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Noncovalent molecular imprinting of a synthetic polymer with the herbicide 2,4-dichlorophenoxyacetic acid has been achieved in the presence of the polar solvents methanol and water. Formation of the prearranged complex relied on hydrophobic and ionic interactions between the template and the functional monomer 4-vinylpyridine. The polymer obtained binds the original template with an appreciable selectivity over structurally related compounds. The potential use of micrometer-sized imprinted polymer particles as the recognition element in a radioligand binding assay for 2,4-dichlorophenoxyacetic acid is demonstrated.

A chemiluminescent microformat enzyme linked immuno sorbent assay (ELISA) has been optimized for the simultaneous determination of multiple 2,4-dichlorophenoxyacetic acid (2,4-D) samples. The competitive immunoassay employed a 2,4-DBSA conjugate, anti-2,4-D monoclonal antibodies and alkaline phosphatase (AP) labelled anti-mouse IgG. The bound AP conjugate was determined by quantitating the chemiluminescence emission from the enzymatic decomposition of the luminogenic substrate, CSPD, by AP using a cooled charge coupled device (CCD) camera. The detection limit for the simultaneous determination of multiple samples was 4.3 × 10−10 M corresponding to 96 pg ml−1 or 192 fg well with a coefficient of variation (CV, %) of 12.5%. The linear range of the assay was 4.5 × 10−7–4.5 × 10−10M. The ability of gold coated silicon wafers and glass capillaries to serve as solid phase supports in the imaging ELISA was investigated. The highly reflective gold surfaces improved both the linear range and the sensitivity of the assay, as compared to thick-film patterned surfaces. The capillary supports, on the other hand, lead to a reduction in the linear range and the sensitivity of the assay, as compared to the thick-film patterned surfaces. Initial studies indicate that the capillaries guide the light and may provide a built-in mechanism for collecting the emitted light. Strategies for further development of support materials for imaging-based detectors will be discussed.

Effects of desmopressin (arginine vasopressin V2 receptor agonist) were studied for elucidation of the possible role of the kidneys in the regulation of blood hyaluronidase activity. Similar to vasopressin, intraperitoneal administration of desmopressin to Wistar rats increased blood hyaluronidase activity by 30%. Against the background of water load, nether desmopressin nor vasopressin increased blood hyaluronidase activity. The possibility of renal secretion of hyaluronidase into the blood as a mechanism of antidiuretic activity of vasopressin and its role in the regulation of water metabolism are discussed.

Effect of dehydration and arginine vasopressin treatment (Arg-VP Sigma, USA, 50 ng/100 g b.wt. ip.) on the blood plasma hyaluronidase activity in Wistar rats was studied. It was found that the pH optimum of the enzyme activity was in the range of 3.5-3.7 that is characteristic for the hyaluronidase type 1. Water deprivation for 1 day was followed by significant increase in the blood plasma hyaluronidase activity. The same response was observed under vasopressin treatment. The possible role of increased plasma hyaluronidase activity in the regulation of water balance is discussed.

A flat-well microformat competitive enzyme-linked immunosorbent chemiluminescent assay for the detection of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) is described. Thick-film technology was used to pattern a hydrophobic layer 100 μm thick onto glass microscope slides to form an array of 2 × 2 mm(2) squares. These flat wells were able to hold 2 μL of reagents, corresponding to a height of ∼500 μm, with minimal contamination risk. The hydrophobic ink used to pattern the surfaces allowed significantly larger volumes of samples to be applied when compared with surfaces patterned with nonhydrophobic inks. This reduced evaporation effects and permitted greater pipetting accuracy, thereby improving assay reproducibility. A competitive immunoassay was developed based on the ability of free 2,4-D hapten to inhibit binding of anti-2,4-D monoclonal antibodies to 2,4-D-bovine serum albumin conjugate adsorbed onto the glass support. The support was subsequently incubated with alkaline phosphatase (AP) labeled anti-mouse IgG. The amount of AP conjugate bound was determined by quantitating the chemiluminescent emission produced from the enzymatic breakdown of CSPD substrate by AP using a cooled CCD camera. The detection limit of the single-sample microformat assay was 2.7 × 10(-)(11) M, or 6 pg of 2,4-D. The linear ranges of the single-sample and multisample assays were 4.5 × 10(-)(8)-4.5 × 10(-)(11) and 4.5 × 10(-)(7)-1.66 × 10(-)(10) M, respectively. In comparison, the detection limits of a tube-based chemiluminescent assay using standard luminometer and of a colorimetric ELISA were 45 × 10(-)(11) and 9.9 × 10(-)(8) M, respectively. The ability to scale the thick-film-based microformat assay makes it an ideal candidate for the development of affinity arrays and high-throughput assay formats. Prospects for further improvements of this imaging ELISA strategy will be discussed.

