Chemiluminescence flow sensor for folic acid with immobilized reagents.

A novel chemiluminescence (CL) sensor for folic acid combined flow-injection (FI) technology was presented in this paper. The analytical reagents involved in the CL reaction, including luminol and hexacyanoferrate(III), were both immobilized on an anion-exchange column in FI system. The CL signal produced by the reaction between luminol and hexacyanoferrate(III), which were eluted from the column through sodium phosphate injection, was decreased in the presence of folic acid. The CL emission was correlated with the folic acid concentration in the range from 0.01 to 15 microg ml(-1), and the detection limit was 3.5 ng ml(-1) folic acid (3sigma). At a flow rate of 2.0 ml min(-1), including sampling and washing, could be performed in 2 min with a relative standard deviation of < 2.5%. The flow sensor could be reused more than 300 times and has been applied to the analysis of folic acid in pharmaceutical preparations. and the recovery was from 97.4% to 100.4%.     

Chemiluminescence flow sensor for the determination of ascorbic acid with immobilized reagents

A novel flow sensor based on chemiluminescence (CL) for the determination of ascorbic acid has been proposed. The analytical reagents, luminol and ferricyanide, were both immobilized on an anion-exchange resin column. The CL signal produced by the reaction between luminol and ferricyanide, which were eluted from the column through sodium phosphate injection, was decreased in the presence of ascorbic acid. The CL emission intensity was linear with ascorbic acid concentration in the range 0.01-0.8 mug ml(-1); the detection limit was 5.5 x 10(-3) mug ml(-1). The whole process, including sampling and washing, could be completed in 1 min with a relative standard deviation of less than 5%. The sensor could be reused more than 100 times and has been applied successfully to the analysis of ascorbic acid in pills and vegetables.     

Use of immobilized enzymes in chemical analysis

Immobilized enzymes are becoming increasingly popular as analytical reagents because of their reusability, stability, and sensitivity to many inhibitors that would seriously interfere in assays using soluble enzymes. In this article, some of the kinetic and catalytic effects of immobilized enzymes in analysis will be discussed. The shift of the activity-pH profile curves on immobilization, the changes in temperature dependence, the inhibitor constants (K_i), Michaelis constants (K_m), and the maximum velocity ( V_max), plus others, will be discussed. Finally, the use of these immobilized enzymes in fluorometric and electrochemical monitoring systems will be shown, and the future of these reagents in various areas will be discussed. A survey of enzyme electrodes will be presented as an example of the use of immobilized enzymes. Application of immobilized enzyme technology to the assay of BUN, glucose, uric acid, amino acids, ethanol, and other metabolites will be discussed.     

Determination of phenol and catechol concentrations with oxygen probes coated with immobilized enzymes or immobilized cells

The enzyme phenol 2-hydroxylase was immobilized on Sepharose and used in conjunction with an O₂ electrode for quantitating phenol. Similarly, catechol 1,2-oxygenase was used for quantitating catechol. A third probe was prepared by immobilization ofTrichosporon cutaneum cells rather than purified phenol 2-hydroxylase for phenol quantitation. The whole cell system gave results comparable to the immobilized enzyme system.     

Characterization of immobilized enzymes by flow-injection techniques with variable forward flow

The use of variable forward flow with small packed enzyme reactors is shown to be valuable for improving the efficiency of enzymatic conversion. Designs with stopped flow, oscillating flow and variable (fast/slow/fast) flow are compared for the spectrophotometric determination of glucose with glucose oxidase and horseradish peroxidase immobilized on controlled-pore glass in the same reactor. Variable forward flow increased the sensitivity considerably without excessive time consumption. The technique is also useful for characterizing the activity of immobilized enzyme reactors, e.g., peroxidase reactors for hydrogen peroxide determinations.     

Integrated reaction and photometric detection with enzymes immobilized in the flow cell of a flow-injection system

A biosensor based on the integration of reaction and detection with a conventional photometric detector is proposed. An enzyme is immobilized on the walls of the flow cell and reaction-rate measurements of the enzymatic reaction are made by halting the flow when the sample-coenzyme plug is in the flow cell and monitoring the increase in the reduced form of the coenzyme (λ_max = 340 nm).     

