Enzyme Electrode for Amplification of NAD+/NADH Using Glycerol Dehydrogenase and Diaphorase with Amperometric Detection

Abstract

An enzyme electrode is made from a glassy carbon electrode covered with a gelatin membrane containing entrapped glycerol dehydrogenase (GDH) and diaphorase, and protected with a dialysis membrane. Based on amplification by the recycling reaction catalyzed by the two-enzyme systems, NAD⁺ and NADH can be determined with 800–1200 times higher sensitivity than for the same electrode in a substrate sensing mode when the flow rate was 0.08 ml/min. The detection limit was about 0.03 μM for NADH. The amplification factors were around 1000 for 0.08 ml/min, with quite large variations between electrodes. They had decreased to about 70% of the original value after 7 days. The biosensor is intended for detection in immunoassays with alkaline phosphatase as a marker.     

A Dual-Enzyme Electrode for the Determination of Flavin Adenine Dinucleotide in the Presence of Riboflavin and Flavin Mononucleotide

Abstract

An electrode method has been developed for the determination of flavin adenine dinucleotide (FAD) using a potentiometric gas sensor and commercially available enzyme preparations. The construction of the FAD-sensitive electrode is based on immobilizing alkaline phosphatase (E.C. 3.1.3.1) and adenosine deaminase (E.C. 3.5.4.4) on the sensing tip of an ammonia gas sensor. Alkaline phosphatase enzymatically catalyzes the hydrolysis of FAD to adenosine which is subsequently converted to ammonia by adenosine deaminase. The response of the dual-enzyme electrode is linear between 8 × 10^?5 M and 4 × 10^?3 M FAD with a slope of 43 mV/decade concentration at pH 8.5 and 37[ddot]C. The optimum buffer system is 0.5 M diethanolamine, 1 × 10^?3 M Tris-HCl and 1 × 10^?3 M MgCl₂. Electrodes constructed with enzymes immobilized by cross-linking with glutaraldehyde and bovine serum albumin showed longer life times than electrodes using enzymes entrapped by a dialysis membrane. The electrode is highly selective over riboflavin and flavin mononucleotide, but it does respond to other nucleotides.     

Photovoltaic Determination of Hydrogen Peroxide with A Biophotodiode

Abstract

A novel biosensor, biophotodiode, is proposed for the determination of substances of biochemical and clinical importance. The biophotodiode is a unique combination of a photodiode and matrix-bound peroxidase. Since the enzyme catalyzes the luminescent reaction of the luminol-H₂O₂ system, this assembly is effective on the determination of hydrogen peroxide. The photocurrent of the sensor is correlated with the hydrogen peroxide concentration ranging from 1mM to 10 mM. A biophotodiode for glucose is also constructed by assembling a glucose oxidase-peroxidase bienzyme membrane and a photodiode.     

Flow Injection Zymography - A Novel Procedure For the On-Line Detection of Enzyme Activity

Abstract

This paper describes a new method - Flow Injection Zymography - for the detection of enzyme activity during purification procedures using flow injection analysis (FIA). High sampling frequency (3 samples per minute), small sample volume requirement and a wide range of linearity (up to 70 IU) of enzyme activity was achieved. Reagent costs were minimized using the merging zones mode. the zone sampling technique for sample dilution was successfully incorporated into this system to maintain a reliable signal/response ratio. Most importantly, this new FIA method allowed on the spot identification of the active fractions.     

A Highly Sensitive Enzyme Immunoassay of Anti-Insulin Antibodies in Guinea Pig Serum

Abstract

A highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. Insulin bound to anti-insulin antibodies was separated from free insulin by precipitation with polyethylene glycol. Anti-insulin antibodies in the precipitates were dissociated from insulin and inactivated by incubation with 0.1 mol/1 HCl. The amount of insulin dissociated was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab′-horseradish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was improved 1,000-fold as compared with that of the enzyme immunoassay previously described, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab′-horseradish peroxidase conjugate.     

