Amperometric flow-injection system with an immobilized enzyme reactor for the highly selective detection of phosphate and on-line amplification by substrate recycling

Bio-amperometric flow-injection systems are proposed for the highly selective and sensitive determination of phosphate. One system studied is based on the use of a co-immobilized purine nucleoside phosphorylase-xanthine oxidase reactor, which responds to phosphate with high selectivity, and a detection limit of 3 × 10^?7 M for a 20-μl injection. Another system with a co-immobilized purine nucleoside phosphorylase-xanthine oxidase-alkaline phosphatase reactor gives responses amplified by substrate recycling during passage through the enzyme reactor. Phosphate can be determined with 12 times the sensitivity in the latter system compared with the former, but the latter system responds to nucleotides and pyrophosphate in addition to orthophosphate.     

Flow-injection system for the fluorimetric determination of fructose with an immobilized mannitol dehydrogenase reactor

Immobilized mannitol dehydrogenase is used for the determination of D-fructose in a flow-injection system. The enzyme is immobilized on poly(vinyl alcohol) beads. The oxidation of NADH occurs simultaneously and the disappearance of NADH is measured fluorimetrically. The response is linearly related to fructose concentration in the range 6–600 μM; 30 samples per hour can be analysed. The immobilized enzyme retains over 80% of its initial activity after repetitive use for 2 months.     

The use of an immobilized enzyme nylon tube reactor incorporating a four enzyme system for creatinine analysis

A single immobilized enzyme nylon tube reactor was produced incorporating a four enzyme system for the analysis of creatinine. The enzyme activity ratios in the coupling solution used to prepare the reactor were found to be of extreme importance in governing the activity of the latter. The reactor was incorporated into a continuous flow analysis system used to assay creatinine in urine samples and the results were correlated with a manual technique employing the same enzyme system in solution. The precision, correlation, high specificity, simplicity, and speed of the analysis were concluded to be factors in favor of the method's suitability for urine creatinine determinations.     

Rapid determination of cephalosporins with an immobilized enzyme reactor and sequential subtractive spectrophotometric detection in an automated flow-injection system

A flow-injection system is described for the rapid automated determination of different cephalosporins in aqueous solution. Measurement is based on a selective enzymatic cleavage of the cephalosporin-β-lactam ring in a small immobilized enzyme reactor, which contains highly purified cephalosporinase covalently bound to activated glass beads. Samples are injected into a phosphate buffer stream. The opening of the β-lactam ring is accompanied by a significant decrease in the ultraviolet (u.v.) absorbance. The difference between the absorbances of untreated and degraded cephalosporin is measured at 254 nm and is related linearly to cephalosporin concentration over the range 10–800 μg ml^?1. Several commercially available cephalosporins including some β-lactamase-resistant types were examined. The accuracy of the enzyme reactor/flow injection system was evaluated by comparison with h.p.l.c. results; the correlation was good. The relative standard deviation, evaluated from 15 consecutive injections of the same sample, was 0.7%; 2 μg ml^?1 cephalosporin C was the minimal detectable concentration. A single determination took about 2 min; sample throughput is 30 h^?1. Different β-lactamases were examined for enhancement of substrate selectivity.     

Rapid measurement of fish freshness indices by an amperometric flow-injection system with a 16-way switching valve and immobilized enzyme reactors

A novel flow-injection system is proposed for the rapid measurement of the fish freshness indices K₁ and K₂: K₁=[([HxR+[Hx])×100/([IMP]+[HxR]+[Hx])] and K₂=[[Hx]×100/([HxR]+[Hx])], where [IMP], [HxR] and [Hx] are inosine-5′-monophosphate, inosine and hypoxanthine concentrations, respectively. For the estimation of index K₁, 5′-nucleotidase immobilized reactor and nucleoside phosphorylase (NP)/xanthine oxidase (XO) coimmobilized reactor were incorporated in series in the flow-injection line made up by a 16-way switching valve with two sample loops. For the estimation of index K₂, NP and XO immobilized reactors were also incorporated in the similar flow-line. Two sample portions passed through the flow-line with different residence times so that two peaks were obtained. The first and second peaks obtained in the K₁-determining system corresponded to the total of HxR and Hx and the total of Hx, HxR and IMP, respectively. Similarly, the first and second peaks obtained in the K₂-determining system corresponded to Hx and the total of Hx and HxR, respectively. Therefore, the indices K₁ and K₂ can be estimated by

where i₁ and i₂ present the peak current of the first and second peaks, respectively, and f the ratio of the peak currents of the first and second peaks for a Hx standard solution. A sea bream was selected as a model fish and it was stored at 4 °C after death. A good correlation was found between the index K₁ and the storage time over a period of 400 h, with a correlation coefficient of 0.992, but no correlation between the index K₂ and the storage time. The measurements could be performed at a rate of 22 samples per hour with satisfactory precision (0.6–1.2% R.S.D.), without calibration for each species, accurate weighing of fish meat and any interferences in fish meat.     

