PubMed Health⌕ Search

Biomedical subjects

A A Stomakhin

Publications and source records attributed to A A Stomakhin.

5 recordsLinked to original sources
PubMed2005

Quantitative immunoassay of biotoxins on hydrogel-based protein microchips.

Three-dimensional gel-based microchips with immobilized proteins were used for quantitative immunoassay of a series of plant (ricin and viscumin) and bacterial (staphylococcal enterotoxin B, tetanus and diphtheria toxins, and lethal factor of anthrax) toxins. It was shown that different types of immunoassays (direct, competitive, and sandwich type) could be carried out on gel microchips. As shown by confocal microscope studies, antigen-antibody interactions involving the formation of tertiary antibody-antigen-antibody complex occur in the whole volume of microchip gel elements. Sandwich assay on microchips with immobilized antibodies provided the highest sensitivity of detection (0.1 ng/ml for ricin). Antibodies labeled with fluorescent dyes, horseradish peroxidase conjugates, or biotinylated antibodies with subsequent treatment with labeled avidin were used as developing antibodies. The results of immunoassays were recorded using fluorescence, chemiluminescence, or matrix-assisted laser desorption ionization mass spectrometry directly from microchip gel elements. Gel microchips with immobilized capture antibodies were used to analyze the sample simultaneously for the presence of all six biotoxins with the same sensitivity as that for any single toxin.

Animals↗
PubMed2003

Hydrogel-based protein microchips: manufacturing, properties, and applications.

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.

Equipment Design↗
PubMed2000

DNA sequence analysis by hybridization with oligonucleotide microchips: MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides.

Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has been applied to increase the informational output from DNA sequence analysis. It has been used to analyze DNA by hybridization with microarrays of gel-immobilized oligonucleotides extended with stacked 5mers. In model experiments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overlapping 8mers. Then, in a second round of hybridization DNA-8mer duplexes were hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 10-15 degrees C as a result of stacking interaction with 8mers. Contiguous 13 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA-8mer duplex formed on the microchip. Incorporating a mass label into 5mers optimized MALDI MS monitoring. This procedure enabled us to reconstitute the sequence of a model DNA fragment and identify polymorphic nucleotides. The application of MALDI MS identification of contiguously stacked 5mers to increase the length of DNA for sequence analysis is discussed.

Animals↗
PubMedDate not supplied

[Synthesis of conjugates of bovine serum albumin with water-soluble ytterbium porphyrins].

The ytterbium complex of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin was synthesized as an IR-fluorescent label and covalently bound to bovine serum albumin. The resulting conjugate fluoresces at 985 nm and is of interest for use in IR-fluorescent tumor diagnostic, immunoassay, and energy transfer studies. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

Fluorescent Dyes↗