Loop-mediated isothermal amplification (LAMP) is the method of DNA amplification which has a high potential for "on-site" testing, with peculiar amplicons which consist of stem-loop inverted DNA repeats varying in number and resembling cauliflower-like structures (LAMPlicons). Here, the electrochemical approach to detect LAMP products was developed. The approach is based on a combination of LAMP, 2 '-deoxyuridine-5 '-triphosphate modified with tyrosine aromatic group (dUTP-Y1), and direct voltammetric detection of tyrosine labeled LAMPlicons (dsDNA-Y1). The practicality of the developed approach was demonstrated on the example of plant pathogen Clavibacter sepedonicus, causing ring rot disease of potato. The optimal yield of dsDNA-Y1 was at 60 % substitution of dTTP with dUTP-Y1 in the LAMP reaction mixture with primers targeting a section of C. sepedonicus genome with a unique sequence. The square wave voltammetry allowed us to reliably detect purified dsDNA-Y1 at the potential of about 0.6 V by depositing a few microliters sample on a surface of disposable carbon screen printed electrode. The limit of voltammetric detection of dsDNA-Y1 was estimated as 0.05 g L- 1. The optimized procedure provided the detection of dsDNA-Y1 produced in a single LAMP reaction and purified with silica coated iron oxide magnetic beads. The overall sensitivity of C. sepedonicus detection by the established method can be as low as 10 copies of bacterial genome per amplification reaction or 130 colonyforming units (CFU) per 1 g of potato tissue (about 8 CFU per reaction). In total, amplification, purification, and electrochemical measurements take about 3.5-4 h. The accuracy, speed, diminutiveness, low-cost, and lowpower requirements of voltammetric analysis provide basis for the development of portable devices which would integrate LAMP with electroactive modified nucleotides, magnetic bead DNA purification, and direct voltammetric detection of modified DNA for the purpose of "on-site" pathogen detection.

Objective: The biochip method allows microscale multiparametric analysis of macromolecular samples using a matrix of immobilized molecular probes. Selection of materials for biochip fabrication, functionalization of the carrier surface, and the method of immobilization of molecular probes are the key tasks of biochip technology. Methods: Methods of obtaining polymer coating from polyvinyl acetate on the surface of polyethylene terephthalate polymer substrates and subsequent production of brush polymers by photoinduced radical copolymerization of acrylate monomers have been studied. Cell matrices with numerous reactive chemical groups for subsequent immobilization of proteins were formed by photolithography method. Methods of activation of carboxyl groups on brush polymers attached to the surface of polyethylene terephthalate were tested. Immobilization of model protein streptavidin labeled with fluorescent dye Su3 was performed to test the method of activation of carboxyl groups. Results and Discussion: A variant of the immunofluorescence assay in a biological microarray format was tested on the model “streptavidin–biotinylated immunoglobulin.” Conclusions: Streptavidin immobilized in brush polymer cells retains functionality and spatial accessibility for binding to biotinylated immunoglobulin and subsequent manifestation by antibodies fluorescently labeled with Cy5 dye, which opens prospects for the use of biological microarrays with brush polymer cells on polyethylene terephthalate substrates for immunofluorescence analysis of various protein targets.

Three novel 2 '-deoxyuridine-5 '-triphosphates modified with 4-nitrophenyl groups via various linkers (dUTP-N1, dUTP-N2, and dUTP-N3) were tested as bearers of reducible electroactive labels as well as substrates suitable for enzymes used in polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) with a potential application to direct electrochemical detection of double-stranded deoxyribonucleic acid (dsDNA). In cyclic and square wave voltammograms on carbon screen printed electrodes, the labeled dUTP have demonstrated distinct reduction peaks at potentials of -0.7 V to -0.9 V (phosphate buffer, pH 7.4). The reduction peak currents of dUTP-N derivatives were found to increase with their molar concentrations. The dUTP-N3 with a double bond in the linker had the lowest reduction potential (about 100 mV less negative) among the derivatives studied. Further, dUTP-N nucleotides were tested as substrates in PCR and RPA to incorporate the electroactive labels into 90, 210, or 206 base pair long dsDNA amplicons. However, only a dUTP-N1 derivative with a shorter linker without the double bond demonstrated satisfactory compatibility with both PCR and RPA, though with a low reaction output of modified dsDNA amplicons (at 100% substitution of dTTP). The dsDNA amplicons produced by PCR with 85% substitution of dTTP by the dUTP-N1 in the reaction mixture were successfully detected by square wave voltammetry at micromolar concentrations at high square wave frequency.

