Chemiluminometric method for the determination of glycerol in wine by flow-injection analysis with co-immobilized glycerol dehydrogenase/NADH oxidase

A flow-injection method for the determination of glycerol in wine is described. Glycerol dehydrogenase and NADH oxidase were co-immobilized on poly (vinyl alcohol) beads and incorporated in a flow-injection system. The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate (III) reaction. Wine was diluted 1000-fold with water and sample solution (50 microl) was injected into the carrier stream. The calibration graph was linear in the range 3 x 10(-7)-3 x 10(-4) M; the detection limit was 7 x 10(-8) M and the sample throughout was 30 h(-1) without carryover.     

Flow-injection determination of thyroxine using immobilized enzyme with tris(2,2'-bipyridyl)ruthenium(III) chemiluminescence detection.

A flow-injection method is reported for the determination of thyroxine based on its enhancement effect on the tris(2,2'-bipyridyl)ruthenium(III) chemiluminescence reaction in the presence of NADH using immobilized alcohol dehydrogenase purified from baker yeast. The limit of detection (3 sigma blank) was 5.0 x 10(-8) M with a sample throughput of 80 h(-1). The calibration graph was linear over the range 0.5 - 10 x 10(-7) M (r2= 0.9988) with the relative standard deviation in the range 1.4 - 3.5% (n = 4). The effect of common excipients used in pharmaceutical preparations, some organic compounds and metal ions was studied. The method was applied to pharmaceutical thyroxine tablets, and the obtained results were not significantly different from the amount quoted.     

Amperometric determination of urea using an NADH-dependent coupled enzyme

Enzyme electrodes for the amperometric measurement of urea were prepared by co-immobilizing l-glutamate dehydrogenase and urease onto an Immobilon-AV affinity membrane with attachment to a glassy carbon electrode. Reduced nicotinamide adenine dinucleotide (NADH) was used as the electroactive species. The electrochemical oxidation of NADH was monitored at +1.0 V vs. Ag/AgCl. The enzyme immobilized electrode was linear over the range of 2.0 x 10(-5) to 2 x 10(-4)M. The response time of the electrode was 3 min and the optimum pH of enzyme immobilized membrane was pH 7.4-7.6 (Dulbecco's buffer solution). It was stable for at least two weeks and 50 assays. There were no interferences from other physiological material, except for high levels of ascorbic acid.     

A chemiluminometric method for NADPH and NADH using a two-enzyme bioreactor and its application to the determination of magnesium in serum

Chemiluminometric methods are described for the automated flow injection analysis of NADPH and NADH using an immobilized enzyme column reactor and serum magnesium. This application is for the clinical analysis of NADPH and NADH. The reactor for NADPH and NADH contains immobilized L-glutamate dehydrogenase and L-glutamate oxidase, and that for serum magnesium immobilized hexokinase, glucose-6-phosphate dehydrogenase, L-glutamate dehydrogenase and L-glutamate oxidase. When the sample is introduced into the four-enzyme bioreactor, hydrogen peroxide is produced in proportion to the concentration of serum magnesium by the successive reactions. A co-immobilized hexokinase/glucose-6-phosphate dehydrogenase/glutamate dehydrogenase column reactor gave better efficiency compared with an enzyme column which was prepared by packing co-immobilized hexokinase/glucose-6-phosphate dehydrogenase and immobilized glutamate dehydrogenase to make two layers. Magnesium in serum was determined with 1 microL of the sample without carry-over and for an assay time of approximately 15 s. The present method is sensitive (detection limit 0.1 nmol) because Mg2+ is recycled in a column, and gives perfect linearity of the data up to 3.0 mmol/L with satisfactory precision, reproducibility, and accurate reaction recoveries.     

