamate in seasoning can be determined very selectively with < 0.7% r.s.d.     

Determination of l-amino acids and alcohols with oxidase enzymes and a tubular iodide-selective electrode

L-Amino acids and alcohols (10^-2–10^-4 M) can be determined in a flow system by using a special tubular iodide-selective electrode, after reaction with L-atnino acid oxidase and alcohol oxidase, respectively. The hydrogen peroxide produced by both enzymatic reactions reacts with iodide in the presence of a molybdate catalyst, and the change in iodide concentration is monitored. The ranges of concentration that can be determined can be varied by adjusting parameters such as iodide concentration and buffer pH.     

Amperometric determination of total cholesterol in serum with use of immobilized cholesterol esterase and cholesterol oxidase

Cholesterol esterase and cholesterol oxidase were immobilized on octyl-agarose gel, activated with cyanogen bromide and placed in a reactor. The sensor system for total cholesterol was assembled with the immobilized enzyme reactor, a hydrogen peroxide electrode and a peristaltic pump. Characteristics of the sensor system were investigated by using cholesterol palmitate as a standard substrate. A linear relationship was obtained between peak current and cholesterol palmitate concentration below 1000 mg dl^-1 (10.3 mM). A 10-μl sample could be assayed in 5 min. Total cholesterol in human serum was determined in the range 100–400 mg dl^-1. The standard deviation for the determination of 50 samples of 300 mg dl^-1 was 6 mg dl^-1 (2%). The system was used for 300 assays without loss of enzymatic activity. The correlation coefficient was 0.94 for 27 samples of human sera analyzed by the system proposed and by the conventional chemical method.     

Amperometric determination of phosphatidyl choline in serum with use of immobilized phospholipase d and choline oxidase

Phospholipase D (EC 3.1.4.4.) and choline oxidase (EC 1.1.99.1.) are immobilized together on a hydrophobic agarose gel and used to convert the phospholipid to betaine and hydrogen peroxide, which is measured amperometrically at + 0.60 V vs. SCE. The response time of the sensor is 2 min, and the calibration curve for 0–3 g l^-1 of phosphatidyl choline is linear. Different methods of insolubilizing the enzymes are compared.     

An efficient and practical system for the catalytic oxidation of alcohols, aldehydes, and alpha,beta-unsaturated carboxylic acids

Upon exposure to commercial bleach (approximately 5% aqueous sodium hypochlorite), nickel(II) chloride or nickel(II) acetate is transformed quantitatively into an insoluble nickel species, nickel oxide hydroxide. This material consists of high surface area nanoparticles (ca. 4 nm) and is a useful heterogeneous catalyst for the oxidation of many organic compounds. The oxidation of primary alcohols to carboxylic acids, secondary alcohols to ketones, aldehydes to carboxylic acids, and alpha, beta-unsaturated carboxylic acids to epoxy acids is demonstrated using 2.5 mol % of nickel catalyst and commercial bleach as the terminal oxidant. We demonstrate the controlled and selective oxidation of several organic substrates using this system affording 70-95% isolated yields and 90-100% purity. In most cases, the oxidations can be performed without an organic solvent, making this approach attractive as a "greener" alternative to conventional oxidations.     

A new enzyme electrode based on ascorbate oxidase immobilized in gelatin for specific determination of l-ascorbic acid

A biosensor for the specific determination of l-ascorbic acid in fruit juices and vitamin C tablets was developed using ascorbate oxidase (EC 1.10.3.3) from cucumber (Cucumis sativus L.) in combination with a dissolved oxygen probe. Ascorbate oxidase immobilized with gelatin using glutaraldehyde and fixed on pretreated teflon membrane served as an enzyme electrode. The phosphate buffer (50 mM, pH 7.5) and 35 degrees C were established as providing the optimum conditions. The biosensor response depends linearly on l-ascorbic acid concentration between 5.0x10(-5) and 1.2x10(-3) M with a response time 45 s. The biosensor is stable for more than 2 months, while more than 200 assays were performed. The results obtained for fruit juices and tablets were compared with DCIP (2,6 dichlorophenolindophenol) method.     

Amperometric determination of bonded glucose with an MnO(2) and glucose oxidase bulk-modified screen-printed electrode using flow-injection analysis

A screen-printed amperometric biosensor based on carbon ink double bulk-modified with MnO₂ as a mediator and glucose oxidase as a biocomponent was investigated for its ability to serve as a detector for bonded glucose in different compounds, such as cellobiose, saccharose, (-)-4-nitrophenyl-β-d-glucopyranoside, as well as in beer samples by flow-injection analysis (FIA). The biosensor could be operated under physiological conditions (0.1 M phosphate buffer, pH 7.5) and exhibited good reproducibility and stability. Bonded glucose was released with glucosidase in solution, and the free glucose was detected with the modified screen-printed electrode (SPE). The release of glucose by the aid of glucosidase from cellobiose, saccharose and (-)-4-nitrophenyl-β-d-glucopyranoside in solution showed that stoichiometric quantities of free glucose could be monitored in all three cases.The linear range of the amperometric response of the biosensor in the FIA-mode flow rate 0.2 mL min⁻¹, injection volume 0.25 mL, operation potential 0.48 V versus Ag/AgCl) extends from 11 to 13,900 μmol L⁻¹ glucose in free form. The limit of detection (3σ) is 1 μmol L⁻¹ glucose. A concentration of 100 μmol L⁻¹ yields a relative standard deviation of approximately 7% with five injections. These values correspond to the same concentrations of bonded glucose supposed that it is liberated quantitatively (incubation for 2 h with glucosidase).Bonded glucose could be determined in beer samples using the same assay. The results corresponded very well with the reference procedure.