Anatoli Dzgoev

An enzyme-linked molecularly imprinted sorbent assay

Based on a molecularly imprinted polymer, a competitive binding assay analogous to competitive enzyme immunoassay has been developed. The assay is specific for the herbicide 2,4-dichlorophenoxyacetic acid and uses, for the first time, an enzyme-labelled conjugate as a tracer. The label tobacco peroxidase allowed for colorimetric and chemiluminescence detection. The molecularly imprinted polymer was synthesised in the form of microspheres by precipitation polymerisation. The polymer efficiently and selectively bound the analyte in aqueous solution. Calibration curves were obtained corresponding to analyte concentrations ranging from 40–600 μg mL−1 for the colorimetric assay, and from 1–200 μg mL−1 for chemiluminescence assay.

ENANTIOSELECTIVE MOLECULARLY IMPRINTED POLYMER MEMBRANES

Molecular imprinting is a technology that allows for the creation of molecular recognition sites in synthetic polymers via the use of templates. Imprinted materials have been used in various applications, including stationary phases for chromatography, recognition elements in sensors, or antibody binding mimics in immunoassay-type analyses. In the present work, molecularly imprinted enantioselective polymer membranes are described. The amino acid derivative CBZ-tyrosine was chosen as the target compound. A CBZ-l-tyrosine-imprinted polymer was first tested in HPLC mode, and efficient resolution of racemic CBZ-tyrosine was obtained. Imprinted polymer membranes were prepared by casting the polymer in the pores of a polypropylene membrane, using a specifically adapted polymer recipe. In diffusion experiments across the imprinted membranes, enantioselectivity was observed with the diffusion of the l-enantiomer of CBZ-tyrosine (the original template) being faster than that of the d-enantiomer. Chirality 11:465–469, 1999.

Microformat imaging ELISA for pesticide determination.

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.

Surface plasmon resonance based pesticide assay on a renewable biosensing surface using the reversible concanavalin A monosaccharide interaction

A competitive immunoassay based on surface plasmon resonance (SPR) for the detection of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) is reported. The novelty of the assay is based on the regeneration of the chip surface by the reversible interaction between monosaccharide (D-glucose) and lectin (Concanavalin A). Concanavalin A-2,4-D conjugate was chemically synthesized, purified and used for binding to the SPR chip modified with covalently bound alpha-D-glucose. The interaction between anti-2,4-D antibody and the surface-bound concanavalin A-2,4-D conjugate was monitored by surface plasmon resonance and the response was used for the quantification of 2,4-D. The dynamic range of the calibration curve was between 3 and 100 ng/ml. The demonstrated principle of surface regeneration based on the reversible sugar-lectin interaction may be of more general applicability in immunoassays.

The development and applications of thermal biosensors for bioprocess monitoring

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.

Optimization of a charge coupled device imaging enzyme linked immuno sorbent assay and supports for the simultaneous determination of multiple 2,4-D samples

Abstract 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.

Biomaterials for molecular electronics development of optical biosensor for retinol.

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.

USE OF A CCD-BASED IMAGING DETECTOR AND CAPILLARY SAMPLiNG TECHNIQUES FOR ENVIRONMENTAL TESTING

A microformat ELISA has been developed for the simultaneous determination of multiple 2,4-dichlorophenoxyacetic acid (2,4-D) samples using a CCD-camera and highly effficient chemiluminescent enzyme substrates. The linear range was 4.5×10−10 – 4.5×10−7 M with a lower detection limit of 4.3×10−10 M, corresponding to 96 pg/ml or 192 pg/well with a CV of 12.5%. The use of gold coated silicon wafers and glass capillaries as solid phase supports in the imaging ELISA is also described. The highly reflective gold surfaces improved the linear range and the sensitivity as compared to thick-film patterned surfaces. Sampling in capillary tubes reduces the sensitivity and the linear range, but there are indications that the capillaries can act as light guides and thereby improve the light collection efficiency.