Diffusion coefficient measurements in microfluidic devices

A glassy carbon electrode (GCE) modified with Pd/IrO(2) provides excellent electrocatalytic oxidation of hydrogen peroxide. Glucose oxidase (GOD) and xanthine oxidase (XOD) were co-immobilized on the modified electrode with a thin film Nafion coated on the enzyme layer to form a glucose (Glu)/hypoxanthine (Hx) sensor, without interference from electroactive species such as ascorbic acid (AA) and uric acid (UA). Its response was evaluated with respect to the enzyme amount on the electrode, pH and temperature of the electrolyte. The prepared bienzymic biosensor, used as the detector of HPLC gave a detection limit of 1.0x10(-6) mol l(-1) Glu and 2.0x10(-7) mol l(-1) Hx (Hx) with a linear concentration range of 5.0x10(-6)-2.5x10(-3) mol l(-1) and 1.0x10(-6)-5.0x10(-4) mol l(-1), respectively. Coupled with microdialysis, it was used to monitor the concentrations of Glu and Hx in rat brain.     

Simultaneous determination of D-glucose and 3-hydroxybutyrate by chemiluminescence detection with immobilized enzymes in a flow injection system.

A chemiluminometric flow-through sensor for the simultaneous determination of glucose (Glu) and 3-hydroxybutyrate (HB) in a single sample has been developed. Coimmobilized 3-hydroxybutyrate dehydrogenase/NADH oxidase/peroxidase, a support material, and coimmobilized glucose dehydrogenase/NADH oxidase/peroxidase were packed sequentially in a transparent PTFE tube. The tube was then placed in front of a photomultiplier tube as a flow cell. A two-peak recording was obtained by one injection of the sample solution. The peak heights of the first and second peaks were dependent on the concentrations of HB and Glu, respectively. The calibration graphs for HB and Glu were linear at 0.05-10 and 0.1-30 microM, respectively. The maximum sample throughput was 30 h(-1). The sensor was stable for two weeks.     

Amperometric determination of lactate dehydrogenase based on a carbon fiber microcylinder electrode modified covalently with Toluidine Blue O by acylation

A method has been developed for the modification of a carbon fiber microcylinder electrode with acylation. The stability and surface coverage of the Toluidine Blue O-modified microelectrode were studied by cyclic voltammetry. The modified electrode showed significant activity for the electrocatalytic oxidation of NADH in pH 6.8-7.8 solution. The catalytic current increased linearly with increasing concentration of NADH from 4.0 x 10(-5) to 1.5 x 10(-3) M. A simple amperometric determination based on electrochemical detection of NADH produced from the enzymatic reaction of lactate with NAD(+) under the catalysic effect of lactate dehydrogenase (LDH) is reported. The experimental factors which had primary influence on the analytical performance were studied. The sensor had a linear response over a range of LDH concentrations from 5.0 U l(-1) to 200 U l(-1) at -0.2 V vs. SCE under optimum conditions. A satisfactory result was obtained for the determination of LDH in clinical blood samples.     

A simplified enzyme-based fiber optic sensor for hydrogen peroxide and oxidase substrates

A simplified enzyme-based fiber optic sensor system has been developed for selective determination of hydrogen peroxide. Horseradish peroxidase (HRP) is immobilized on bovine albumin matrix with glutaraldehyde. A new fluorimetric substrate, thiamine is used to indicate the sensing process. Under optimized condition the measuring range of sensor is up to 1 x 10(-4)M hydrogen peroxide with a limit of detection of 5 x 10(-7)M in a 5 min response period. It can be easily incorporated in multienzyme sensors for biochemical substances which produce hydrogen peroxide under catalytic oxidation by their oxidase. This possibility has been tested for the determination of uric acid, D-amino acid, L-amino acid, glucose cholesterol, choline and acetylcholine, respectively, using a membrane with co-immobilized oxidase and horseradish peroxidase.     

Electroanalytical properties of aldehyde biosensors with a hybrid-membrane composed of an enzyme film and a redox Os-polymer film.

Aldehyde biosensors were constructed by cross-linking formaldehyde dehydrogenase (FDH) or aldehyde dehydrogenase (ADH) and bovine serum albumin on the surface of a redox Os-polymer-coated electrode. The prepared aldehyde biosensors responded rapidly (within 30 s) to aldehydes without the addition of a soluble mediator, because the inner redox Os-polymer film effectively mediated the electron transfer from NADH generated enzymatically into the outer enzyme film to a glassy carbon electrode. An FDH/Os-polymer electrode responded linearly over the concentration range of 2 x 10(-6)-5 x 10(-4) M for formaldehyde, while an ADH/Os-polymer electrode, though responding similarly to long chain aldehydes, such as propionaldehyde and butylaldehyde, responded linearly over the concentration range of 4 x 10(-6)-2 x 10(-4) M for acetaldehyde.     

