A. S. Zasedatelev

Diagnostic Microarrays in Hematologic Oncology

Microarrays have become important tools for high-throughput analysis of gene expression, chromosome aberrations, and gene mutations in cancer cells. In addition to high-density experimental microarrays, low-density, gel-based biochip technology represents a versatile platform for translation of research into clinical practice. Gel-based microarrays (biochips) consist of nanoliter gel drops on a hydrophobic surface with different immobilized biopolymers (primarily nucleic acids and proteins). Because of the high immobilization capacity of the gel, such biochips have a high probe concentration and high levels of fluorescence signals after hybridization, which allow the use of simple, portable detection systems. The notable accuracy of the analysis is reached as a result of the high level of discrimination between positive and negative gel-bound probes. Different applications of biochips in the field of hematologic oncology include analysis of chromosomal translocations in leukemias, diagnostics of T-cell lymphomas, and pharmacogenetics.

Evaluation of a low-density hydrogel microarray technique for mycobacterial species identification

ABSTRACT In addition to the obligatory pathogenic species of the Mycobacterium tuberculosis complex and Mycobacterium leprae, the genus Mycobacterium also includes conditionally pathogenic species that in rare cases can lead to the development of nontuberculous mycobacterial diseases. Because tuberculosis and mycobacteriosis have similar clinical signs, the accurate identification of the causative agent in a clinical microbiology laboratory is important for diagnostic verification and appropriate treatment. This report describes a low-density hydrogel-based microarray containing oligonucleotide probes based on the species-specific sequences of the gyrB gene fragment for mycobacterial species identification. The procedure included the amplification of a 352-nucleotide fragment of the gene and its hybridization on a microarray. The triple-species-specific probe design and the algorithm for hybridization profile recognition based on the calculation of Pearson correlation coefficients, followed by the construction of a profile database, allowed for the reliable and accurate identification of mycobacterial species, including mixed-DNA samples. The assay was used to evaluate 543 clinical isolates from two regions of Russia, demonstrating its ability to detect 35 mycobacterial species, with 99.8% sensitivity and 100% specificity when using gyrB, 16S, and internal transcribed spacer (ITS) fragment sequencing as the standard. The testing of clinical samples showed that the sensitivity of the assay was 89% to 95% for smear-positive samples and 36% for smear-negative samples. The large number of identified species, the high level of sensitivity, the ability to detect mycobacteria in clinical samples, and the up-to-date profile database make the assay suitable for use in routine laboratory practice.

The effect of chromophore charge on the incorporation efficiency of fluorescence-labeled nucleotides catalyzed by Taq DNA polymerase in matrix synthesis

The effect of the chromophore charge on the efficiency of incorporation of fluorescent-labeled nucleotides into DNA during PCR was studied using three dUTP derivatives that contain different fluorescent labels, i.e., electroneutral, positively charged, and negatively charged Cy5 analogs. dUTP labeled with an electroneutral Cy5 analog was shown to be most efficiently incorporated into DNA when Tag polymerase was used in PCR.

Immunoassay of nine serological tumor markers on a hydrogel-based microchip

A prototype test-system for simultaneous quantitative assay of nine tumor markers in blood serum was developed. The main constituent of the test-system is an OM-9 biochip containing immobilized antibodies against nine oncomarkers: α-fetoprotein (AFP), carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), cancer antigen 15-3 (CA 15-3), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), total and free forms of prostate-specific antigen (PSAtot and PSAfree), and neuron-specific enolase (NSE). The biochip-based two-step sandwich immunoassay procedure for carrying out simultaneous quantitative determination of nine tumor markers in patients’ blood serum was proposed. The main analytical characteristics of the method were obtained. The results suggest that the prototype of the test-system could be a promising instrument for clinical application. The test-system prototype was tested using blood serum samples of oncological patients (252 samples) and healthy donors (185 samples). Increased concentrations of one or more tumor markers above the normal level were found in 76.6% cases of oncological patients and only in 6% cases of healthy donors. For colorectal cancer patients, application of modern statistical methods of data processing in medical research, i.e., receiver operating characteristics analysis (ROC curve) and logistic regression, indicated that the simultaneous assay of nine markers on biochips showed much more diagnostic significance (area under the ROC curve, AUC, was 0.84) than a traditional assay of two tumor markers, CEA and CA 19-9 (AUC = 0.59). The developed biochip-based test-system can be recommended for both the estimation of people’s health, e.g., for standard medical examination, and tracking the tumoral process in the postsurgical period or after specific tumor treatment.

Method for automated extraction and purification of nucleic acids and its implementation in microfluidic system

A method and a microfluidic device for automated extraction and purification of nucleic acids from biological samples have been developed. The method involves disruption of bacterial cells and/or viral particles by combining enzymatic and chemical lysis procedures followed by solid-phase sorbent extraction and purification of nucleic acids. The procedure is carried out in an automated mode in a microfluidic module isolated from the outside environment, which minimizes contact of the researcher with potentially infectious samples and, consequently, decreases the risk of laboratory-acquired infections. The module includes reservoirs with lyophilized components for lysis and washing buffers; a microcolumn with a solid-phase sorbent; reservoirs containing water, ethanol, and water-ethanol mixtures for dissolving freeze-dried buffer components, washing the microcolumn, and eluting of nucleic acids; and microchannels and valves needed for directing fluids inside the module. The microfluidic module is placed into the control unit that delivers pressure, executes heating, mixing of reagents, and movement of solutions within the microfluidic module. The microfluidic system performs extraction and purification of nucleic acids with high efficiency in 40 min, and nucleic acids extracted can be directly used in PCR reaction and microarray assays.