A Trypsin Immobilized Sol-Gel for Protein Indentification in MALDI-MS Applications

The proteolytic enzyme trypsin was chemically immobilized to an amine-functionalized sol-gel using adipoyl chloride under nonaqueous conditions and a nitrogen atmosphere. In the synthesis of the sol-gel, tetraethyl orthosilicate (TEOS), and 3-(2-aminoethylamino) propyldimethoxymethylsilane (AEAPMS) (50:50, v/v) were used, which provided convenient physical and chemical conditions to maintain catalytic activity of immobilized trypsin molecules for the digestion of proteins in proteomics applications. Bovine serum albumin was used as a model protein to perform enzymatic digestion using the trypsin immobilized sol-gel. The resulting peptides were analyzed by matrix-assisted laser desorption/ionization-mass spectrometry to evaluate the digestion performance and specificity of the sol-gel material. The trypsin immobilized sol-gel showed superior enzymatic activity in protein digestion and it was determined that the sol-gel material could be repeatedly used at least 25 times without significant activity loss in long-term use. Additionally, autocatalysis was prevented by immobilization of trypsin. The peptide digest having the highest purity was obtained for protein identification studies.     

A Bioelectrochemical Method for the Determination of Acetate with Immobilized Acetate Kinase

Abstract

Flow injection determinations of acetate were carried out using immobilized acetate kinase, pyruvate kinase and lactic dehydrogenase with an amperometric method. Two acetate kinases from E. coli and B. stearothermophilus were tested. It was found that the immobilized acetate kinase from B. stearothermophilus was more stable than that from E. coli., but it is much more expensive and less available. Acetate kinase coupling at pH 7.4 using CPG aminopropyl and glutaraldehyde seems to be superior to other immobilization methods. A high immobilization yield can be obtained by immobilization of the three enzymes separately giving high conversions of all the three. Plots of current versus concentration show a useful operating range from 0.3 to 2 mM acetate with a linear response. The detection limit was 0.2 mM at a flow rate of 0.3 ml/min with 200 μl injections. The method is therefore well suited for monitoring of the level of acetate in fermentations with acetate as the carbon source.     

The Application of an Enzyme Electrode for Blood Glucose Determination in the Automated Flow System

Abstract

An enzyme electrode containing immobilized GOD was utilized in a continuous flow system for the automatical determination of glucose. The advantages of kinetic measurement of H₂ O ₂ are used. Characteristic parameters of device and results in routine are shown. Particular advantages are: low requirement on material (20 μl blood, < l μg GOD/measurement) and time (60 samples/h at 15 sec response time) as well as sufficient precision and accuracy (S % - serial: < 2 %).     

A New Immunoassay Separation Technique Using Reversibly Soluble Polymers

Abstract

A new immunoassay separation technique was developed based on the use of antibody-polymer conjugates with reversible solubility characteristics. Antibody was coupled to sodium alginate with water-soluble carbodiimide. These conjugates could be used in place of unmodified antibody in various assay formats and removed from solution when desired by converting the polymer to an insoluble form through pH adjustment or addition of metal ions. An enzyme immunoassay for theophylline was used to demonstrate the usefulness of the new approach for both manual and partially automated procedures.     

Effect Of Temperature on the Sensitivity of Sandwich Enzyme Immunoassay with Fab'-Horseradish Peroxidase Conjugate

Abstract

Effect of temperature was examined on the sensitivity of sandwich enzyme immunoassay for human chorionic gonadotropin (hCC) with anti-hCG Fab'-horseradish peroxidase conjugates prepared by using three different reagents (N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-l-carboxylate, glutaraldehyde and metaperiodate). The non-specific bindings of the conjugates to anti-hCC IgG-coated polystyrene balls were much lower at 20°C than at 37°C, and the specific bindings were slightly higher at 20°C than at 37°C. The lowest non-specific binding and the highest specific binding were obtained by incubation at 20°C with the maleimide conjugate. As a result, the sensitivity could be more easily improved by incubation at 20°C than at 37°C and the highest sensitivity was obtained with the maleimide conjugate. Similar results were also obtained for other macromolecular antigens such as human ferritin and α-fetoprotein.