Chemical Shift Reference for MASSCP-13C NMR Studies of Reagents Immobilized on Silica Gel

Abstract

This report demonstrates that immobilized trimethylsilane is an appropriate reference standard for MASSCP-¹³C NMR studies of reagents immobilized on silica gel. An example is presented which shows that the immobilized trimethylsilane exhibits many of the advantages as a reference for these solid samples as does tetramethylsilane for liquid samples. It is further shown that pure solids may interact with an immobilized phase and produce significant spectral changes and thus are not appropriate as reference standards.     

A Selective Determination of Hypoxanthine,Xanthine and Inosine by Reversed-Phase Liquid Chromatography Coupled with Enzyme Reactors

Abstract

A Selective and sensitive assay of hypoxyanthine, xanthine and inosine by reversed-phase liquid chromatography coupled with immobilized enzyme reactors is described. The flourometric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of hypoxanthine, xanthine and inosine, which were oxidized to hydrogen peroxide in the presence of the immobilized enzymes (purine nucleoside phosphorylase and/or xanthine oxidase. The enzymes were immobilized the the intermolecular cross-linking method on controlled pore-glass. The method established was applied to serum and urine samples. The detection limits of hypoxanthine, xanthine and inosine were approximately 130, 300 and 650 pg per injection, respectively.     

Amperometric Determination of Lactic Acid. Applications on Milk Samples

Abstract

A hydrogen peroxide electrochemical sensor, coupled with immobilized lactate oxidase and covered with a cellulose acetate dialysis membrane has been applied in flow analysis of lactate in milk samples. The hydrogen peroxide produced by the enzymatic reaction is measured with a platinum electrode polarized at +650 mV versus Ag/AgCl. Milk samples were analyzed and compared with a spectophotometric reference method. The developed procedure is very simple and the short response time allows its use in assaying milk samples on dairy farms.     

Amperometric Enzyme Biosensor Based on the Controlled Pore Glass

Abstract

A new amperometric biosensor based of glucose oxidase immobilized in aminopropyl-controlled pore glass (CPG) is reported. The glucose oxidase was linked to the CPG by covalent bonds with glutaraldehyde. The effect of analytical variables on the biosensor response was studied using experimental design methodology. Analytical properties such as linearity, detection limit, quantitation limit, range, and precision are reported. Interferences caused by compounds usually present in biological samples were eliminated.     

Detection of Cocaine Using the Flow Immunosensor

Abstract

A continuous flow immunosensor has been designed for the detection of cocaine in aqueous samples. The continuous flow immunosensor relies on the displacement of fluorophore-labeled antigen from immobilized monoclonal antibody. The sensitivity and accuracy of the flow immunosensor were investigated while varying the parameters of immobilized antibody density, flow rate, amount of antibody-coated Sepharose used in each column, and the saturation of antibody binding sites with fluorophore-labeled antigen. Using a low density of immobilized anti-benzoylecgonine antibody, as little as 5 ng/ml cocaine could be detected. Small amounts of antibody-coated Sepharose could be used repeatedly and the lifetime of the column was proportional to the amount of Sepharose used. Results were obtained in less than a minute and cross-reactivity against various other drugs was negligible.     

Quantitation of Monosodium Glutamate Using Immobilized Glutamate Oxidase/Peroxidase and Flow Injection Analysis

ABSTRACT

A rapid and sensitive flow injection method is developed for the quantitation of glutamate in food samples. The method incorporates a covalently immobilized glutamate oxidase and peroxidase bioreactor linked in tandem. The H₂O₂ liberated from the glutamate samples as a result of enzymatic action is monitored spectrophotometrically at 552 nm using 4-aminoantipyrine and 3-dimethylaminobenzoic acid as a new chromogenic reagent. Glutamate calibration curves are linear up to 140 μM with a detection limit of 1 μM. Recovery yields from soup matrices are in the range 97 – 101%. Inter- and intra-day precision studies gave CV's of less than 3.5%. The immobilized enzymes show good storage and operational stabilities. Up to 40 samples h^?1 can be manually analyzed. Excellent correlation is obtained from a comparison of the glutamate content of various soup brands and matrix type (solid, condensed and broths) obtained by the proposed FI method with those obtained for the same samples analyzed by the standard reference method (y 0.99x + 0.034).     

