Spectrophotometric and fluorimetric enzymatic determination of serum creatine kinase mb isoenzyme by using immuno-inhibition

Two fully enzymatic methods, colorimetric and fluorimetric, are reported for the determination of creatine kinase (EC 2.7.3.2) MB isoenzyme after immune-inhibition with the use of goat anti-human CK-M IgG antibodies. The residual creatine kinase activity is assayed by using hexokinase and glucose-6-phosphate dehydrogenase systems; the resulting NADPH is determined spectrophotometrically by reaction with p-iodonitro-tetrazolium violet in the presence of diaphorase as an intermediate electron carrier, or fluorimetrically by coupling the NADPH with resazurin/diaphorase to form resorufin. Both assays take only 12 min and require only 100 or 25μl of serum. The calibration plots of enzyme activities are linear up to 580 and 435 U l^-1 of CK-MB in serum for the spectrophotometric and fluorimetric assays, respectively, the coefficients of variation being 2.3% and 4.6%, and the recovery values 103% and 100% respectively. The results correlate very well with those obtained by the Helena electrophoresis—fluoridensitometric methods. As little as 4 U l^-1 and 2 U l^-1 CK-MB could be measured reproducibly.     

An improved enzymatic assay for glucose determination in blood serum using a l,1′-dimethylferricinium dye

l,l′-dimethylferricinium (DMFe⁺),a stable and pH-insensitive blue dye, was prepared via enzymatic oxidation of a 1,1′-dimethylferrocene (DMFe):2-hydroxypropyl-β-cyclodextrin (HPCD) watersoluble inclusion complex, using bilirubin oxidase immobilized onto porous aminopropyl glass beads via glutaraldehyde activation. In the presence of glucose, DMFe⁺ was reduced to DMFe by reacting with the reduced glucose oxidase (FADH₂), and the absorbance decrease was followed at 650 nm. In acetate pH 5.2 buffer, the response to glucose in blood serum was nonlinear, especially in the low concentration range, because of a competition for the reduced glucose oxidase between the DMFe⁺ dye and oxygen. At this pH, endogenous ceruloplasmin was also observed to oxidize residual DMFe (16%) in the dye preparation, causing an increase in absorbance at 650 nm. An assay protocol was then developed using maleate buffer, pH 6.5, to overcome these interferences as well as mutarotation of α-D-glucose. The results obtained for glucose in the blood serum samples agreed well with those of the reference hexokinase/glucose-6-phosphate dehydrogenase method.     

An Electrochemical Flow Analysis System for Putrescine Using Immobilized Putrescine Oxidase and Horseradish Peroxidase

An electrochemical flow analysis system was optimized together with immobilized putrescine oxidase and horseradish peroxidase for putrescine measurement. Four coupling agents, suberic acid bis(N‐hydroxysuccinimide ester), γ‐maleimidobutyric N‐hydroxysuccinimide ester, 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide and glutaraldehyde, were used for immobilizing the two enzymes on porous aminopropyl glass beads to form a bienzymic detection column. Although the glutaraldehyde crosslinking procedure offered the highest response, the immobilized bienzyme system was responsive to putrescine, spermidine (123% of the putrescine response at 250 μM) and cadaverine (98% of the putrescine response). In contrast, the enzymes immobilized on the glass beads using suberic acid bis(N‐hydroxysuccinimide ester) offered significantly better selectivity towards putrescine at the same concentration. For comparison, cadaverine and spermidine only provoked a response of 4.7% and 27.5% of the putrescine signal. The response to cadaverine and spermidine was further suppressed by lowering the detection pH from 8 to 7. At 250 μM, the response obtained for cadaverine and spermidine was only 1.5% and 3.9%, respectively, of the signal obtained for putrescine. A linear response to putrescine was obtained from 5 to 75 μM (0.629 μAs μM^?1, R²=0.997) with a detection limit of 5 μM (S/N=3). The amperometric response retained 75% of its initial value after 600 repeated injections. The immobilized PUO/HRP (putrescine oxidase/horseradish peroxidase) was successfully demonstrated for measuring putrescine in fish extracts as an indicator of fish spoilage.     