A method has been developed for manufacturing biological microchips on an aluminum substrate with hydrophilic cells from brush copolymers with the formation of a matrix of cells using photolithography. The surface of aluminum substrates was previously coated with a thin, durable, moderately hydrophobic layer of cross-linked polymer to prevent contact with the aluminum surface of the components used in the analysis of nucleic acids. Aluminum biochip substrates have high thermal conductivity and low heat capacity, which is important for the development of methods for multiplex PCR analysis on a chip. Oligonucleotide probes were covalently immobilized in the cells of the biochip. The preservation of the hybridization activity of the immobilized DNA probes was demonstrated in a hybridization analysis with a synthetic DNA target representing a section of the sequence of the seventh exon of the human ABO gene. The methods developed can be used in the development of a technology for parallel multiple rapid microanalysis of nucleic acids “lab on a chip” for the detection of human somatic and infectious diseases.

A general approach is presented for synthesizing alkyne-modified nucleoside triphosphates via the Sonogashira cross-coupling reaction of unprotected halogenated 2ʹ-deoxynucleoside, followed by monophosphorylation and the reaction of the corresponding phosphoromorpholidate with tributylammonium pyrophosphate. A highly efficient approach for the milligram-scale synthesis of base-modified nucleoside triphosphates with an amino acid-like side chain was developed. The present chemical method outweighs the other reported methods of a base-modified nucleoside triphosphates synthesis in terms of it being a protection-free strategy, the shortening of reaction steps, and increased yields (about 70%). The resulting 8-alkynylated dATP was tested as a substrate for DNA polymerases in a primer extension reaction.

The development of rapid analysis of human serum for the presence of allergen-specific Immunoglobulin E (IgE) is currently important. Consequently, we developed two types of three-dimensional (3D) protein biochips. The first one is a 3D hydrogel biochip containing hydrogel droplets with protein molecules (allergens, immunoglobulins and others). These droplets are disposed on elements consisting of short polymer brushes grafting from a surface of polybutylene terephthalate polymer. The immobilization of proteins was induced by short-wave ultraviolet (UV) radiation. On such a biochip, the kinetics of allergen-sIgE complex formation reached 60% of saturation for 6 h. Also, we developed a 3D brush microchip containing on the surface of a polyethylene terephthalate polymer the brush elements with protein molecules covalently immobilized by opening oxirane cycles by amino and thiol nucleophilic groups contained in proteins. In the case of the 3D brush microchip, the kinetics of allergen-sIgE complex formation reached 100% of saturation for 3 h, and fluorescent signals were 2-3 times higher than those of the 3D hydrogel biochip for some allergens. Thus, the comparative analysis revealed that 3D brush biochips are more useful for further studies of protein-protein interaction than 3D hydrogel ones.

The methods of obtaining a polymer coating from polyvinyl acetate on the surface of polyethylene terephthalate polymer substrates and subsequent production by photoinduced radical copolymerization of acrylate monomers of brush polymers have been studied. Cell matrices with numerous reactive chemical groups were formed by photolithography for subsequent immobilization of proteins. Methods of activation of carboxyl groups on brush polymers attached to the surface of polyethylene terephthalate have been tested. Immobilization of the streptavidin model protein labeled with fluorescent dye Cy3 was performed to test the activation method of carboxyl groups. A variant of immunofluorescence analysis in the format of a biological microchip was tested on the streptavidin – biotinylated immunoglobulin model. Streptavidin, immobilized in brush polymer cells, retains functionality and spatial accessibility for binding to biotinylated immunoglobulin and subsequent manifestation by antibodies fluorescently labeled with Cy5 dye, which opens up prospects for the use of biological microchips with brush polymer cells on polyethylene terephthalate substrates for immunofluorescence analysis of various protein targets.

