A bifunctional molecularly imprinted polymer (MIP): analysis of binding and catalysis by a thermistor.
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Acetates↗
Biomedical subjects
Bengt Danielsson
Publications and source records attributed to Bengt Danielsson.
Development of a plasma panel test for detection of human myocardial proteins by capillary immunoassay.
A chemiluminescence immunoassay for the detection of four heart marker proteins: myoglobin, creatine kinase mb [CKmb], troponin I [TnI], and fatty acid-binding protein [FABP], was designed. The immunoassay was based on enzyme-linked immunosorbent assay [ELISA] and antibodies immobilized in glass capillaries pre-treated with 3-aminopropyltriethoxysilane. The protein bound to the antibody was detected by using an anti-protein-horseradish peroxidase [HRP] conjugate. The reaction of the HRP with luminal and hydrogen peroxide-based substrate generated the chemiluminescence and a photodiode detector was used to measure the light intensity. The same assay protocol was used to detect all four proteins. Ultrasound waves were used to improve the silanization of glass and the antibody immobilization process. The optimization of the duration and intensity of the ultrasound was performed for the myoglobin assay. Ultrasound improved the silanization procedure and the capillaries gave an approximately 2.5 times greater ELISA response. Ultrasound also improved the sensitivity by approximately 100% when monoclonal antibody was immobilized on a glass capillary. Calibration curves corresponding to analyte concentrations ranging from 2.4 to 2400 ng/ml in plasma samples were recorded. The detection limits were in the region of 1.2 myoglobin, 0.6 CKmb, 5.6 TnI, and 4 ng/ml FABP in plasma with a coefficient of variation of 3-9.9%.
Antibodies↗
Extraction of rye bran by supercritical carbon dioxide: influence of temperature, CO2, and cosolvent flow rates.
Process parameter optimization for the supercritical CO(2) extraction of rye bran to obtain alkylresorcinols (AR) was studied by carrying out a two-level fractional design experiment. Four parameters, temperature, CO(2) flow rate, cosolvent percentage, and extraction time, presumed to influence the extraction process, were analyzed. A tentative fractionation of the crude extract was also carried out and is discussed. The best extracts were achieved when the CO(2) flow rate and extraction time or temperature and cosolvent addition were kept high. It was found that temperature increase was not statistically significant within the range of the study performed, and the extraction time was thus the most important factor. A preliminary fractionation process in two cyclone separators yielded two fractions, one rich in AR components with higher molecular weights and the other rich in AR components with low molecular weight.
Carbon Dioxide↗
Fluorometric broad-range screening of compounds with affinity for nucleic acids.
The potential of a nucleic acid-based optical bioprobe for environmental measurements and drug monitoring is described. The sensor employs the long-wavelength intercalating fluorophore TO-PRO-3 (TP3). Compounds that interact with the TP3-DNA complex are indirectly detected by a decrease in the fluorescence intensity. We found that the configuration and length of the DNA dramatically affected the intensity of the fluorescence emitted from the TP3-DNA complex. We compared nucleic acids from different sources and optimized the system for pBR322 plasmid DNA (4363 bp) digested by HindIII restriction endonuclease. This endonuclease has a single recognition site in plasmid pBR322. In the proposed method, we attempt to combine broad-range detection with rapid and simple operation. A fiber-optic capillary fluorescence system was used to analyze toxic aromatic amines, antibiotics, and several kinds of antitumor drugs, using small amounts of sample, down to 10 muL, with a sensitivity comparable to that of current electrochemical methods. The detection limit can be as low as a few ppb or submicromolar. This approach is useful for routine screening in environmental monitoring or for controlling cytotoxic drug administration. The ease of operation and the rapid response allow high-throughput screening.
Amines↗
The cytotoxic and genotoxic effects of conjugated trans-2-nonenal (T2N), an off-flavor compound in beer and heat processed food arising from lipid oxidation.
