Selective Amplification of SPR Biosensor Signal for Recognition of rpoB Gene Fragments by Use of Gold Nanoparticles Modified by Thiolated DNA.

An experimental approach for improving the sensitivity of the surface plasmon resonance (SPR) DNA hybridization sensor using gold nanoparticles (GNPs), modified by specific oligonucleotides, was elaborated. An influence of the ionic strength on the aggregation stability of unmodified GNPs and GNPs modified by the thiolated oligonucleotides was investigated by monitoring a value of light extinction at 520 nm that can be considered as a measure of a quantity of the non-aggregated GNPs. While the unmodified GNPs started to aggregate in 0.2 × saline-sodium citrate (SSC), GNPs modified by the negatively charged oligonucleotides were more stable at increasing ionic strength up to 0.5 × SSC. A bioselective element of the SPR DNA hybridization sensor was formed by immobilization on the gold sensor surface of the thiolated oligonucleotides P2, the sequence of which is a fragment of the rpoB gene of Mycobacterium tuberculosis. The injections into the measuring flow cell of the SPR spectrometer of various concentrations of GNPs modified by the complementary oligonucleotides T2-18m caused the pronounced concentration-dependent sequence-specific sensor responses. The magnitude of the sensor responses was much higher than in the case of the free standing complementary oligonucleotides. According to the obtained experimental data, the usage of GNPs modified by specific oligonucleotides can amplify the sensor response of the SPR DNA hybridization sensor in ~1200 times.

Surface Plasmon Resonance Investigations of Bioselective Element Based on the Recombinant Protein A for Immunoglobulin Detection.

The developed surface plasmon resonance (SPR) biosensor based on the recombinant Staphylococcal protein A with an additional cysteine residue (SPA-Cys) used as a biorecognition component showed a good selectivity and sensitivity for the immunoglobulin detection. The developed biosensor with SPA-Cys-based bioselective element can also be used as a first step of immunosensor creation. The successful immobilization of SPA-Cys on the nanolayer gold sensor surface of the SPR spectrometer was performed. The efficiency of blocking nonspecific sorption sites on the sensor surface with milk proteins, gelatin, BSA, and HSA was studied, and a rather high efficiency of using gelatin was confirmed. The SPR biosensor selectively interacted with IgG and did not interact with the control proteins. The linear dependence of the sensor response on the IgG concentration in the range from 2 to 10 µg/ml was shown. Using the calibration curve, the IgG concentration was measured in the model samples. The determined concentrations are in good agreement (r 2 = 0.97) with the given concentration of IgG.

SPR Detection and Discrimination of the Oligonucleotides Related to the Normal and the Hybrid bcr-abl Genes by Two Stringency Control Strategies.

In this study, we applied two stringency control strategies for surface plasmon resonance (SPR) detection of DNA hybridization and discrimination of completely and partially complementary 24-mer sequences. These sequences are specific to the human normal bcr and the hybrid bcr-abl genes, protein products of which are responsible for some leukemia. SPR sensors based on resonance phenomena in nanoscale gold films are well suited for label-free, real-time investigations of the macromolecule interactions. Thermodynamic parameters obtained using the web server DINAMelt allowed supposing the possibility for realization (a) stringency control based on the ionic strength of the hybridization buffer and (b) stringency control based on the temperature elevation. The first one resulted in that the discrimination index of completely complementary and partially complementary oligonucleotides depending on the target concentration varied from 1.3 to 1.8 in 2 × SSC and from 2.0 to 2.9 in 0.5 × SSC. For implementation of the second stringency control strategy, SPR spectrometer measuring flow cell with built-in high-precision temperature control and regulation as well as corresponding software was created. It is shown that the duplexes formed by the immobilized probes mod-Ph and completely complementary oligonucleotides P1 remained without significant changes until ~50 °C, while the duplexes formed with partially complementary oligonucleotide Bcrex14 almost entirely disrupted at 40 °C. Thus, the absolutely effective thermodiscrimination of this pair of oligonucleotides was achieved in this temperature range (40-50 °C).

Discrimination of single base mismatched oligonucleotides related to the rpoB gene of Mycobacterium tuberculosis using a surface plasmon resonance biosensor.

Single base mismatched oligonucleotides related to the rpoB gene of Mycobacterium tuberculosis, the mutations of which cause drug resistance of the infectious agent, were detected and discriminated using a surface plasmon resonance biosensor system. Thiol-modified oligonucleotides of the selected sequence (the probe) and 1-mercapto-6-hexanol were immobilized on a gold sensor surface. Hybridization between immobilized probe P2 and perfectly matched target T2 as well as a single base mismatched target TN was investigated in buffer solutions of various stringencies. Discrimination of perfectly matched and single base mismatched targets is achieved due to a difference in the level of their hybridization with the immobilized probe depending on stringency of the buffer solution. In 0.5×SSC buffer solution (7.5 mM sodium citrate, pH 7, containing 75 mM NaCl), sensor response at T2 injection into the measuring sensor cell was 16 times that at TN injection. The experimental results on surface hybridization between the studied oligonucleotides demonstrated a good correlation with theoretical calculations of thermodynamic parameters of these interactions in the solution. The described approach could be proposed as a basis for creating a biosensor for real-time label-free diagnostics of drug-resistant tuberculosis.