Objective: Polyethylene terephthalate (PET) is thermally stable, biocompatible, transparent in visible and near-infrared light. The study of grafting conditions and the distribution of reactive amino groups on the PET surface without affecting the polymer array makes it possible to change the surface properties in a directed manner. Methods: A method for obtaining active amino groups on the surface of polyethylene terephthalate (PET) substrate by reaction with ethylenediamine was developed. A method for quantitative estimation of the concentration and distribution of chemically accessible amino groups on the surface of PET substrate using cyanine dye Cy5 and digital fluorescence microscopy was developed. Results and Discussion: The PET surface during chemical modification remains without visible damage up to the concentration of amino groups 8 pmol/cm2, while surface degradation is observed at higher concentrations. Chemically available amino groups capable of covalently binding to Cy5 dye are distributed unevenly, which is probably due to the presence of amorphous and crystalline areas on the surface of PET substrates. Amino groups can be used for further chemical modification of the PET surface, grafting of various functional groups, and covalent binding to biomolecules, which opens up prospects for the wide use of inexpensive PET as functional substrates in biochips, biosensors, lab-on-a-chip devices, and other biotechnological applications.

Objective: The introduction of fluorescently labeled nucleotides in the process of DNA amplification in molecular genetic analysis is a very attractive alternative to post-labeling or the use of labeled primers in the reaction (RCA). Methods: To study the substrate efficiency, the kinetic index (amplification efficiency) was studied, which allowed us to evaluate the inhibitory effect of modified substrates; as well as the yield of the isothermal amplification reaction product and the density of embedding the label into the growing DNA chain. Results and Discussion: Two pairs of Cy5-labeled dU and dC triphosphates with similar electroneutral fluorophore structures, differing in the length of the hydrocarbon linker between the fluorophore and the nitrogenous base were synthesized. A comparative analysis of their substrate behavior in the rolling circle amplification (RCA) reaction using Bst 3.0 DNA polymerase was carried out. It was found that nucleotides with a long linker between the fluorophore and the pyrimidine base are more efficiently incorporated into the growing DNA chain, while the nucleotides with a short linker cause lesser inhibition of RCA. In each of the pairs consisting of dU and dC with similar fluorophores and linkers, the fluorescently labeled uridine derivatives demonstrated a higher incorporation density. It was revealed that, under simultaneous incorporation of the labeled dU and dC, the inhibitory effects do not sum up. This gives grounds for a more careful study of different Cy5-dC variants with a view to increasing the sensitivity of the analysis under simultaneous introduction of the labeled dU and dC. Conclusions: The work presents the results of comparative study of the substrate properties of fluorescently labeled deoxynucleoside triphosphates in RCA. The influence of the chemical nature of the nucleotide (dU or dC) and the structure of the fluorophore on the substrate properties of modified deoxynucleoside triphosphates has been studied. Deoxyuridines with a long linker between the fluorophore and the nitrogenous base were found to be the most effective substrates. In addition, the possibility of simultaneous use of labeled dU and dC in the process of isothermal amplification has been shown, which is potentially capable of increasing the sensitivity of the analysis method.

The genus Trichoderma comprised important antagonists of pathogenic fungi and can be used in agriculture to combat various plant diseases. In the course of the present work, two micromycete strains were isolated from wood cuts in Eastern Siberia, which were identified by morphological and molecular genetic characteristics as Trichoderma atroviride and Trichoderma harzianum. These Trichoderma strains efficiently inhibited the development of the pathogenic fungi studied (by up to 80%). The data presented in the paper indicate that Trichoderma atroviride and Trichoderma harzianum may be promising for further study of the means of biocontrol of plant diseases.