Multiplexed Determination of Amino‐Terminal Pro‐B‐Type Natriuretic Peptide and C‐Reactive Protein Cardiac Biomarkers in Human Serum at a Disposable Electrochemical Magnetoimmunosensor

A rapid magnetoimmunosensor for the simultaneous determination of two cardiac biomarkers, amino‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) and C‐reactive protein (CRP), in human serum is described. Specific capture antibodies were covalently immobilized onto carboxylic acid‐modified magnetic beads. The quantification of NT‐proBNP and CRP was performed by using indirect competitive and sandwich configurations, respectively, and horseradish peroxidase‐labeled tracers. The use of dual screen‐printed carbon electrodes allowed the achievement of simultaneous independent amperometric readout for each cardiac biomarker. The developed methodology showed very low limits of detection (0.47 ng mL^?1). An international standard for CRP serum spiked with NT‐proBNP was analyzed to evaluate the usefulness of the magnetoimmunosensor.     

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.     

聚ε-己内酯的微米硅球固定化猪胰脂肪酶促合成

本文采用微米硅球固定化猪胰脂肪酶为催化剂合成聚ε-己内酯, 以期获得具有较高分子量、 良好生物相容性和使用安全性的生物可降解医用高分子材料.     

Covalent binding of genetically engineered microorganisms to porous glass beads

Several covalent immobilization methods, which have been routinely used with proteins and antibodies, were studied for their ability to immobilize genetically engineered Escherichia coli cells to glass beads. The cells used in this study expressed a metal binding peptide that binds cadmium (Cd) and mercury (Hg). The initial work focused on a method employing 2.5% aminopropyltrimethoxy silane and 2.5% glutaraldehyde for covalent immobilization of cells onto porous glass beads. Scanning electron microscopy (SEM) demonstrated cell attachment (average of 3.0×10⁸ cells per bead) to the irregular surface. Columns containing cells immobilized with the 2.5% aminosilane and 2.5% glutaraldehyde removed more than 90% of the Cd from solutions with 50 ppb and 1 ppm levels. Following removal of the bound Cd with HCl elution and regeneration to pH 6.0, the columns were shown to effectively bind additional cadmium. Various concentrations of aminosilane and glutaraldehyde were tested for improved cell density.Glutaraldehyde is a universal and convenient cross-linker, but there are some concerns with its effects on the cells and proteins, therefore, two additional covalent techniques were examined. One method employed the aminopropyltrimethoxy silane and carbodiimide, and the other used mercaptopropyltrimethoxy silane and the heterobifunctional cross-linker GMBS. Some comparisons of these two immobilization methods to the method employing glutaraldehyde are described.     

Characterization of the Binding Performance of Silica-Based Immunoaffinity Adsorbents Prepared via Online Coupling Reactions

Antibodies against a conjugate of daidzein and bovine serum albumin (BSA) were immobilized onto dihydrazide-activated silica beads via online coupling reactions. The binding performances of the obtained immunoaffinity adsorbents to the corresponding hapten and carrier protein were all optimized and characterized. It was found that online coupling reactions offered a convenient and practical way to generate the requisite immunoaffinity columns. The optimum binding buffers for BSA and daidzein were found to be pH 5.0, 0.01 mol L^?1 phosphate-buffered saline (PBS) and 0.05% Tween-20 in pure water, indicating different interactions between the immobilized antibodies with different parts of the antigen. Under the optimum operating conditions, the silica-based immunoaffinity adsorbents showed specific and selective binding properties to the target protein; while for small molecules, a mixed adsorption mechanism caused by both specific and non-specific interactions was observed. For comparison, offline coupled silica-antibody immunoaffinity adsorbents and Sepharose 4B support immobilized with the same antibody were also prepared and tested. The experimental results of this study may provide useful information for further development of other high-performance immunoaffinity chromatographic methods for different purposes.     