Surface plasmon resonance detection of oligonucleotide sequences of the rpoB genes of Mycobacterium tuberculosis.

Oligonucleotide sequences related to the normal and mutated rpoB genes of Mycobacterium tuberculosis are detected using a surface plasmon resonance (SPR) biosensor system. A bioselective element was prepared by immobilizing the thiol-modified oligonucleotides of the selected sequence (the capture probe P2) that contains the mutated TCG→TTG codon 531 (evoking drug resistance) of the rpoB gene of M. tuberculosis on a gold sensor surface. Specific hybridization between immobilized probe P2 and complementary target T2 gave the highest sensor response, single-base mismatched oligonucleotide TN (corresponding to the normal gene sequence) produced somewhat smaller response and no response was observed at injection of noncomplementary oligonucleotide TC. The P2-T2 hybridization efficiency is calculated ca. 30% (5 × 10(12) molecules cm(-2)), and the lowest detection limit of T2 was 10nM. An extended T2E oligonucleotide sequence consisting of T2 sequence and additional 24 nucleotides was shown to cause more pronounced sensor response (at least 5 nM T2E was easily detected). Injection into the sensor cell of the oligonucleotides complementary to the free additional part of T2E after P2-T2E hybridization gave a significant additional SPR response, thus showing that the sandwich hybridization format further improves the sensor sensitivity and decreases the lowest detection limit. The experimental results on surface hybridization between the studied oligonucleotides were in good agreement with thermodynamic parameters of the hybridization calculated for solution conditions. The described approach could be proposed as a basis for creating a biosensor for real-time and label-free diagnostics of drug resistant tuberculosis.

Towards molecularly imprinted polymers selective to peptides and proteins. The epitope approach.

In this paper, we describe the epitope approach to molecular imprinting. The applicability of molecular imprinting, a method that allows the preparation of biomimetic compounds (artificial receptors and antibodies), is extended by this approach. Our approach makes it possible to obtain imprinted polymers selective to peptides and proteins whereas, to date, molecular imprinting has been used primarily for the preparation of polymers that selectively bind to relatively low molecular weight substances. The epitope approach is based on using (as a template) a short peptide that represents only part of a larger peptide or protein (as an epitope represents an antigen), which in turn can be recognized by the synthesized polymer. It is demonstrated that although other parts of peptides can influence the process of molecular recognition, the polymers imprinted with a short peptide efficiently recognize both the template and larger peptides (for example, oxytocin) that possess the same C-terminal part of the structure.

Study of the factors influencing peak asymmetry on chromatography using a molecularly imprinted polymer prepared by the epitope approach.

Investigations of the effect of sample load on peak asymmetry during chromatography on molecularly imprinted polymer prepared by the epitope approach showed that the shape of the peaks for the template Tyr-Pro-Leu-Gly-NH2 and for acetyl-L-tyrosine ethyl ester changed considerably until a split was observed. In contrast, the asymmetry of the peaks corresponding to oxytocin, which possesses the same C-terminus tripeptide as the template and interacts with the imprinted polymer, remained essentially unaltered. The circular dichroism (CD) spectra of these peptides showed significant dependence on peptide concentration, and the dependence was nearly the same for all the tested peptides. The addition of acetic acid influenced the CD spectra of YPLG and oxytocin but had no influence on the spectrum of acetyl-L-tyrosine ethyl ester. The shape differences in the chromatographic peaks seem to be associated with a solvation mechanism rather than with solute-solute complexation in solution. However, the observed differences in peak asymmetry cannot be completely explained by the mechanisms that have been postulated previously. Our results suggest the formation of triple complexes between a solute molecule (or molecules), an already adsorbed solute molecule, and an adjacent region of the polymeric stationary phase. These triple complexes may influence the retention of analytes and contribute to peak asymmetry.

Recognition of oxytocin and oxytocin-related peptides in aqueous media using a molecularly imprinted polymer synthesized by the epitope approach.