Bioluminescent Flow Sensors: L-Alanine Determination in Serum and Urine

Abstract

A continuous flow bioluminescent method for L-alanine analysis in serum and urine has been developed. Serum can be analyzed directly after simple filtration. Response is linear from 50 to 1500 pmoles in biological matrix. Alanine dehydrogenase is immobilized onto a nylon coil separated from the reactor coil containing bioluminescent enzymes. The stability of nylon immobilized enzymes is high (over three months) and more than 900 samples can be analyzed with few mg of enzymes. The results obtained with the bioluminescent sensor agree well with those obtained by ion exchange chromatography (amino acid analyzer).     

Determination of Glycerol and Serum Triglycerides with Collagen-Immobilized Glycerol Dehydrogenase and Amperometric Detection

Abstract

An amperometric procedure is described for the determination of glycerol and triglycerides in aqueous samples and in serum using glycerol dehydrogenase immobilized on a collagen membrane. Glycerol is determined by measurement of the steady-state oxidation currents generated at a platinum electrode by NADH produced in the enzyme-catalyzed reaction. The triglycerides were first hydrolyzed by the enzyme lipase in solution and the resulting glycerol determined similarly. Olive oil, determined to contain 78 % triolein, was used as the source of triglycerides in this study. For both glycerol and triglycerides the calibration plots are linear in the range from 0 to 12 μM, with detection limits of 0.2 and 0.7 μM, respectively. The immobilized glycerol dehydrogenase retained high operational activity for a period longer than 30 days.     

Drop Size Distribution of O/W Emulsions by Drop Immobilization with Agarose

ABSTRACT

A method for the determination of the drop size distribution of oil-in-water (O/W) emulsions is presented. Water-based coolant emulsions used in rolling mill operations were studied. The emulsions were gelled in agarose so that the oil droplets were immobilized and samples of these gels were measured by confocal laser scanning microscopy (CLSM) and image processing. The influence of the addition of CaCl₂ as an emulsion destabilizer on the size distributions was also studied. The experimental data obtained were compared to those obtained using photon correlation spectroscopy (PCS).     

Detection of Oxidase Generated Hydrogenperoxide by a Solid State Peroxyoxalate Chemiluminescence Detector

Abstract

The compatability of a solid state peroxyoxalate chemiluminescence detector for hydrogen peroxide with an immobilized oxidase reactor is investigated. As a model system glucose oxidase immobilized by electrostatic forces on an ion-exchanger or chemically bonded to glass beads were chosen. The former support is less suitable for immobilization of oxydases due to strong retention of hydrogenperoxide on the ion exchanger.

The relatively little flow dependence of these systems renders them suitable for low-cost manual sample injection monitors as well as in a flow injection analyses (FIA) mode with low-cost pumping systems. The system was operated with 80% acetonitrile water solutions. A detection limit of 8 × 10^?7M of glucose was achieved in directly injected samples.

Enzymes more sensitive to organic solvents can be operated with pure water and adjustment for optimal chemiluminescence condition is achieved with a make-up flow prior to detection. A detection limit of 5 × 10^?8M glucose is achieved under these conditions. The feasability of this approach to other oxidase based monitors and to detection in liquid chromatography is discussed.     

Continuous-Flow Determination of Phenol With Chemically Immobilized Polyphenol Oxidase (Tyrosinase)

Abstract

The use of covalently bound mushroom polyphenol oxidase (tyrosinase, EC 1.10.3.1) for the determination of μg/mL and ng/mL concentrations of phenol in water samples with use of continuous-flow sample/reagent processing is described. Immobilization on controlled-pore glass, CPG, was accomplished via diazo coupling. Detection was effected with hexacyanoferrate(II) as a redox mediator and was either spectrophotometric or amperometric. the immobilized enzyme preparation was part of an open tubular reactor (CPG thermally embedded on Tygon tubing). the redox mediator was used either in solution or as part of a thin-layer cell and immobilized on poly(4-vinylpyridine) incorporated in a carbon paste electrode. Different spectrophotometric and amperometric strategies are compared and the method is applied to the determination of phenol in water samples and quality control standards.     