The effective incorporation of electroactive labeled nucleotides into DNA during its enzymatic amplification provides the ground for developing assays based on direct electrochemical detection of amplification products. Yet, whether the template sequence can affect the incorporation efficiency of modified nucleotides is still poorly understood. Here we examined the effects of a poly(dA) stretch in template sequence on the rate and yield of polymerase chain reaction (PCR) in the presence of modified 2 '-deoxyuridine-5 '-triphosphates (dUTP) in the reaction mixture. Four dUTP derivatives carrying 4-hydroxyphenyl (tyrosine; dUTP-Y1 and dUTP-Y2), 4-nitrophenyl (dUTP-N1), or indolyl (tryptophan; dUTP-W1) aromatic groups were tested as substrates for Taq and KTN (lacking 3 '-5 ' exonuclease activity) polymerases at 100 % substitution of 2 '-deoxythymidine-5 '-triphosphate (dTTP) in the PCR mixture. The 120 base pair long synthetic DNA templates contained poly(dA)& sdot;poly(dT) tracts of various length (dAn & sdot;dTn, where n = 2-10). In all cases, full-size amplicons have been obtained. Overall, in the presence of modified dUTP, the PCR yield was higher with KTN polymerase than that with Taq polymerase. The PCR yield slightly (for dUTP-Y1 and dUTP-Y2) or significantly (for dUTP-N1 and dUTP-W1) decreased with an increase of n in the dAn & sdot;dTn tract. In most cases, in the presence of modified dUTP, the PCR rate substantially decreased with n >= 4 in dAn & sdot;dTn. No changes in amplicon sequences due to potential incorrect nucleotide pairing during amplification with the complete substitution of dTTP with dUTP-Y2 were revealed. Electrochemical activity of tyrosine labeled PCR products dsDNA-Y2 differing in the length of stretches of modified nucleotides was similar. The findings demonstrate that the incorporation efficiency for base-modified nucleotides can in general depend on template sequence, in particular on the presence of long poly(dA) stretches when the modified dUTP are used. That has to be taken into account when developing electrochemical or other DNA assays with a specific modified nucleotide.

The approach based on a combination of isothermal recombinase polymerase amplification (RPA), 2'-deoxyuridine-5'-triphosphate modified with tyrosine aromatic group (dUTP-Y1), and direct voltammetric detection of RPA product carrying electroactive labels was successfully applied to the potato pathogen Dickeya solani. The artificial nucleotide dUTP-Y1 demonstrated a good compatibility with RPA, enabling by targeting a section of D. solani genome with a unique sequence to produce the full-size modified products at high levels of substitution of dTTP by dUTP-Y1 (up to 80-90 %) in the reaction mixture. The optimized procedure of square wave voltammetry allowed to reliably detect the product generated by RPA at 80 % substitution of dTTP by dUTP-Y1 (dsDNA-Y1) in microliter sample volumes on the surface of disposable carbon screen printed electrodes at the potential of about 0.6 V. The calibration curve for the amplicon detection was linear in coordinates 'Ip, A vs. Log (c, M)' within the 0.05-1 μM concentration range. The limit of detection for dsDNA-Y1 was estimated as 8 nM. The sensitivity of the established electrochemical approach allowed to detect amplicons generated in a single standard 50 μL RPA reaction after their purification with silica-coated magnetic beads. The overall detectability of D. solani with the suggested combination of RPA and voltammetric registration of dsDNA-Y1 can be as low as a few copies of bacterial genome per standard reaction. In total, amplification, purification, and electrochemical detection take about 120-150 min. Considering the potential of direct electrochemical analysis for miniaturization, as well as compliance with low-cost and low-power requirements, the findings provide grounds for future development of microfluidic devices integrating isothermal amplification, amplicon purification and detection based on the tyrosine modified nucleotide for the purpose of 'on-site' detection of various pathogens.