An artificial polymeric receptor prepared by the epitope approach of molecular imprinting was shown to recognize the peptide hormone, oxytocin, in aqueous media. The proposed approach is based on using (as a template) a compound, whose structure represents a small exposed fragment of a larger molecule (as an epitope represents an antigen). A HPLC study has demonstrated the important role of ionic interactions and the N-terminal amino group of oxytocin and oxytocin-related peptides in the process of their recognition by the molecularly imprinted polymer in the aqueous-rich media. However, the specificity of the process is considered to be defined by hydrophobic interactions and hydrogen bonding. Moreover, it was shown that the selectivity of the molecularly imprinted polymer can be attenuated by water content, ionic strength and pH of the chromatographic mobile phase: depending on these factors the template, Tyr-Pro-Leu-Gly-NH2, or, for example, oxytocin, a larger peptide, which possesses the same three amino-acid C-terminal parts of the structure, can be preferentially retained by the molecularly imprinted polymer.

An approach to conductometric immunosensor based on phthalocyanine thin film.

A new approach to conductometric biosensors utilizing iodine-sensitive phthalocyanine thin films has been proposed. The excellent sensitivity of the tetra-tert-butyl copper phthalocyanine (ttb-CuPc) to free iodine was used for the first time to detect a peroxidase-initiated reaction in an aqueous medium. To minimize the interfering effect of aqueous electrolytes on the impedance responses of the ttb-CuPc film itself, Au/Cr interdigitated planar electrodes bearing ttb-CuPc thin films were protected with hydrophobic gas-permeable membranes, namely thermally evaporated calixarene or plasma polymerized hexamethyldisiloxane films. Impedance spectroscopy data were analyzed in order to define the optimal operating frequency. An enzyme sensor with peroxidase immobilized in a cross-linked albumin matrix was tested. Its impedance responses were studied under variation of the substrate concentration, pH, ionic strength and buffer capacity. These results were used to define conditions for peroxidase-linked immunoassay in subsequent tests. With the developed sensor, concentrations of IgG in 0.2-2 micrograms/ml range were measured in a competitive mode with satisfactory accuracy. The detection of IgG in both test solutions and blood serum samples has been demonstrated.

[The dynamics of erythrocyte metabolism in severe forms of meningococcal infection and suppurative meningitis in children]. / Dinamika metabolizma éritrotsitov pri tiazhelykh formakh meningokokkovoi infektsii i gnoinykh meningitov u detei.

In 63 children with severe meningococcal infection (MI) and meningitides of another origin red cell metabolism was studied: levels of ATP, ADP, AMP, ATP/ADP, ATP/AMP, energetic charge, 2,3-DPG, FAD, piruvate, lactate, activity of lactate dehydrogenase, piruvate kinase, glucose-6-phosphate dehydrogenase, glutatione reductase, Mg2+, Na+, K(+)-dependent ATPase. All the disease periods were characterized by combined pathobiochemical shifts of different degree typical for varying metabolic systems and correlating with the infection severity. The discussion covers pathogenetic and clinical significance of red cell metabolism shifts in patients with MI and purulent meningitides.

Adaptogenic and cardioprotective action of Galleria mellonella extract in rats and frogs.

Pharmacological and metabolic effects of Galleria mellonella larvae extract used in Russian folk medicine to treat cardiovascular and senile diseases were studied. It was shown that the extract possesses adaptogenic, cardiotropic, cardioprotective, and hypocoagulant properties. The extract possesses low toxicity and does not cause significant changes in biochemical parameters in the blood serum of laboratory animals. Increase in catecholamine content in the heart and aortic tissues and their decrease in adrenal glands are unfavourable effects of high doses of the extract.

[The effect of heparin in vasorenal hypertension on blood electrolyte balance, on the abdominal aortic walls and on the erythrocyte rheological properties in rats]. / Vliianie geparina pri vazorenal'noi gipertenzii na balans élektrolitov krovi i stenki briushnoi aorty i reologicheskie svoistva éritrotstov krys.

The paper provides the results of experimental studies of the effects of heparin given to albino rats with vasorenal hypertension in a dose of 150 U/kg on electrolyte levels in plasma, erythrocytes, cardiac and abdominal aortic tissues, the viscosity coefficient and charge of erythrocytic suspension, transmural difference of abdominal aortic potentials. Heparin has been shown to correct hypertension-induced changes in the transmural difference of abdominal aortic potentials, the charge and viscosity coefficient of erythrocytic suspension, the gradient of sodium, potassium, and calcium in the system of erythrocyte-plasma-abdominal aortic wall.

[Determination of the optimal condition of hemoprotein immobilization on the polystyrene surface]. / Opredelenie optimal'nykh uslovii immobilizatsii gemoproteidov na poverkhnosti polistirolovykh plashek.