Rapid and reagent-saving immunoassay using innovative stirring actions of magnetic beads in microreactors in the sequential injection mode

We developed new ELISA techniques in sequential injection analysis (SIA) mode using microreactors with content of a few microliters. We immobilized antibodies on magnetic beads 1.0 μm in diameter, injected the beads into microreactors and applied rotating magnetic fields of several hundred gauss. Magnetic beads, suspended in liquid in density of approximately 10⁹-10¹⁰ particles per millilitre, form a large number of thin rod clusters, whose length-wise axes are oriented in parallel with the magnetic field. We rotate the Nd magnets below the center of the microreactor by a tiny motor at about 2000-5000 rpm. These rotating clusters remarkably accelerate the binding rate of the antibodies with antigens in the liquid. The beads are trapped around the center of the rotating magnetic field even in the flowing liquid. This newly found phenomenon enables easy bead handling in microreactors. Modification of reactor walls with selected blocking reagents was essential, because protein-coated beads often stick to the wall surface and cannot move freely. Washing steps were also shortened.     

Phenylboronic acid immunoaffinity reactor coupled with flow injection chemiluminescence for determination of α-fetoprotein

A reusable and sensitive immunoassay based on phenylboronic acid immunoaffinity reactor in combination with flow injection chemiluminescence (CL) for determination of glycoprotein was described. The reactor was fabricated by immobilizing 3-aminophenylboronic acid (APBA) on glass microbeads with γ-glycidoxypropyltrimethoxysilane (GPMS) as linkage. The α-fetoprotein (AFP) could be easily immobilized on the APBA coated beads through sugar-boronic interaction. After an off-line incubation, the mixture of the analyte AFP with horseradish peroxidase-labeled AFP antibody (HRP-anti-AFP) was injected into the reactor. This led the trapping of free HRP-anti-AFP by the surface coated AFP on glass beads. The trapped HRP-anti-AFP was detected by chemiluminescence due to its sensitizing effect on the reaction of luminol and hydrogen peroxide. Under optimal conditions, the chemiluminescent signal was proportional to AFP concentration in the range of 10-100 ng mL⁻¹. The whole assay process including regeneration of the reactor could be completed within 31 min. The proposed system showed acceptable detection and fabrication reproducibility, and the results obtained with the present method were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. The described method enabled a low-cost, time saving and was potential to detect the serum AFP level in clinical diagnosis.     

A Multi‐electrochemical Competitive Immunosensor for Sensitive Cocaine Determination in Biological Samples

A multi‐electrochemical competitive immunosensor for the rapid determination of unmetabolized cocaine (COC) in urine, saliva and human serum matrices is reported. Anti‐cocaine polyclonal antibodies were immobilized in an oriented way onto protein‐G functionalized magnetic beads. The immunosensor is based on an array of eight carbon‐based screen‐printed electrodes for simultaneous electrochemical determinations. The treatments of the biological samples were simplified and optimized for avoiding matrix interferences. The immunosensor was sensitive (EC₅₀≈2.92–3.88 ng mL^?1 COC), required a very small volume of sample (200 µL), was reproducible (%RSD was lesser than about 18 %), and accurate (recovery percentages ranged 88–117 %).     

Sensitive flow-injection determination of l-lactate in human blood with immobilized enzyme columns and fluorimetric detection

Enzyme columns prepared by packing l-lactate oxidase and horseradish peroxidase immobilized chemically on controlled-pore glass beads are connected in series. Hydrogen peroxide formed in the enzymatic conversion of l-lactate in the first column is mixed with 3-(p-hydroxyphenyl)propionic acid before passage through the peroxidase column and fluorimetric measurement. Linear calibration was obtained for 0.5–500 pmol of L-lactate in 20 μl of 1000-fold diluted, deproteinated whole blood. A rapid sampling rate (60 h^?1) was possible.     