Amperometric enzyme electrodes for the determination of l-glutamate

Electrodes for amperometric measurement of l-glutamate were prepared by immobilization of l-glutamate oxidase on an Immobilon-AV Affinity membrane and attachment to an oxygen/hydrogen peroxide sensor. The response of the hydrogen peroxide sensor was linear over the concentration range 5.0 x 10(-8)-5.0 x 10(-4)Ml-glutamate, with a limit of detection of 35nM. Attachment of a size-exclusion membrane (cut-off for molecular weight > 100) or of a hydrophobic oxygen membrane eliminated electro-oxidizable interferences, but the response was attenuated by a factor of 2-3. The response may be amplified 10-fold by co-immobilizing l-glutamate dehydrogenase with the l-glutamate oxidase. The electrode initially lost 25% of its activity but was then stable for more than 320 days and at least 200 assays. The electrode was successfully used to assay glutamate in a protein tablet and in several food products. A flow-injection system was assembled for the continuous assay of l-glutamate.     

Electrochemical behavior and electrocatalytic study of the methylene green coated on modified silica gel

The electrochemical behavior of methylene green (MG) adsorbed on a silica surface modified with niobium oxide (SN) was investigated, using modified carbon paste electrodes. It was also used in an electrocatalytic study of NADH oxidation. The electrode showed a high stability attributed to the presence of SN, which avoids the leaching of the mediator from the electrode surface. The formal potential (E(0')) of the adsorbed MG was -35 mV vs SCE, showing a shift of 30 mV toward more positive potential values, compared to the MG dissolved in aqueous solution. This shift was assigned to the interaction between the basic nitrogen of MG and the acid sites of SN. The variation of the solution pH between 4 and 8 did not affect the stability nor the formal potential. However, for solution pH lower than 4 the formal potential was affected by the acidity of the medium. The electrocatalytic oxidation of NADH at the electrode was investigated. In the solution pH between 5 and 8 the electrocatalytic activity remained almost constant, giving a response signal of 13.3 nA L micromol(-1) cm(-2) and a K(Mapp) of 1.4 x 10(-5) mol L(-1). The electrode gave a linear response range between 5.0 x 10(-4) and 4.0 x 10(-3) mol L(-1) NADH concentration at pH 7.0 at an applied potential of 50 mV vs SCE. Applying a flow injection analysis system, the electrode showed a better analytical performance for NADH detection, presenting a linear response range between 6.0 x 10(-5) and 1.0 x 10(-3) mol L(-1), with an analytical frequency of 30 determinations/h, a detection limit of 8.2 x 10(-6) mol L(-1), and a precision for 25 replicates of 1% expressed as a relative standard deviation.     

Enzyme electrodes with substrate and co-enzyme amplification

The enzyme couples horseradish peroxidase/glucose dehydrogenase, glucose oxidase/glucose dehydrogenase, and cytochrome b₂/lactate dehydrogenase are applied in enzyme electrodes. Based on amplification by the recyclization reactions catalyzed by these two-enzyme systems, NADH, NAD⁺, glucose, lactate and pyruvate, are determined with 8–40-fold increased sensitivity compared to the unamplified reactions. Detection limits are 1.0 × 10^?6 M NADH, 1.2 × 10^?6 M NAD⁺, 8 × 10^?7 M glucose, and 3 × 10^?7 M lactate or pyruvate.     

l- and d-Lactate assay in real milk samples with immobilized enzyme reactors and graphite electrode