Detection of Aflatoxins in Capsicum Spice Using an Electrochemical Immunosensor

Abstract

Electrochemical immunosensor based on immobilized aflatoxin B1 (AFB)–albumin conjugate and polyclonal antibody against AFB1 was performed for a competitive assay of AFB1 in capsicum spice. Spiked samples were used for construction of calibration curve. The proved limit of detection was 2.4 ppb. Immunosensor long-term stability was also estimated. Decrease of immunosensor sensitivity was less than 10% when stored in a refrigerator and approximately 22% for the immunosensor preserved at laboratory temperature for two weeks. Consequent performance of immunosensors for assay of real capsicum spice samples with proven aflatoxins presence produced good correlation with the data from valid method (high-performance liquid chromatography with fluorescence detector).     

1,1′-Dimethylferrocene Mediated L-Glutamate Electrodes Modified With Electropolymerized 1,3-Diaminobenzene Film

Abstract

A mediated L-glutamate biosensor was constructed by incorporating 1,1′-dimethylferrocene in electropolymerized 1,3-diaminobenzene and immobilizing L-glutamate oxidase on the electropolymerized film. Stabilizers (1% DEAE-dextran, 1% MgCl₂ and 10% sucrose) were added to the immobilized enzyme to improve the long-term storage stability. The electrode responded linearly to L-glutamate concentration between 0.1 and 2.0 mM and the response of the electrode did not interfere with electroactive species and oxygen. The useful lifetime of the sensor was at least 3 weeks. When stored in dry form at 28°C, the sensor with stabilizers was stable at least 6 months. The electrode was applied to determine L-glutamate in fermentation broth samples. Good correlations were achieved between results obtained with the sensor and by enzymatic analysis using glutamate dehydrogenase.     

The Successful Use of Cellulose Acetate Membrane for Very Low Density Lipoprotein Isolation and Cholesterol Quantitation

Abstract

The lipoproteins have been successfully isolated electrophoretically from the blood samples of patients having suffered from hyperlipoproteinemia type III but having survived myocardial infarction. The VLDL fraction from the cellulose acetate membrane is cut out and the total cholesterol and cholesterol of the VLDL extracted. The cholesterol is determined using an immobilized cholesterol esterase/oxidase column using a flow injection analysis system.     

Amperometric Determination of Galactose,Lactose and Dihydroxyacetone Using Galactose Oxidase in a Flow Injection System with Immobilized Enzyme Reactors and On-Line Dialysis

Abstract

A flow injection manifold containing a dialyzer and reactors with immobilized galactose oxidase and peroxidase was used for the determination of galactose in urine, lactose in milk and dihydroxyacetone in a biotechnological reaction medium. The hydrogen peroxide which is formed by the galactose oxidase reaction was detected by amperometric reduction of a mediator. The latter had been produced from hydrogen peroxide in a peroxidase catalyzed reaction. The hydrogen peroxide detection step was studied with several mediators and hexacyanoferrate (II) was selected. An ion exchange HPLC procedure was used to purify the galactose oxidase, in particular from catalase, and the kinetics and the selectivity of a reactor containing the immobilized enzyme was investigated. Columns for removal of certain interferents such as ascorbic acid were used in the determination of galactose in urine. The response to galactose standards was linear from the detection limit of 2 μM to 60 mM. The throughput was 45 samples per hour and the relative standard deviation 0.4%.     