The effect of pH, buffer composition, salts concentration and protein content on the immobilization efficiency of peroxidase, hemoglobin and their conjugates with bovine serum albumin (BSA) have been studied. All the substances were immobilized on the surface of polystyrene matrix. When using the 0.01 M carbonate-bicarbonate buffer (pH 9.6) immobilization of soluble substances is optimal. It was shown that the sorption efficiency of hemoproteins and their conjugates with BSA is considerably enhanced by drying solution containing ammonium bicarbonate. The increase of ammonium bicarbonate concentration from 0.13 M to 0.5 M leads to the process intensification.

[Effect of vasorenal hypertension and neodicoumarin on the rheological properties of erythrocytes and the balance of blood electrolytes, heart and abdominal aorta wall in rats]. / Vliianie vazorenal'noi gipertenzii i neodikumarina na reologicheskie svoistva eritrotsitov, balans elektrolitov krovi, serdtsa i stenki briushnoi aorty krys.

It has been shown that with vasorenal hypertension, erythrocyte suspension viscosity coefficient increases, the abdominal aorta transmural potential difference decreases with lower levels of Na+, K+, Ca2+, and Mg2+ in plasma, cardiac and abdominal aorta tissue and higher red blood cell Na+ levels. Neodicumarinum, 3 mg/kg, modified Na+ and K+ imbalance in the red blood cell-plasma-abdominal aorta wall system, which had been caused by vasorenal hypertension. At the same time the changes in Ca2+ and Mg2+ levels in the abdominal aorta and heart tissue, as well as the value of red blood cell suspension viscosity coefficient were demonstrated to be effectively abolished with neodicumarinum in a dose of 30 mg/kg.

[State of the hemostasis system in patients with malignant III-stage neoplasms of the maxilla and larynx]. / Sostoianie sistemy gemostaza u bol'nykh so zlokachestvennymi novoobrazovaniiami verkhnei cheliusti i gortani III stadii.

Hemostasis of patients with III stage malignant neoplasms of the maxilla and larynx was investigated. We examined 40 patients, 8 of which had maxillary tumor and 32 had laryngeal tumor. The age of the patients varied from 44 to 68 years. It was found that the patients with malignant neoplasms of the maxilla and larynx had hemostatic disorders which formed the thrombohemorrhagic syndrome. During the surgical intervention they displayed the hypocoagulation phase of the syndrome, which indicated the most dangerous prethrombotic state of the blood system.

[Effect of acute hemorrhage on hemostasis in dogs not adapted to high-altitude conditions]. / Vliianie ostroi krovopoteri na gemostaz u neadaptirovannykh k usloviiam vysokogor'ia sobak.

Acute blood loss was easier after 3-day stay high in the mountains than after a stay in a low place. This is explained by increased blood fibrinolytic activity and high blood antithrombin level recorded by the third day of the animals' stay in the mountains.

[Synthesis of ovalbumin and globin during simultaneous translation of their mRNA in the presence of tRNA of different origin in the cell-free protein-synthesizing system from wheat germ]. / Sintez oval'bumina i globina pri odnovremennoi transliatsii ikh mRNK v prisutstvii tRNK razlichnogo proiskhozhdeniia v beskletochnoi beloksinteziruiushchei sisteme iz zarodyshei pshenitsy

Competition among ovalbumin and globin mRNA under their simultaneous translation in the tRNA-dependent cell-free system from wheat germ was studied. One of the mRNAs was added to samples in a constant amount, that provided 50% protein synthesis level of the maximum, other--in increasing amount (from 0 to the maximum). The ration of ovalbumin and globin synthesis rates has been shown to depend essentially (1.5-2 fold) on the nature of tRNA, being added to the system. The obtained data suggest that functional adaptation of tRNA is a part of the mechanism of protein biosynthesis regulation and it is possible to modulate some mRNAs translation selectivity on the elongation by different sets of tRNA.

[Modulation of the selective translation of mRNA in cell-free system exposed to tRNA]. / Moduliatsiia izbiratel'noi transliatsii mRNK v beskletochnoi sisteme pod vliianiem tRNK.

The homo- and heterologous tRNA were studied for their influence on the synthesis rate of globin and ovalbumin under simultaneous translation of their mRNAs in the tRNA-dependent cell-free system from wheat germ. In the presence of the fixed concentrations of one of the mRNAs the different amounts of the other mRNA (from 6 up to saturation of the system) were added to the samples. The ratio of the synthesis rate of the translation products after their electrophoretic characterization is determined on the basis of the autoradiogram scanning. The competition among mRNAs in question for some components of translation apparatus has been found: globin mRNA has a higher competitiveness than mRNA for ovalbumin. Modulation of the simultaneous translation of these mRNAs has been shown to depend on the nature of tRNA. The presence of homologous tRNA in the system increases a competitiveness of the corresponding mRNA, but heterologous tRNA reduces it. This regularity is retained in a wide range of the concentration ratios for the both mRNAs.