A sensitive flow system for the determination of l- and d-lactate in milk samples is described. l- and d-Lactate dehydrogenase, LDH, were immobilized on aminopropyl-controlled pore glass beads. l- and d-Lactate are oxidized to pyruvate in the presence of NAD(+) and NADH is produced. The electrochemical determination of NADH allows the measurement of the substrate involved in the reaction. We used a graphite-based anode sensor without any mediator at +500 mV vs. Ag/AgCl. The analytes were measured, in standard solutions, in the concentration range from 1 x 10(-6) to 4 x 10(-4)M using 1 mM NAD(+) concentration and 0.1M Tris buffer pH 9. Experiments with real milk samples showed large values of currents probably due to electroactive substances usually contained in milk. To eliminate interfering compounds a microdialysis probe coupled with a pre-oxidizing cell was used. This method of pre-treatment removes the interfering substances, but leaves the analytes under study unaffected. The procedure allows the determination of l- and d-lactate in milk samples in the concentration range from 1 x 10(-5) to 5 x 10(-4)M. The assay was applied to monitor continuously the bacterial fermentation of Staphylococcus aureus in UHT milk as an example of possible contamination detection in the manufacturing process.     

Inhibitory effects of glycyrrhetic acid derivatives on 11 beta- and 3 alpha-hydroxysteroid dehydrogenases of rat liver.

Glycyrrhetic acid (GA), an aglycone of glycyrrhizin (GL), is a potent inhibitor of 11 beta- and 3 alpha-hydroxysteroid dehydrogenases. 11 beta-Hydroxysteroid dehydrogenase activity of rat liver microsomes was potently inhibited by GA, 3-deoxyglycyrrhetic acid (3-deoxyGA), 3-ketoglycyrrhetic acid (3-ketoGA), 3-epiglycyrrhetic acid (3-epiGA) and 11-deoxoglycyrrhetic acid (11-deoxoGA), with I50 values of 2-4 x 10(-7) M. However, 18 alpha-stereoisomers (I50 = 3-7 x 10(-6) M) of GA, 3-deoxyGA and 11-deoxoGA were one tenth less inhibitory on the enzyme activity than the corresponding 18 beta-isomers. On the other hand, 18 alpha-stereoisomers of GA, 3-deoxyGA and 11-deoxoGA inhibited 3 alpha-hydroxysteroid dehydrogenase activity of rat liver cytosol more potently than the corresponding 18 beta-isomers. I50 values of 18 alpha- and 18 beta-isomers were 2 and 7 x 10(-6) M, respectively, in the case of GA, 8 and 20 x 10(-6) M in 3-deoxyGA, 3 and 20 x 10(-6) M in 11-deoxoGA. These results indicate that the 18 beta-conformation of oleanane is important for the inhibition of 11 beta-hydroxysteroid dehydrogenase but on the contrary the 18 alpha-conformation is important for the inhibition of 3 alpha-hydroxysteroid dehydrogenase.     

Properties of polyaniline/carbon nanotube multilayer films in neutral solution and their application for stable low-potential detection of reduced beta-nicotinamide adenine dinucleotide

A conducting polymer, polyaniline (PANI), was successfully assembled with commercially available poly(aminobenzenesulfonic acid)-modified single-walled carbon nanotubes (PABS-SWNTs) via the simple layer-by-layer method. PABS-SWNTs inside the multilayer film can dope PANI effectively and shift its electroactivity to a neutral pH environment, pointing to their potential biological applications. The obtained PANI/PABS-SWNTs multilayer films are very stable and show a high electrocatalytic ability toward the oxidation of reduced beta-nicotinamide adenine dinucleotide (NADH) at a much lower potential (about +50 mV vs Ag/AgCl), which makes it an ideal substrate for NADH detection and offers great promise for developing dehydrogenase-based biosensors depending on NADH as a cofactor. For a six-bilayer sample, the detection limit can go down to 1 x 10(-6) M as detected by the simple cyclic voltammetry method, with a linear detection range for NADH at concentrations between 5 x 10(-6) and 1 x 10(-3) M. The substrate can be used repeatedly for consecutive detection cycles of NADH with a very stable signal.     

An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a beta-type zeolite modified electrode

A new biosensor for the amperometric detection of hydrogen peroxide was developed based on the coimmobilization of horseradish peroxidase (HRP) and methylene blue on a beta-type zeolite modified glassy carbon electrode without the commonly used bovine serum albumin-glutaraldehyde. The intermolecular interaction between enzyme and zeolite matrix was investigated using FT-IR. The cyclic voltammetry and amperometric measurement demonstrated that methylene blue co-immobilized with HRP in this way displayed good stability and could efficiently transfer electrons between immobilized HRP and the electrode. The sensor responded rapidly to H2O2 in the linear range from 2.5 x 10(-6) to 4.0 x 10(-3) M with a detection limit of 0.3 microM. The sensor was stable in continuous operation.     