Amplified Detection of NAD+ and NADH by the Enzymatic Cycling with the Use of Immobilized Enzymes in a Flow System

Abstract

The enzymatic cycling for the detection of NAD⁺ and NADH in low level was carried out in the flow system with immobilized enzymes. Alcohol dehydrogenase and glutamate dehydrogenase immobilized on Sepharose 4B were used for the cycling reaction. The compound produced in the enzymatic cycling reaction was subjected to an enzymatic reaction to yield NADH, which was detected fluorometrically.     

Immunosensor for the Measurement of Human Serum Albumin in Urine Based on the Spreeta Surface Plasmon Resonance Sensor

The application of SPR for measurement of the concentration of human serum albumin (HSA) in urine was studied using the compact integrated SPR sensing system Spreeta. HSA was immobilized via cystamine and glutaraldehyde onto the gold sensing area and a competitive assay for HSA was developed using a limited amount of the monoclonal antibody AL-01 in solution. Measurements were carried out in the flow-through mode and the interaction between immobilized HSA and antibody was observed in real time. To obtain reproducible results, different conditions of the measurement (method of immobilization of HSA, data evaluation, concentration of antibody, regeneration procedure) were tested. The calibration curve for clinically relevant concentrations of HSA in urine samples was constructed using 300-times diluted antibody in the form of ascites fluid. The measuring range was between 0.1 and 5 mg/l of HSA, the sensing surface was successfully regenerated and suitable for more than 20 assays. The developed method was tested on real samples of urine; to overcome the non-specific adsorption of urine components, the differential approach was adopted and the measured signal was corrected by subtraction of the response observed in the absence of the antibody.     

Amperometric Choline Sensor with Enzyme Immobilized by Gamma-Irradiation in a Biocompatible Membrane

Abstract

An amperometric choline biosensor was constructed using choline oxidase immobilized on poly(2-hydroxyethylmethacrylate) membranes obtained by gamma radiation-induced polymerization at low temperature. The measurements were carried out by Clark-type oxygen or hydrogen peroxide electrodes. Calibration curves were linear in the 10-200 umol · 1^?1 range for the oxygen probe and 5-250 umol · 1^?1 for the H₂O₂-based probe. Temperature and pH effects on the activity of immobilized enzyme are described and the response characteristics of the sensor are summarized. The immobilized enzyme membranes stored in glycine buffer or in a dry state were very stable and no significant decrease in the electrode response was observed after three months. The biosensor was employed also to analyse a choline-containing pharmaceutical product and the results were compared to those obtained by enzymatic-spectrophotometric detection.     

Enzymatic Assay by Flow Injection Analysis with Detection by Chemiluminescence: Determination of Glucose,Creatinine, Free Cholesterol and Lactic Acid Using an Integrated Fia Microconduit

Abstract

A miniaturized flow injection system for the determination of D-glucose, L-lactic acid, creatinine and free cholesterol is described. All substrates are degraded enzymatically by means of oxidases which, along with ancillary coenzymes (creatinine assay), are immobilized on controlled porosity glass and incorporated into small PVC column reactors. The hydrogen peroxide generated by the individual oxidases is determined by chemiluminescence with an alkaline reagent containing luminol and hexacyanofer rate (III). The injection valve, flow channels, enzyme reactor and light detector are integrated into a FIA microconduit. The detection limits were 0.03 mg glucose/dl, 0.03 mg lactate/dl, 0.3 mM creatinine and 0.5 mg cholesterol/dl. The enzyme reactors all showed little change in activity over a 3 months period of operation and were found fully compatible with serum samples.     

A Dedicated Instrument for the Analysis of Blood Urea Nitrogen Using an Immobilized Enzyme Reagent

Abstract

A dedicated instrument for the quantitative determination of urea in blood serum or plasma has been developed. The instrument uses urease immobilized on a porous alumina support and a gas-detecting electrode to measure ammonia resulting from hydrolysis of urea. Reproducibility and recovery studies show a high degree of accuracy, sensitivity, and specificity. Comparison of patient samples (plasma and serum) show no statistical difference when compared to the diacetyl monoxime, Auto Analyzer method.