Characterization and application of silylated substrates for the preconcentration of cations

Chelating functional groups immobilized on silica and controlled pore glass beads are used to preconcentrate cations from aqueous solution in the concentration region of ng ml^?1. The functional groups are chemically bonded to the substrates by silylation reagents which may be employed as received or further modified. For example, commercially available 1,2-diamines may be used or converted to the dithiocarbamate after immobilization. This report includes studies of ionic strength and sodium chloride effects on the recovery of transition metal ions. Batch extraction of transition metal ions is accomplished with silica as the substrate for solutions containing transition metal ions at μg ml^?1 concentrations. Column extraction of solutions pumped at 50 ml min^?1 is accomplished at concentrations of 25 ng ml^?1. Recovery is good in both cases. The cations were determined directly on pelletized substrates by x-ray fluorescence. However, the ions may be eluted from the substrate and determined by other methods. Examples of determinations of several cations in lake water and high-purity industrial water are given.     

Analysis of Early Medieval Glass Beads – The Raw Materials to Produce Green, Orange and Brown Colours

 Monochrome coloured glass beads of the Merovingians (5^th–7^th cent. AD) have been examined by different analytical methods. The elemental composition of a large number of mostly unprepared beads have been measured non-destructively by X-ray fluorescence analysis. After subtracting the content of the colouring oxides of the glass beads and normalising the residual values to 100% an identical soda-lime-glass matrix was obtained. X-ray diffraction was used for the identification of the crystalline colouring and opacifying pigments (SnO₂, Cu, Cu₂O, PbSnO₃), and scanning electron microscopy as well as electron probe microanalysis were applied to study the microstructure and the composition of white, brown, green, orange and yellow coloured glass beads. Oxidised metals, alloys (lead, copper, bronze, brass and mixtures of them) and iron smelting slag have been identified as raw materials to colour the soda-lime-glass.     

Label-free quantification of cystatin C as an improved marker for renal failure

A label-free biosensor has been developed, allowing quantification of cystatin C in human serum. This was achieved by using reflectometric interference spectroscopy as detection method. Cystatin C is a small serum protein that allows detection of renal failure more reliably than established parameters as creatinine. The protein was immobilized on the surface of a glass transducer, forming the sensitive layer of the sensor chip. Based on a binding-inhibition assay, two different types of monoclonal cystatin C antibodies were compared, by their behavior and their obtained working range in buffer and serum as matrix. Both antibodies allowed quantification of the protein in serum as matrix within the required clinical ranges of 0.53–1.02 mg/L. Detected recovery rates are in a range between 84.8% and 116.1%. The developed sensor shows high inner chip reproducibility and low cross-sensitivity.     

Cholera toxin subunit B detection in microfluidic devices

Fluorescence and electrochemical microfluidic biosensors were developed for the detection of cholera toxin subunit B (CTB) as a model analyte. The microfluidic devices were made from polydimethylsiloxane (PDMS) using soft lithography from silicon templates. The polymer channels were sealed with a glass plate and packaged in a polymethylmethacrylate housing that provided leakproof sealing and a connection to a syringe pump. In the electrochemical format, an interdigitated ultramicroelectrode array (IDUA) was patterned onto the glass slide using photolithography, gold evaporation and lift-off processes. For CTB recognition, CTB-specific antibodies were immobilized onto superparamagnetic beads and ganglioside GM₁ was incorporated into liposomes. The fluorescence dye sulforhodamine B (SRB) and the electroactive compounds potassium hexacyanoferrate (II)/hexacyanoferrate (III) were used as detection markers that were encapsulated inside the liposomes for the fluorescence and electrochemical detection formats, respectively. Initial optimization experiments were carried out by applying the superparamagnetic beads in microtiter plate assays and SRB liposomes before they were transferred to the microfluidic systems. The limits of detection (LoD) of both assay formats for CTB were found to be 6.6 and 1.0 ng mL⁻¹ for the fluorescence and electrochemical formats, respectively. Changing the detection system was very easy, requiring only the synthesis of different marker-encapsulating liposomes, as well as the exchange of the detection unit. It was found that, in addition to a lower LoD, the electrochemical format assay showed advantages over the fluorescence format in terms of flexibility and reliability of signal recording.     