This study investigates the toxic effect of E(2)nonenal (trans-2-nonenal, T2N) and its conjugate with horse muscle myoglobin (Mb) tested on murine cell line L929 and human cell line A549, as well as the genotoxic effect of these compounds assayed by measuring of micronuclei in human cells K562. It is an aldehyde, which is occurring as the substance responsible for an off flavour in aged beers, but originates also from lipid oxidation in heat processed food. T2N is an aldehyde formed from linoleic acid as a secondary oxidation product. The modification of Mb with T2N was analyzed with the use of SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and electrospray ionization mass spectrometry (ESI-MS). Results from SDS-PAGE suggest that T2N substitutes Mb and additionally causes cross-linking with polymerization of Mb resulting in an insoluble fraction. The ESI-MS spectrum of the soluble fraction used in the toxicity tests, demonstrated that conjugation of T2N with Mb yielded Mb adducts with one residue of trans-2-nonenal per myoglobin molecule as the major fraction and adducts with different numbers of T2N molecules as minor fractions. In the cytotoxicity assay the T2N and its Mb conjugate causes 50% destruction of cells at the concentration 95-125 microg/ml and 200 microg/ml respectively, when L929 and A549 cell lines were used, whereas Mb control tested up to 2000 mg/ml was without any cytotoxic effect. In genotoxicity in vitro assay we have observed that the T2N and its Mb conjugate expressed the genotoxicity. The number of micronuclei in human K562 cells reached 26 +/- 2.16 promille (MN/1000 cells), comparing to 62 +/- 8.64 MN/1000 cells for the reference free T2N, whereas a control value was 10.33 +/- 1.25 MN/1000 cells. The studied compounds expressed also the apoptotic effect in K562 cells as the number of apoptotic cells increased to 44.67 +/- 4.92 promille for T2N-Mb, comparing to 168.67 +/- 37.28 promille for free T2N, whereas a control value was 30.33 +/- 1.36 promille for Mb. In these assays the T2N-Mb conjugate is several times more toxic in relation to control protein. Results indicate that T2N adducts with protein are potent to induce various cytotoxic and apoptotic effects when assayed in vitro tests. It suggests that higher level of such aldehyde might create in organism severe potential of toxicity.
Aldehydes↗
Electrochemical study of the XNA on Gold microarray.
A novel electrode array was developed based on the XNA on Gold trade mark microarray platform. The platform combines self-assembling monolayers, thick film patterning and streptavidin based immobilization to provide a robust, versatile platform capable of analysing virtually any biomolecule including nucleic acids, proteins, carbohydrates and lipids. Electrochemical analysis of the self-assembling monolayer/streptavidin (SAMS) XNA on Gold coating revealed that the ferrocene redox current for the SAMS modified electrode was greater than that with a bare Gold electrode. The electrochemical reaction of K4Fe(CN)6 was inhibited by the SAMS coating, but was reactivated upon addition of ferrocene. These results indicate that ferrocene is involved as a mediator in the electron transfer of K4Fe(CN)6 to the SAMS modified electrode. Addition of DNA to the SAMS resulted in only a minor change in the electrochemical signal, indicating that XNA on Gold can be used for electrochemical based bioanalysis. After cycling a SAMS electrode 50 times, no signs of deterioration were detected showing that coating has excellent stability. In addition to the biosensing applications, the scheme provides a non-invasive method for accessing the quality of the SAMS coatings which is of industrial interest. These studies show that the XNA on Gold microarray platform can be used for electrochemical studies, thus providing an additional alternative for developing multianalyte biosensors as well as expanding the range of detection methods available for microarray analysis.
DNA↗
Novel peptide surface for reversible immobilization of concanavalin A.
Concanavalin A (Con A) was spontaneously adsorbed on polymyxin B surface. This peptide-lectin interaction was strong, K(D)=1.9 x 10(-10), based predominantly on creation of hydrophobic bonds, and was completely reversible. Concanavalin A on polymyxin B (PmB) retained higher binding capacity for yeast mannan, compared with covalently immobilized lectin. Kinetics of mannan-concanavalin A interaction were significantly different in dependence on type of concanavalin A immobilization.
Adsorption↗
Mannan-penicillin G acylase neoglycoproteins and their potential applications in biotechnology.
Mannan-penicillin G acylase neoglycoproteins were prepared by the conjugation of Saccharomyces cerevisiae mannan with enzyme penicillin G acylase using the reductive amination method. Eight neoglycoproteins preparations were obtained after gel chromatography. The preparations contained from 42 to 67% (w/w) saccharides and their molar masses varied from 283 to over 1000 kDa. Significant biospecific interaction of separated fractions with the lectin concanavalin A was evaluated by the precipitation and sorption method (equilibrium constants) and further characterized using surface plasmon resonance to determine kinetic association and dissociation constants. K (D) was determined over the range 10(-7) M. High-molar-mass preparations appeared to be more suitable for preparation of stable and active complexes with concanavalin A for prospective use as a penicillin G acylase biocatalyst in enzyme reactors. The enzyme stability of such complexes was significantly increased compared with the original neoglycoprotein. Lower-molar-mass preparations were more suitable for applications such as biocatalysts in bioanalytical devices.