The abundance, biomass, size-morphological structure, growth rate and production of bacterioplankton, the intensity of primary phytoplankton production and dark fixation of CO2, as well as the abundance and biomass of heterotrophic nanoflagellates were determined in a large plain eutrophic reservoir (Gorky Reservoir, Middle Volga). The abundance, biomass, and production of bacterioplankton were relatively high and averaged 7.6 × 106 cells/mL, 117.9 mg C/m3, and 59.2 mg C/(m3 × day), respectively. Heterotrophic nanoflagellates reached a high level of quantitative development – 6.9 × 103 cells/mL, 47.9 mg C/m3. Their biomass averaged 41.6 ± 18.4% of the bacterioplankton biomass, which indicates that, in addition to bacteria, nanoflagellates used other food sources. Small rods and cocci dominated among the size-morphological bacterioplankton groups and accounted for 36.3 and 33.3% of its total abundance, respectively. Small rods averaged more than a half (56.2%) of the total biomass and were the most stable component of the community. The growth rate and production of bacterioplankton increased in those parts of the reservoir where the medium-sized cocci and coccobacilli accounted for from 18.2 to 29.3% of the total abundance. The highest bacterial activity was recorded in the area affected by the warm waste waters of the Volgorechensk State District Power Plant. Based on the data obtained, the reservoir divided on the upper river section and the lower lake section.

Бактерии выполняют важные и разнообразные функции в экосистемах крупных рек. В среднем и нижнем течении р. Оби в районах городов и населенных пунктов определяли уровень количественного развития и изучали характер пространственного распределения гетеротрофного бактериопланктона и его размерно-морфологических групп. Вода реки характеризовалась высоким содержанием взвешенных частиц (в среднем (2,22 ± 0,21) × 106 мл-1), к которым была прикреплена значительная часть бактерий: в среднем 41,5 % их общей численности и 35,7 % биомассы. Установлено, что концентрация взвешенных частиц и электропроводность воды находятся среди главных факторов, влияющих на количественное развитие и структуру бактериопланктона. Существенное возрастание его количества, в основном за счет мелких одиночных клеток, зарегистрировано ниже впадения в р. Обь ее крупнейшего притока - Иртыша, и расположенного на его берегах г. Ханты-Мансийска. На этом участке Оби средние значения численности и биомассы бактериопланктона составляли (6,87 ± 4,99) × 106 кл/мл и 142 ± 13 мг С/м3 соответственно, что в 1,6-1,7 раза выше, чем в зонах влияния других городов. Пространственное распределение гетеротрофного бактериопланктона в р. Оби определяется главным образом динамикой водных масс, а также влиянием притоков и городов Bacteria perform important and diverse functions in the ecosystems of large rivers. The abundance, biomass and spatial distribution of heterotrophic bacterioplankton and its size-morphological groups were studied in the middle and lower reaches of the Ob River in the areas impacted by cities. The river water was characterized by a high content of suspended particles (on average (2.22 ± 0.21) × 106 ml-1), to which a significant part of bacteria was attached: on average, 41.5 % of their total number and 35.7 % of their biomass. The concentration of suspended particles and water conductivity were found to be among the main factors affecting the abundance and structure of bacterioplankton. A significant increase in bacterial abundance, mainly due to small free-living bacteria, was recorded below the mouth of the largest tributary, the Irtysh River, and the city of Khanty-Mansiysk. On this section of the Ob River, the average values of bacterioplankton abundance and biomass were (6.87 ± 4.99) × 106 cells/mL and 142 ± 13 mg C/m3, respectively, which were 1.6-1.7 times higher than in the other sections. The spatial distribution of heterotrophic bacterioplankton in the Ob River is mainly determined by the dynamics of water masses and the influence of tributaries and cities.

Fluorescently labeled nucleotides that contain a zwitterionic indodicarbocyanine dye have been synthesized. The dye has been attached at the C5 position of the pyrimidine base through a trans-alkene spacer. The substrate efficiency of the labeled nucleotides has been tested in recombinase polymerase amplification (RPA). As a result of RPA, full-sized target products of the ebpS gene fragment of the causative agent of bacterial pneumonia ( Staphylococcus aureus ) have been formed with a high density of fluorescent label.