Potentiometric Biosensing of Penicillins Using a Flow-Through Reactor with Penicillinase or Penicillin Amidase Immobilized by Gamma-Irradiation

Abstract

Penicillinase and penicillin amidase were immobilized, with similar yield, by physical entrapment in porous beads of poly-2-hydroxyethyl methacrylate matrix formed by gamma-irradiation. Flow-injection analysis of penicillins was performed by using a column reactor filled with enzyme-loaded polymer beads and a wall-jet pH-metric micro-cell with a combined flat-membrane glass electrode as the sensing element. With both enzymes it was possible to have either a linear response dynamic range or a logarithmic one by appropriate selection of buffer capacity and flow rate of the carrier solution.     

Comparative Evaluation of Pumice Stone as an Alternative Immobilization Material for 1,3-Propanediol Production from Waste Glycerol by Immobilized Klebsiella pneumoniae

In this study, pumice stone (PS), which is a vastly available material in Turkey, was evaluated as an alternative immobilization material in comparison to other commercially available immobilization materials such as glass beads and polyurethane foam. All immobilized bioreactors resulted in much better 1,3-propanediol production from waste glycerol in comparison to the suspended cell culture bioreactor. It was also demonstrated that the locally available PS material is as good as the commercially available immobilization material. The maximum volumetric productivity (8.5?g?L^?1?h^?1) was obtained by the PS material, which is 220?% higher than the suspended cell system. Furthermore, the immobilized bioreactor system was much more robust against cell washout even at very low hydraulic retention time values.     

An ELIME assay for the rapid diagnosis of coeliac disease

Coeliac disease (CD) is a gluten-induced autoimmune enteropathy found in genetically susceptible subjects. Because of the high number of undetected cases, rapid and cheaper screening methods are needed. Currently, the CD diagnosis involves the detection of anti-transglutaminase IgA antibodies (anti-tTG IgA) in blood serum through the use of ELISA systems with confirmation by histology of the intestinal mucosa. A new, rapid magneto-electrochemical immunosensor for CD diagnosis has been developed and applied to serum sample analysis. The system uses magnetic beads coated with tTG antigen to detect anti-tTG antibodies in positive serum samples and an alkaline phosphatase-conjugated anti-human IgA as label. An electrochemical readout, using magnetized screen-printed electrodes coupled with a portable instrument, is made after the addition of α-naphtyl phosphate, which is enzymatically converted into the electrochemically active α-naphthol product. The work involved the following considerations: (1) optimization of analytical parameters; (2) recovery evaluation, adding known concentrations of anti-tTG IgA to “blank” sera; (3) analysis of 107 blood serum samples; (4) calculation of the ROC curve, resulting in a cut-off of 1.0 U/ml, 100% of clinical sensitivity and 98.36% of clinical specificity; evaluation of the agreement between electrochemical and ELISA kit values (r ² of 0.943). The system developed could be an useful tool for a correct and rapid CD diagnosis. This method is simple, cheap, rapid, and suitable for screening analyses performed outside of the classical diagnostic laboratory.     

Improvement of Bioethanol Productivity of Immobilized Saccharomyces Bayanus with Using Sodium Alginate-Graft-Poly(N-Vinyl-2-Pyrrolidone) Matrix

In this study, immobilization conditions and bioethanol production characteristics of immobilized Saccharomyces bayanus were investigated into sodium alginate-graft-poly(N-vinyl-2-pyrrolidone; NaAlg-g-PVP) matrix. The matrix that crosslinked with calcium clorid was used for immobilization of S. bayanus. Bioethanol productivity of the NaAlg-g-PVP matrix was found to increase from 4.21 to 4.84?gL^?1?h^?1 when compared with the convential sodium alginate matrix. The production of bioethanol was affected by initial glucose concentration and percentage of immobilized cell beads in fermentation medium. Bioethanol productivity was increased from 3.62 to 4.84?gL^?1?h^?1 while the glucose concentration increasing from 50 to 100?gL^?1. Due to the increase in percentage from 10 to 20?% of immobilized cell beads in the fermentation medium, bioethanol productivity was increased from 4.84 to 8.68?gL^?1?h^?1. The cell immobilized NaAlg-g-PVP beads were protected 92?% of initial activity after six repeated fermentation.