Amperometric sensor for pyruvate with immobilized pyruvate oxidase

Several procedures for immobilization of pyruvate oxidase by chemical bonding are reported. Attachment to nylon net was unsatisfactory in terms of yield and stability. Polyazetidine, a new commercially available prepolymer and a new nylon membrane with surface carboxyl groups provided good long-term stability, up to 30 days in the case of the nylon membrane. Under optimal conditions of solution preparation (0.1 mmol 1^-1 thiamine pyrophosphate, 0.5 mmol 1^-1 phosphate, 2.5 mmol 1^-1 calcium and 0.04 mol 1^-1 Tris buffer pH 7.0), linear calibration graphs were obtained until the lowest concentration of 10^-3 mmol 1^-1 pyruvate and amperometric sensors based on these membranes. With careful standardization, the procedures were suitable for application to blood serum.     

Automatic flow systems based on sequential injection analysis for routine determinations in wines

Sequential injection systems for wine analysis have been developed in recent years for determination of more than 20 species. Several aspects of these systems are reviewed in the present paper. Special focus is given to implementation of in-line sample treatment and adaptation of system operation through software control to enable determination in different types of wine. The strategies used to enhance selectivity and the capacity for multi-parameter determination are also addressed.     

Use of flow injection analysis based on iodometry for automation of dissolved oxygen (Winkler method) and chemical oxygen demand (dichromate method) determinations

Summary Two analytical methods for water analysis were miniaturized and partly automated by the flow injection analytical approach. In the standard Winkler method for dissolved oxygen determination, the final step, the titration of liberated iodine with Na₂S₂O₃, was replaced by amperometric detection in a flowing stream. The concentration of dissolved oxygen was determined by the standard addition method using KIO₃ as a standard reagent. The dichromate method for chemical oxygen demand determinations was also miniaturized. Oxidation of organic substances by K₂Cr₂O₇ in H₂SO₄ (1+1) medium was carried out under standard conditions and the excess of dichromate ions was determined electrochemically via measurement of released I₂ after addition of KI in a continuous stream. The chemical oxygen demand of a sample was calculated on the basis of the current without standard addition.

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Anwendung der iodometrischen Fließinjektionsanalyse zur Automation der Bestimmung von gelöstem Sauerstoff (Winkler-Methode) und chemischem Sauerstoffbedarf (Dichromat-Methode)

     

Fructose determination using immobilized enzymes in a flow system with special emphasis on the effect of isomerism

A flow injection method for the determination of fructose has been developed using immobilized hexokinase, glucose phosphate isomerase and glucose-6-phosphate dehydrogenase. The produced NADH was monitored amperometrically with a graphite electrode modified by an adsorbed Nile-blue derivative. The response was linear from the detection limit, 1M to 3 mMD-fructose for a sample volume of 25l. Glucose, the only interfering substrate, was eliminated with a reactor containing immobilized glucose oxidase, mutarotase and catalase. The experiments indicate that hexokinase is selective for the-fructo-furanose isomer. The other isomers will spontaneously isomerize to-fructo-furanose with time, if the latter is consumed. The fructose response factor of an enzyme reactor or biosensor will therefore depend on the isomer distribution in the original sample solution. The effect of pH and temperature can be controlled, but complexing ligands like borate interfere in a complex way and should be absent.     

Chemiluminescence flow sensor with immobilized reagent for the determination of pyrogallol based on potassium hexacyanoferrate(III) oxidation

A novel chemiluminescence (CL) flow-through sensor for the determination of pyrogallol has been developed. The method is based on the reaction between pyrogallol and potassium hexacyanoferrate(III) in sodium hydroxide solution. Potassium hexacyanoferrate(III) involved in the CL reaction was electrostatically immobilized on anion-exchange resin packed in a column. Pyrogallol was sensed by the CL reaction between pyrogallol and potassium hexacyanoferrate(III) which was eluted from the ion-exchange column through sodium phosphate injection. The CL emission allows quantitation of pyrogallol concentration in the range 0.01-3.8 microg/mL with a detection limit (3 sigma) of 0.003 microg/mL and a sample throughput of 118 h(-1). The relative standard deviation (n=7) was 2.2% for 0.2 microg/mL of pyrogallol. The influence of foreign compounds was tested.     