Microfluidic module for automated isolation and purification of nucleic acids from biological samples

291 A device for isolation and purification of nucleic acids from biological specimens using a microfluidic module comprising vessels with reagents, microchan nels, and valves, which are required for movement of solutions and reaction mixtures within the module, was developed. The procedure is automated and com pletely excludes the contact of a researcher and a potentially pathogenic specimen. The obtained nucleic acid preparation can be immediately used in molecular genetic analysis.

New indodicarbocyanine dyes for the biological microchip technology

New indodicarbocyanine dyes with the carboxybutyl group in position-3 of the indolenine fragment bearing methyl and sulfonic groups in positions 5 and 7 of the cycle were synthesized in order to find the most effective fluorescent labels for the biological microchip technology. The position of absorption and fluorescence maxima, the total charge of the dye molecule, and water solubility depend on the location and the total amount of methyl and sulfonic groups. The spectral characteristics of the dyes synthesized were determined. The relative fluorescence efficiencies of the dyes at equal concentrations were measured at excitation wavelengths of 635 and 655 nm and emission wavelengths of 670 and 690 nm, respectively.

Multiplex assay of allergen-specific and total immunoglobulins of E and G classes in the biochip format

289 Allergy is an acute immune response of the body to a normally harmless substance, allergen, which devel ops under a repeated exposure. Allergic reactions are mediated primarily by immunoglobulins of classes E (IgE) and G (IgG). IgE is responsible for the mani festation of the immediate type allergy (type I, the so called IgE mediated hypersensitivity of the body); as much as 20–50% of the population of developed countries is prone to it. In addition to determining the levels of specific IgE (sIgE), for differential diagnosis of food allergy and food intolerance, which can be the cause of many chronic diseases, it is also necessary to determine the levels of specific IgG4 (sIgG4) to numerous food allergens. In addition, the determina tion of the level of sIgG4 and the IgG4/IgE ratio is important to monitor immunotherapy. The existing in vitro allergy diagnostic methods are aimed at semiquantitative and quantitative determina tion of sIgE and sIgG4 in patient’s blood serum. The majority of methods make it possible to determine the level of only sIgE or sIgG4 in a single assay. Modern diagnosis of allergic diseases requires par allel (multiplex), highly sensitive analysis that allows simultaneous determination of different sIgE and sIgG4 in a single assay. A promising tool for developing such methods are biological microchips (biochips)— arrays of cells containing a large set of immobilized allergens and allowing determination of a variety of different immunoglobulins in a single experiment using a minimal number of samples. The ImmunoCAP ISAC microchip (Thermo Fi scher) includes 103 natural and recombinant aller gens, which are immobilized on a substrate in the form of spots (two dimensional microchip). This micro chip makes it possible to determine the levels of sIgE in a sample and classify them in accordance with the four classes of allergic reactions (negative, low, medium, and high). This chip, however, cannot be used to analyze sIgG4. In this paper, we propose a procedure for a simulta neous determination of 32 different sIgE and sIgG4 in a single assay on a hydrogel based biochip (three dimensional microchip). Biochips are produced by the polymerization immobilization technology devel oped at the Institute of Molecular Biology, Russian Academy of Sciences [1, 2]. This technology is based on the photoinduced copolymerization of molecular probes (proteins, oligonucleotides, or glycans) with gel forming monomers during the formation of hydrogel cells of the biochip. The molecular probes immobilized in hemispherical gel cells (microdrop lets) are uniformly distributed throughout the volume of the drop; they are sufficiently distant from one another (1000 Å) and do not come into contact with the hydrophobic substrate. This is especially impor tant for immobilization of protein probes, which can undergo partial or complete denaturation as a result of such contacts. In addition, the use of three dimen sional gels raises their capacity, increasing the possible amount of the immobilized protein, which leads to an increase in the sensitivity of the assay [1–5]. Biochip structure. A biochip for a simultaneous determination of 32 sIgE and sIgG4 in blood serum contains different classes of allergens (including recombinant) immobilized in hydrogel elements: (1) the class of tree pollen allergens (t2, t3, bet v11, and bet v21; (2) the class of weeds and flowers (w1, w6, and w5); (3) the class of meadow grass allergens (g3, g4, g5, g6, g12, phl p11, phl p51, phl p71, and phl p121); (4) the class of fungal allergens, molds and yeasts (m5 and m6); (5) the class of epidermal allergens (e1 and e5); (6) the class of household allergens, mites (d1 and d2);

Cyanine-dye-modified 2′-deoxyuridine-5′-triphosphates: Synthesis, applications, and linker effect on substrate properties for Taq DNA polymerase

A number of fluorescently labeled nucleoside triphosphates containing electroneutral indodicarbocyanine dye (Cy) have been synthesized. The dye has been attached to the C5 position of pyrimidine through the transalkene linkers of different structure. The synthesized labeled nucleoside triphosphates have been tested as substrates for Taq polymerase in PCR using the “TB-biochip” test system.

Structural and functional analysis of biopolymers and their complexes: Enzymatic synthesis of high-modified DNA

Here, we describe the synthesis and purification of six deoxyuridine triphosphate derivatives that contain protein-like functional groups and alkene linkers of various lengths. Using KOD XL and Deep Vent polymerases, these derivatives have been incorporated into single-stranded DNA, achieving a high degree of DNA modification. These polymerases are able to utilize highly modified DNA strands as templates for synthesizing unmodified DNA. The synthesized deoxyuridine triphosphate derivatives are promising as substrates for producing modified aptamers to various target proteins using, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) methodology.