Functionalized biosensing surfaces were developed for chemiluminescent immunoassay of pesticides. Two approaches to construct functionalized surfaces were tested: (i) pesticide is immobilized to the surface and interacts with a labeled antibody; (ii) antibody is immobilized and interacts with a labeled pesticide. As labels alkaline phosphatase and peroxidase were used with their corresponding substrates CSPD and luminol, respectively. Light produced by chemiluminescent substrate was detected by a thermoelectrically cooled CCD camera or a photomultiplier. The best detection limit 0.00001 ng/ml was obtained using antibodies immobilized to dextran-enhanced surface. Completely renewable surface was obtained using reversible lectin-monosaccharide interaction, one surface was used for 200 analyses without any loss of binding capacity. Most favorable stability and cost per analysis was achieved with molecularly imprinted polymer (MIP) instead of antibody. The functionalized biosensing surfaces were prepared to detect 2,4-dichlorophenoxyacetic (2,4-D) acid as a model pesticide. The developed concepts are, however, generally applicable to other pesticides and to other optical formats, e.g. optical fiber.

In this paper, a novel strategy for multicomponent analysis of two classes of pesticides such as triazines (atrazine and simazine) and phenoxyalkanoic acids (2,4-dichlorophenoxy acetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-chlorophenoxyacetic acid (CPOAc), phenoxyacetic acid (POAc)) employing immuno-arrays is demonstrated. The approach is based on cross-reactive arrays of specific antibody pairs coupled to chemometric pattern recognition. The monoclonal antibody pairs employed in this work (atrazine–simazine and 2,4-D) are specific towards a set of analytes and preclude a particular set of others present in the sample matrix. Antibody pairs of atrazine, simazine, and 2,4-D are used to discriminate and quantify analyte of interest. Atrazine was quantified in presence of trace concentration of simazine and that of 2,4-D. The combinatorial cross-reactivity of antibody pairs towards simazine, atrazine and 2,4-D is used to distinguish among different classes of analytes and their influence on the signal suppression in immuno-techniques. These sensors exclude recognition by carbamates such as carbaryl and carbofuran.

Results of treatment of 76 patients with gunshot wounds of the colon received during local military conflicts and peacetime are analyzed. The patients were hospitalized within 30-120 min since the injury moment. Clinical examination, primary surgical treatment and laparoscopy were the best diagnostic methods. This order of diagnostic methods permitted to reduce time of examination from 60-80 to 20-40 min. Resection of injured part of the colon over the limits of contusion zone with "extraabdominal partial anastomosis" creation, Hartman's or Lachey's operation were the optimal surgery. In injury of contramesenteric part of the colon it makes sense to create loop colostoma by original method. The number of intraabdominal inflammatory complications decreased from 57 to 6.9%, lethal outcomes--from 17.6 to 6.9%.

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.

Molecular electronics involves expertise from several branches of science. Various biomaterials and electronics are involved in the fabrication of such devices. While passive biomaterials are involved in anchoring the active biomolecules, the latter are involved in switching and/or signal transduction. In the present investigation we have used a glass-capillary-based approach to design a biosensor for retinol. The sensing element is retinol-binding protein (RBP). The affinity of retinoic-acid-horseradish peroxidase (conjugate) to RBP is tested using a surface plasmon resonance technique. A simple photomultiplier-tube-based system is exploited to monitor the chemiluminescent signal generated upon reaction of hydrogen peroxide and luminol with the conjugate bound to RBP. The photomultiplier tube is directly coupled to a computer for data logging.

Enzyme thermistors are biosensors that use thermal resistors to measure the heat change caused by an enzymatic reaction. They combine the selectivity of enzymes with the sensitivity of biosensors and allow continuous analysis in a flow-injection mode. They can be used to monitor fermentation systems, biocatalysis, enzyme-catalysed synthesis and clinical and food technology. This article gives an overview of the general principles of enzyme thermistors, the sampling process and the ongoing developments in the field of bioprocess monitoring.

Specific vasopressin-sensitive phosphorylation of the collecting duct proteins with molecular mass 15, 20, 25, and 33 kDa, was revealed. The proteins implemented the hormonal effect of vasopressin by means of qualitative molecular modifications with subsequent phosphorylation.