A stopped-flow instrument for reaction-rate measurements with immobilized enzymes

A stopped-flow instrument that provides direct and continuous monitoring of reactions catalyzed by enzymes immobilized inside open tubes is described. The instrument allows complete reaction progress curves to be obtained during a single experiment. Because the reaction occurs under static conditions, the kinetics are controlled only by diffusion and the inherent enzyme reaction rate. The kinetics constants for lactate dehydrogenase covalently bound inside nylon tubing and in aqueous solution were determined and compared.     

Fiber-optic fluorescence sensor for dissolved oxygen detection based on fluorinated xerogel immobilized with ruthenium (II) complex

A fiber-optic sensor based on fluorescence quenching was designed for dissolved oxygen (DO) detection. The fluorinated xerogel-based sensing film of the present sensor was prepared from 3, 3, 3-trifluoropropyltrimethoxysilane (TFP–TriMOS). Oxygen-sensitive fluorophores of tris (2, 2′- bipyridine) ruthenium (II) (Ru(bpy)₃²⁺) were immobilized in the sensing film and the emission fluorescence was quenched by dissolved oxygen. In the sensor fabrication, a two-fiber probe was employed to obtain the best fluorescence collection efficiency and the sensing film was attached to the probe end. Scanning electron microscope (SEM), UV–Vis absorption spectroscopy (UV–Vis) and fourier transform infrared spectroscopy (FTIR) measurements have been used to characterize the sensing film. The sensor sensitivity is quantified by I _deoxy/I _oxy, where I _deoxy and I _oxy represented the detected fluorescence intensities in fully deoxygenated and fully oxygenated environments, respectively. Compared with tetramethoxysilane (TMOS) and methyltriethoxysilane (MTMS)-derived sensing films, TFP–TriMOS-based sensor exhibited excellent performances in dissolved oxygen detection with short response time of 4 s, low limit of detection (LOD) of 0.04 ppm (R.S.D. = 2.5%), linear Stern–Volmer calibration plot from 0 to 40 ppm and long-term stability during the past 10 months. The reasons for the preferable performances of TFP–TriMOS-based sensing film were discussed.     

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.     

A lactate electrode with lactate oxidase immobilized on nylon net for blood serum samples in flow systems

Commercially available lactate oxidase from Mycobacterium smegmatis is immobilized on a nylon net which is fixed on an oxygen probe to provide a simple l-lactate sensor. A citrate buffer, pH 6.0, is the only reagent required. The high activity of the enzyme obtained with this immobilization process permits the use of only 20–100 μl of plasma; diluted with citrate buffer to 2 ml, the sample is pumped through a flow cell. The high dilution offsets inhibitory effects of some anions present in blood such as oxalate, hydrogencarbonate and chloride. The response of the sensor is linear over the range 0.2– 2 × 10^?4 M l-acetate. The lifetime is about two months. Effects of pH, temperature and different buffers are described and results on serum samples are reported.     

Selective detection in flow analysis based on the combination of immobilized enzymes and chemically modified electrodes

The combination of immobilized enzymes and amperometry to build selective detection devices in flow-injection analysis and liquid chromatography is described. The pros and cons of enzyme electrodes and of immobilized enzyme reactors are discussed. The paper concentrates on the use of immobilized dehydrogenases, oxidases, peroxidases, and on electrodes on which these enzyme reactions can be selectively followed. The work in the field by the authors is reviewed.     

Fibre optic sensor with bio-membrane supported immobilized morin for aluminium determination

A new type of fibre optic sensor based on the use of bio-membrane is proposed for the determination of aluminium. Morin is immobilized on mutton membrane pre-treated with formaldehyde and attached to the fibre optic. Fluorescence response is linear from 8 × 10^–7 to 3 × 10–5M. The detection limit is 6 × 10^–7 M. Response time is 30—60 s.     

Enthalpic determinations of aliphatic ester-solvent systems with correlations

Calorimetric measurements were carried out on ester-solvent systems where the esters were H(CH₂)_xCOO(CH₂)_yH, with x and y varying from 1 to 5, and the solvents were n-hexane and 1,2-dichloroethane. Calculation of the enthalpies of cavity formation enabled the enthalpies of interaction to be determined. Both enthalpies correlated with number of carbon atoms N, equal to x+y in the esters, giving for 1,2-dichloroethane