Enzymic determinations with rotating bioreactors and continuous-flow-stopped-flow processing. Determination of choline esters in pharmaceuticals.

The neuromuscular blocking agent, succinylcholine, and the neurotransmitter, acetylcholine, have been determined in pharmaceutical preparations utilizing a sensing unit comprised of a rotating bioreactor and an amperometric detector. The bioreactor consisted of a rotating disk made of Teflon (1 cm id) with cholinesterase (EC 3.1.1.8) and choline oxidase (EC 1.1.3.17) co-immobilized on the top surface of the disk. Amperometric detection of H2O2 and initial rate measurements were employed for quantification.

On-line enzymatic amplification by substrate cycling in a dual bioreactor with rotation and amperometric detection.

The amplification approach centered on the cycling of two reversibly interconvertible chemical species sequentially participating in two different enzyme-catalyzed reactions (enzymatic amplification by substrate cycling) has been implemented on-line into a continuous-flow/stopped-flow/continuous-flow operation. The implementation is illustrated with the determination of L-lactate in a dual enzyme reactor containing immobilized lactate oxidase (LOD, EC 1.1.3.x) to catalyze the oxidation of L-lactate by dissolved oxygen. The immobilized LOD was affixed to a rotating disk in the lower part of the flow-through cell. Immobilized lactate dehydrogenase (EC 1.1.1.27), affixed to the top part of the cell regenerates L-lactate with the mediation of beta-NADH as the hydrogen donor. The substrate cycling permits the generation of H2O2 beyond the stoichiometric limitation, and this is detected at a stationary Pt-ring electrode located at the bottom part of the cell. The stationary Pt-ring electrode is positioned concentrically to the rotating disk containing the immobilized LOD. The resulting amplified response permits, in a simple manner, achievement of detection limits as low as 0.3 fmol.liter-1 and allows the processing of 30 samples per hour.

Continuous-flow/stopped-flow system incorporating two rotating bioreactors in tandem: application to the determination of alkaline phosphatase activity in serum.

Two rotating bioreactors in tandem have been incorporated into a continuous-flow/stopped-flow sample/reagent processing setup for the determination of alkaline phosphatase (EC3.1.3.1) activity in serum samples. The strategy circumvents incompatibility of buffer systems as well as that of the immobilized enzymes utilized in the bioreactors (alkaline phosphatase and alcohol oxidase, EC 1.1.3.13). The determination is indirect in nature although recorded responses are directly related to the enzyme activity in the sample. It couples the following enzyme-catalyzed reactions: (1) hydrolysis of p-nitrophenyl dihydrogen phosphate catalyzed by alkaline phosphatase, (2) enzymatic reaction between unreacted p-nitrophenyl dihydrogen phosphate with methanol, and (3) conversion of the residual methanol to the corresponding aldehyde and H2O2, catalyzed by alcohol oxidase. The H2O2 is amperometrically determined at a stationary Pt-ring electrode (applied potential + 0.600 V vs a Ag/AgCl, 3.0 M NaCl reference).

Simultaneous kinetic-based determination of fructose and ascorbate with a rotating bioreactor and amperometric detection: application to the analysis of food samples.

A recently introduced biosensor comprising a rotating bioreactor and a stationary platinum ring amperometric detector (Anal. Chem. 1993, 65, 636-639) has been utilized for the simultaneous determination of fructose and ascorbate. The approach has been successfully applied to the simultaneous determination of these analytes in fresh food samples. Hexacyanoferrate(II) is the monitored species at +0.380 V vs a Ag/AgCl, 3 M NaCl reference. The determination takes advantage of a fast chemical oxidation of ascorbate by hexacyanoferrate(III) ions and the subsequent slower production of hexacyanoferrate(II) in the D-fructose 5-dehydrogenase-catalyzed reaction between D-fructose and hexacyanoferrate(III) as acceptor. D-Fructose 5-dehydrogenase (EC 1.1.99.11) was incorporated into the rotating disk reactor in immobilized form and on controlled-pore glass via the glutaraldehyde attachment and cross-linking with bovine serum albumin. Sample/reagent processing was accomplished by programmed continuous-flow/stopped-flow/continuous-flow operation.

Continuous-flow performance of carbon electrodes modified with immobilized Fe(II)/Fe(III) centers Amperometric response to N(2)O, NO and NO(2).

The amperometric performance of two types of chemically modified carbon electrodes developed for the determination of oxides of nitrogen in continuous-flow systems is presented. The modification consists in immobilization of reversible Fe(II)/Fe(III) centers. The first type of electrode is a simple modification made by direct mixing of carbon paste with tris-4,7-diphenyl-1,10-phenanthroline-iron(II) perchlorate; the other is a glassy-carbon surface modified by oxidative electropolymerization of tris-[5-amino-1,10-phenanthroline]iron(II) perchlorate. Detection is accomplished by transporting an injected sample plug to the sensing surface with the aid of gravitational flow of an aqueous solution of supporting electrolyte. The polymer-coated electrochemical detector compares favourably with the chemically modified carbon paste. It offers excellent resistance to poisoning and a competitive limit of detection [about 2 ppb (2 parts in 10(9)) v/v], at + 1.0 V vs. Ag/AgCl, and good selectivity for NO(2) when used in a thin-layer cell. Incorporation of the cell in a continuous-flow system allows injection of about 120 samples per hour. The typical concentration range amenable to determination is 2-25 ppb v/v but depends on the thickness of the polymeric film. Nitrogen monoxide can also be detected but only in undiluted, pure form. Dinitrogen oxide gives no amperometric signal at any of the modified surfaces.

Preparation and gas chromatographic characterization of some crown-ether stationary phases.

Three open tubular glass capillary Chromatographic columns containing poly(vinylbenzo-15-crown-5), vinylmethylsila-17-crown-6, and vinylmethylsila-14-crown-5 have been prepared and characterized. The silacrown-ether stationary phases were polymerized inside the capillary and copolymerized with a methacryloxysilane bound to the inner surface. The poly(vinylbenzo-15-crown-5) was prepared and used as a coating in a capillary which had been roughened and deactivated. Characterization was performed by use of gas chromatography and included determination of column bleed character, phase transition changes, polarity and selectivity. Differential scanning calorimetry confirmed the phase transition temperature of the poly(vinylbenzo-15-crown-5) phase. The polarity and selectivity of these phases are comparable to those of Carbowax 20M.

Nylon shavings enzyme reactor for batch determination of urea.

The design and performance of an enzyme reactor (enzyme electrode) which features (i) incorporating nylon shavings onto which an enzyme is covalently bonded, (ii) a flat-surface combination pH electrode for proton monitoring, and (iii) a body providing an injection port for sample injection and washing and stirring capabilities is described. The reactor configuration described here offers good diffusional and partition characteristics which result in relatively fast response, good stability, simplicity of operation, low sample and reagent consumption, and adaptability to flow systems. Application to the determination of urea in standards and physiological salt solutions is demonstrated by use of immobilized urease (EC 3.5.1.5).