The dynamics of the size-morphological groups of heterotrophic prokaryotoplankton of the largest freshwater reservoir in the Caucasus, Lake Sevan (Armenia) has been studied, which makes it possible to explain its spatio-temporal organization and succession. The lake is characterized by an alternation of stable and unstable periods of existence of hydrobionts due to abrupt changes in environmental conditions, mainly caused by anthropogenic impacts. In the community of planktonic prokaryotes of the lake, the following size-morphological groups were distinguished: small cocci and coccobacilli, small rods and vibrios, medium-sized cocci and coccobacilli, large rods and vibrios, filaments, as well as cells associated with detrital particles. The main contribution (on average 55.5%) to the formation of the prokaryotoplankton biomass of the lake was made by small rods and vibrios. The biomass of each of the groups fluctuated in time and space within relatively narrow limits, and the development of the groups occurred in close relationship with each other. Apparently, different size-morphological groups of prokaryotes are adapted to exist within similar ecological and phylogenetic niches, and jointly and consistently perform common functions in the mineralization of organic matter and trophic interactions in the lake. At the same time, these groups implement various ecological strategies that can be successful at different periods of the ecosystem's existence.

The effect of multiple paternity on the development of the humoral immune response in Syrian hamster pups was studied. The paternity type was found to be determined by 10 microsatellite markers. Pups were immunized with T-dependent antigens (fissurella hemocyanin, KLH) at 33 days of age, with the level of specific antibodies (anti-KLH IgG) measured in blood samples at 5, 10, 20 and 25 days after immunization. As much as 24% pups from multiple paternity litters and 25% from single paternity litters were revealed to develop no immune response. At the same time, in all pups that responded to immunization, the level of anti-KLH IgG significantly increased over time, starting with the 5th day after immunization. However, there were significant differences in the concentration of anti-KLH IgG in the blood serum of pups derived neither from litters with multiple paternity nor litters obtained from one male. Therefore, our results fail to support the hypothesis that multiple paternity enhances the immunity of Syrian hamster pups.

A method for the photolithographic fabrication of biochips with hydrogel cells made of brush acrylic acid–acrylamide copolymers fixed by one end on the surface of a polymer substrate has been developed. Hydrogel cells with reactive carboxyl groups were used for covalent immobilization of oligonucleotide probes. The efficiency of the method was demonstrated by the hybridization analysis of DNA targets of different lengths, matching a segment of exon 7 of the human ABO gene.

The approach based on molecular modeling was developed to study dNTP derivatives characterized by new polymerase-specific properties. For this purpose, the relative efficiency of PCR amplification with modified dUTPs was studied using Taq, Tth, Pfu, Vent, Deep Vent, Vent (exo-), and Deep Vent (exo-) DNA polymerases. The efficiency of PCR amplification with modified dUTPs was compared with the results of molecular modeling using the known 3D structures of KlenTaq polymerase–DNA–dNTP complexes. The dUTPs were C5-modified with bulky functional groups (the Cy5 dye analogs) or lighter aromatic groups. Comparing the experimental data and the results of molecular modeling revealed the decrease in PCR efficiency in the presence of modified dUTPs with an increase in the number of non-covalent bonds between the substituents and the DNA polymerase (about 15% decrease per one extra non-covalent bond). Generalization of the revealed patterns to all the studied polymerases of the A and B families is discussed herein. The number of non-covalent bonds between the substituents and polymerase amino acid residues is proposed to be a potentially variable parameter for regulating enzyme activity.

A method has been developed for the immobilization of short DNA sequences in agarose cells fixed on the surface of a polymer substrate, with their subsequent partial cleavage from agarose when heated while protecting functional properties. The effectiveness of the method was demonstrated on oligonucleotides, which, after cleavage from agarose, retained the ability to participate as primers in the PCR amplification of the sequence site 7 of the exon of the human ABO gene.