Spectrophotometric determination of ethanol in blood using a flow-injection system with an immobilized enzyme (alcohol dehydrogenase) reactor coupled to an on-line dialyser

The determination of ethanol in whole blood without pretreatment using spectrotometric detection with an immobilized enzyme reactor coupled to an on-line dialyser is described. Optimum working conditions are given. The effects of the injection volume and the flow-rate on the peak height using the dialyser were investigated. A good linear calibration graph over the ethanol concentration range 3–40 μg ml^?1 was observed; these concentrations correspond to 0.3?4.0 g of ethanol per 1000 g of whole blood prior to dilution. For comparison, whole blood samples were analysed by gas chromatography with direct injection. The effect of temperature on the peak height was also studied in a system without the dialyser.     

Flow-injection determination of 3-hdroxybutyrate in serum with an immobilized 3-hydroxybutyrate dehydrogenase reactor and chemiluminescence detection

A flow-injection system for the determination of 3-hydroxybutyrate in serum is described. 3-Hydroxybutyrate dehydrogenase is immobilized on poly(vinyl alcohol) beads and incorporated in a flow-injection system. 1-Methoxy-5-methylphenazinium methylsulphate reacts with enzymatically generated NADH to give H₂O₂, which is detected chemiluminometrically with the reaction of luminol and hexacynoferrate(III). Serum is diluted and filtered through an ultrafiltration membrane. The system responds linearly to injected samples (80 μl) in the concentration range 0.5–300 μM; the detection limit is 0.1 μM. The within-day relative standard deviation (n = 90) for 58 μM 3-hydroxybutyrate in serum is 0.8%. The maximum throughout is 20 samples per hour. The immobilized enzyme is stable for at least 1 month.     

Exchangeable immobilized enzyme reactor for enzyme inhibition tests in flow-injection analysis using a magnetic device. Determination of pesticides in drinking water

A flow-injection system for rapid automated enzyme inhibition testing is described. Exchange of inactivated enzyme immobilized on magnetic particles was performed with magnetic devices which could be electrically switched off to release all bound material. The flow resistance of the reactor was excellent. Inhibition of immobilized acetylcholinesterase was used to determine pesticides in drinking water. Concentrations of 0.5 μg l^?1 of the pesticides carbofuran and malaoxon were detected. A complete cycle of analysis, including calibration, took 20 min.     

Determination of fructosyl amino acids and fructosyl peptides in protease-digested blood sample by a flow-injection system with an enzyme reactor.

A flow-injection system with an enzyme reactor was proposed for the measurement of fructosyl amino acids and fructosyl peptides in protease-digested blood samples. A fructosyl-amino acid oxidase (FAOX-TE) and two fructosyl-peptide oxidases (FPOX-CE and FPOX-CET) were covalently immobilized onto an inert support. They were used as the enzyme reactor in a FIA system with a hydrogen peroxide electrode. In particular, the FPOX-CET reactor possessed high selectivity for the detection of fructosyl valine (FV) and fructosyl valyl histidine (FVH) and an excellent operational stability. The proposed FIA system with the FPOX-CET reactor responded linearly to the concentration of FV over the dynamic range of 7.8 x 10(-6) to 5.8 x 10(-4) M. The present method could be successfully applied to the assay of FV and FVH in the protease-digested blood samples.     

Determination of glucose in fermentation processes by means of an on-line coupled flow-injection system using enzyme sensors based on chemically modified electrodes

The application of an inexpensive, easy-to-make and disposable biosensor, based on a chemically modified graphite electrode with an adsorbed and cross-linked layer of glucose dehydrogenase, as a detector in a flow-injection system for on-line monitoring of glucose in fermenters is described. The performance of the sensor as a function of time is investigated, and used for assaying glucose in a wine fermentation process. It is concluded that the sensor operates satisfactorily during at least 3 days of continuous use. No interference from ethanol is encountered. With regard to practical applications, special emphasis is placed on the interfacing of the fermenter and the analytical system.     

Amperometric enzyme electrode for the determination of aspartate aminotransferase and alanine aminotransferase in serum

Glutamate oxidase (E.C. 1.4.3.7) was immobilized at a platinized activated carbon electrode and the enzyme electrodes were used for the amperometric determination of L-glutamate in a stirred aqueous solution by the electrochemical detection of enzymically produced hydrogen peroxide at + 320 mV vs. Ag/AgCl. A linear calibration graph was obtained between 2 μM and 2 mM with a steady-state response time of 1 min. The glutamate oxidase electrode was subsequently applied to the measurement of aspartate aminotransferase (AST) (E.C. 2.6.1.1) and alanine aminotransferase (ALT) (E.C. 2.6.1.2) in serum. The performance of the electrode was compared with that of techniques used in the hospital diagnostic laboratory. The responses of the enzyme electrode to AST and ALT activities were linear over the clinically relevant range (5-500 U l ^?1), and correlated well (r=0.99) with the methods used for routine clinical analysis.     