Adsorption↗
Optical lectin based biosensor as tool for bacteria identification.
Biosensor techniques are based on biospecific interaction between the biological parts of biosensor with the analyte. In biosensor construction, antibodies are usually used for the detection of analytes such as microorganism, because of very strong and highly specific interaction. The disadvantages of this assay are a long time needed for antibody isolation and purification as well as difficult regeneration of biosensor chip. The use of lectins instead of antibodies could solve these problems because a several hundred lectins are commercially available and their stability in standard buffers is better compared to monoclonal antibodies. While antibody can only be used to detect that antigen it was designed for, lectin as low affinity molecule may bind several different pathogens. Using the discriminative effect of an artificial neural network the application of a lectin array will compensate for the lower specificity. Microbial surfaces bear many of the sugar residues capable of interacting with lectins. The ability of lectins to react with microbial glycoconjugates means that it is possible to employ them as probes and sorbents for whole cells, mutants and numerous cellular constituents and metabolites, and it makes them useful tools for identification or typing of bacteria. Lectins are attractive reagents for the clinical diagnostic laboratory because of their diverse specificity, commercial availability, a wide range of molecular weights, and their stability in standard buffers. The construction of lectin biosensor could be an advantage method for detection of pathogenic bacteria.
Animals↗
Affinity analysis of lectin interaction with immobilized C- and O- gylcosides studied by surface plasmon resonance assay.
A biosensor based on the surface plasmon resonance (SPR) principle was used for kinetic analysis of lectin interactions with different immobilized saccharide structures. A novel affinity ligands beta-D-glycopyranosylmethylamines derived from common D-aldohexoses linked to the carboxymethyl dextran layer of the SPR sensor surface served for interactions with a wide range of lectins. The method of preparation and use of the beta-D-mannopyranosyl glycosylated sensor surface was described. The results of affinity analysis of lectin-ligand interactions were evaluated and compared with data obtained from measurements using commercially available p-aminophenyl alpha-D-glycopyranosides. Possible applications and advantages of C- and O-glycosylated SPR biosensors are discussed.
Biophysics↗
Gold-coated capillary based 2,4-dichlorophenoxyacetic acid chemi-luminescent assays: possibilities towards multianalysis.
The application of gold-coated glass capillaries for the design of a sensitive chemiluminescent immunoassay for 2,4-dichlorophenoxyacetic acid (2,4-D) is reported. The gold coating on the glass capillaries has been partially characterized and its effect on enhancing the signal intensity has been measured. A simple photo-multiplier tube-based photon detector is used for this purpose. At least three times improvement in the signal intensity is observed compared to uncoated glass capillaries, with a consequent improvement in the sensitivity of detection. Using such gold-coated glass capillaries, 2,4-D in the range 10(-9) to 10(-13) mol/l is detectable at a precision of +/-15% (CV%) and a limit of detection of 10(-15) mol/l is achievable. The possibility of using such gold-coated capillaries with a portable multianalytical set-up for field studies is also demonstrated.
2,4-Dichlorophenoxyacetic Acid↗
ELISA on a microchip with a photodiode for detection of amphetamine in plasma and urine.
A rapid and sensitive assay was developed for the detection of amphetamine in plasma and urine. The method relies on the principle of competitive ELISA (enzyme-linked immunosorbent assay). A flow microchip with a total volume of 7 microL was used for the development of a chemiluminescent ELISA technique. Solutions, samples, and the chemiluminescence substrate were injected by a flow system, and a photodiode detector was used to measure the light intensity. The incubation time of the competitor (competition phase) was reduced to 10 min. Calibration curves corresponding to analyte concentrations ranging from 40 to 1,000 microg/L in urine samples and from 6 to 96 microg/L in plasma samples were obtained. The detection limits were in the region of 20 and 6 microg/L in urine and plasma, respectively. The main focus of the work was on speed, reliability, reproducibility, and operational stability of the assay. This method was proven readily adaptable to automation and provided reproducible results.
Amphetamine↗