Monoclonal antibodies to phenobarbital have been developed. Spleen cells of BALB/c mouse immunized with conjugate keyhole limpet haemocyanin and phenobarbital were fused with P3-X63-Ag8.653 mouse myeloma cells. Three monoclonal antibodies, selected by indirect ELISA, were produced in mouse ascite fluids, purified and analyzed. Antibody 1A9 was selected for use in immunoassay, its association constant being 1.6 x 10(9) M-1.

The influence of the immunogen's tracer's structure on properties of antibodies to phenobarbital was studied by the polarisation fluoroimmunoassay method. It was shown that in homogeneous method of polarisation fluoroimmunoassay the titer of antibodies to phenobarbital (1/1000-1/10,000) is affected by the position of linker binding the antigen with carrier-protein and by the structure of the linker. The affinity constant ((1-4) x 10(9) M-1), cross-reactivity, limit of the detection (0.6-2.6 micrograms/ml) phenobarbital with different combinations of antibodies and tracers were calculated. The antisera grew the more heterogeneous in affinity and specificity, the longer became the linker. The more heterogeneous antibodies are preferable in the class specific assay of barbiturates.

Endogenous phosphorylation of proteins in cell suspensions of collecting tubes was studied. Using SDS disc electrophoresis in polyacrylamide gel with subsequent autoradiography, it was shown that vasopressin increases the 32P incorporation into two proteins with molecular masses of 15 kDa and 33 kDa, which serve as endogenous substrates for cAMP-dependent protein kinase. The hormone-dependent phosphorylation of these proteins was typical of the membrane fraction of collecting tube cells but was absent in the cytosolic fraction. The results obtained are suggestive of the direct involvement of vasopressin in the regulation of membrane protein phosphorylation by cAMP-dependent protein kinase which may increase the permeability of cells for H2O.

Using SDS-PAAG electrophoresis with subsequent autoradiography, several proteins from plasma membranes and cell cytosol of rat kidney papillary zone were identified as substrates for endogenous cAMP-dependent protein kinases. The cAMP-dependent phosphorylation of plasma membrane proteins was made possible only after the destruction of membrane vesicles. Plasma membrane and cytosol fractions were found to contain a 58 kDa protein whose properties are similar to those of the regulatory subunit of cAMP-dependent protein kinase of the second type. It was shown also that the content of endogenous substrates of cAMP-dependent protein kinases in cell cytosol is higher than that in plasma membranes.

The article analyses the results of operations for creating end-to-side extra-intracranial anastomoses between the branches of the external carotid artery and cortical branches of the middle cerebral artery in 62 patients with occlusion of the cerebral vessels and in 3 patients with giant aneurysms of the supraclinoid part of the internal carotid arteries.

The aldosterone, cAMP and ADH receptors were studied in kidneys of 10-14- and 60-day old rats. Concentrations of aldosterone and cAMP receptors were reduced, while ADH binding was increased during the period of maturation of kidney functions. The investigation of the affinity and molecular weights of cAMP and ADH receptors suggests their complex nature and altering of their individual properties during ontogenesis. Aldosterone and cAMP with their receptors seem to participate in the regulation of gene activity in ontogenesis and ADH seems to participate in the formation of its own receptors.

The application of a newly isolated transgenic tobacco peroxidase (TOP) as a chemiluminescent label for immunoassay purposes is described for the first time. The enzyme has been oxidized with m-periodate and subsequently coupled to the model compound 2,4-dichlorophenoxyacetic acid (2,4-D) using a carbodiimide method. As compared to the native horseradish peroxidase used in control experiments, the TOP enzyme showed significantly higher efficiency of coupling to the antigen and no loss of the specific activity was observed. The obtained 2,4-D-TOP conjugate demonstrated unique properties in chemiluminescent detection. The latter allowed the minimization of the conjugate concentration due to the superior chemiluminescent activity of the enzyme. A highly sensitive capillary chemiluminescent immunoassay using the 2,4-D-TOP conjugate as labeled competitor is reported. Direct competitive ELISA has been performed using a specific monoclonal antibody immobilized onto the sol-gel treated glass capillary surface. A modified photomultiplier tube with a special holder for a capillary was used for the resulting chemiluminescent signal detection. The typical standard calibration curve for the 2,4-D pesticide detection is linear between 30 pg and 500 ng/mL.