Differential pulse voltammetric indirect determination of aluminium in drinking waters, blood, urine, hair, and medicament samples using L-dopa under alkaline conditions

The differential pulse voltammetric (DPV) indirect determination of aluminium using L-dopa under alkaline conditions on a glassy carbon working electrode was studied. The proposed method relies on the linear decrease of the DPV anodic peak current of L-dopa with increase in the concentration of aluminium added. Under the optimum experimental conditions (pH 8.5, 0.08 M NH4Cl-NH3.H2O buffer solution, and 4 x 10(-4) M L-dopa), the linear range is 2-18 x 10(-7) M AlIII. The detection limit is 7.6 x 10(-8) M and the relative standard deviation for 8 x 10(-7) M AlIII is 3.5% (n = 8). A number of foreign species were examined as potential interferents. The method was applied to the determination of aluminium in drinking waters, synthetic renal dialysate, sodium chloride injection, sucrafate, hydrothorax, blood, urine and hair samples. The physiological significance is discussed.     

Sensing phenothiazine drugs at a gold electrode co-modified with DNA and gold nanoparticles.

DNA and gold nanoparticles are co-immobilized at a gold electrode through elaborate self-assembly processes. This configuration has proven to be useful as a sensor for phenothiazine drugs, taking advantage of the well-known, relatively large surface area of gold nanoparticles and the strong intercalation between dsDNA and phenothiazine drugs. This modified electrode has demonstrated good sensitivity and stability towards the oxidation of two model phenothiazine drugs: promethazine and chlorpromazine. A linear dependence between the concentration of phenothiazine drugs and the peak current is observed, with a concentration range of 2.0 x 10(-5)-1.6 x 10(-4) M and 1.0 x 10(-5)-1.2 x 10(-4) M, and a detection limit of 1.0 x 10(-5) M and 7.0 x 10(-6) M, for promethazine and chlorpromazine, respectively.     

Immobilization of lactate oxidase in a poly(vinyl alcohol) matrix on platinized graphite electrodes by chemical cross-linking with isocyanate

A new method for development of an electrochemical sensor based on lactate oxidase is dedbed. Platinized spectroscopic-grade graphite electrodes were modified by chemically cross-linking l-lactate oxidase from Pediococcus species into a poly(vinyl alcohol) network through reaction with a tri-isocyanate. The immobilized enzyme exhibits high activity and long-term stability. The sensor provides a linear response to l-lactate over a concentration range of 2 x 10(-5)-4 x 10(-3)M and a sensitivity of 1.71 muA.1. mmole(-1). The response time of the sensor is 10-45 sec and the detection limit is 10muM. Stable response to the substrate was obtained over a period of 3 months. The new sensor was also used for the analysis of some dairy products without any special pretreatment.     

Au nanoparticle-enhanced surface plasmon resonance sensing of biocatalytic transformations

N-(3-Aminopropyl)-N'-methyl-4,4'-bipyridinium is coupled to tiopronin-capped Au nanoparticles (diameter ca. 2 nm) to yield methyl(aminopropyl)viologen-functionalized Au nanoparticles (MPAV(2+)-Au nanoparticles). In situ electrochemical surface plasmon resonance (SPR) measurements are used to follow the electrochemical deposition of the bipyridinium radical cation modified Au nanoparticles on an Au-coated glass surface and the reoxidation and dissolution of the bipyridinium radical cation film. The MPAV(2+)-functionalized Au nanoparticles are also employed for the amplified SPR detection of NAD(+) and NADH cofactors. By SPR monitoring the partial biocatalyzed dissolution of the bipyridinium radical cation film in the presence of diaphorase (DP) NAD(+) is detected in the concentration range of 1x10(-4) M to 2x10(-3) M. Similarly, the diaphorase-mediated formation of the bipyridinium radical cation film on the Au-coated glass surface by the reduction of the MPAV(2+)-functionalized Au nanoparticles by NADH is used for the amplified SPR detection of NADH in the concentration range of 1x10(-4) M to 1x10(-3) M.