Amperometric detection of acetylcholine and choline in a liquid chromatographic system with an immobilized enzyme reactor

Acetylcholinesterase and choline oxidase were co-immobilized by reaction with glutaraldehyde onto alkylamino-bonded silica, which was incorporated as the enzyme reactor in an h.p.l.c. system for the determination of acetylcholine and choline. The hydrogen peroxide produced enzymatically in the enzyme reactor, after the separation of acetylcholine and choline by the reverse-phase column, was monitored amperometrically. The detection limits were 1.2 pmol for choline and 1.8 pmol for acetylcholine.     

A flow-injection system for assay of the activity of an immobilized enzyme chemically-modified electrode

Chymotrypsin, covalently immobilized to the surface of an IrO₂-coated titanium electrode, responds potentiometrically to various substrates. A flow-injection system is described for assay of the activity of the immobilized enzyme with N-benzoyl-l-tyrosine ethyl ester as substrate and an ultraviolet detector. Least-squares fits of peak height vs. time typically yield correlation coefficients of 0.999 and standard errors of estimate of 0.0043 absorbance for a total absorbance change of about 0.130. Slopes of such plots vary linearly with enzyme activity.     

Determination of acetylcholine and choline in the femtomole range by means of HPLC,a post-column enzyme reactor,and electrochemical detection

Summary The measurement of choline and acetylcholine by means of HPLC, a post-column enzyme reactor, and electrochemical detection has been simplified and optimised. The use of a cation exchanger and enzyme reactor fitted in a cartridge holder appeared to result in reproducible, sensitive, and selective measurement of endogenous choline and acetylcholine with a lower detection limit of 50 fmole.     

Immobilized enzyme reactor chromatography: optimization of protein retention and enzyme activity in monolithic silica stationary phases

Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low μL/min range. Using the enzyme γ-glutamyl transpeptidase (γ-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by ∼2-fold with increases in both flowrate (from 250 to 1000 nL/min) and backpressure generated (from 500 to 2100 psi) during the chromatographic activity assay owing to increases in k_cat and decreases in K_M, switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography.     

Flow-injection analysis for hypoxanthine in meat with dissolved oxygen detector and enzyme reactor

A low cost flow-injection analysis (FIA) with a dissolved oxygen (DO) detector and a xanthine oxidase immobilized column for the analysis of hypoxanthine as an index to determine degree of aging in meat was developed for quality control in the food industry. In this system, hypoxanthine is oxidized by an enzyme reaction with xanthine oxidase immobilized on the column to produce xanthine. Then the catalytic reaction between hypoxanthine and DO with xanthine oxidase proceeds with the DO concentration decreasing in the stream of the flow system. Decrease in the DO concentration was monitored by a DO detector located downstream of the flow system. This decrease in DO concentration was proportional to the hypoxanthine concentration. For detecting the decreased DO concentration efficiently a flow-through cell with a polarographic-type DO sensor was specially designed. As a result, a linear working curve was obtained from 3.68 × 10⁻⁵ to 1.84 × 10⁻³ M hypoxanthine concentrations with this FIA system. We applied the present system with a DO detector for the determination of hypoxanthine in meat samples and compared the results with those obtained by the conventional HPLC method. The data obtained with the present FIA method were in fairly good agreement with those obtained by the conventional HPLC method for the meat samples. Correlation factor and regression line between the two methods were 0.998 and Y= 1.51X-32.64 respectively. We concluded that the present FIA system with a DO detector was suitable as a simple, easy to handle and reliable instrument for quality control in the food industry.     

Determination of viscosity with an open-closed flow-injection system

A flow-injection configuration for the determination of the viscosity of water-miscible samples is proposed. The method is based on the use of an open-closed flow-injection system involving a trapped sample (carrier) and injection of a dye plug. The behaviour of the plug is monitored photometrically by a detector included in the circuit. The parameters of the multipeak recording obtained are related to the viscosity, which can be determined in the range 1-28 cp with an r.s.d. of about +/- 1 %.     

Spectrophotometric flow-injection determination of urea in body fluids by using an immobilized urease reactor

A flow system involving a packed-bed enzyme reactor (volume 180 μl) with urease immobilized covalently on poly(glycidyl methacrylate)-coated porous glass is used for determining urea in blood serum and urine. Enzymatically produced ammonia is converted to an indophenolate dye (by oxidative coupling with hypochlorite and sodium salicylate), which is detected spectrophotometrically at 700 nm. The calibration graph is rectilinear for 25–500 μM urea when injecting samples (75 μl) diluted 1:50 for serum or 1:1000 for urine at a frequency of 60 h^?1; the relative standard deviation is 1.1% for ten injections of 300 μM urea. The immobilized urease is stabilized by the addition of disodium EDTA, sodium azide and 2-mercaptoethanol to a 0.2 M phosphate buffer (pH 6.9) used as the carrier stream, which serves also as a preservative for longterm storage of the urease reactor packing at 4°C.     

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.