The detection of hepatitis c virus core antigen using afm chips with immobolized aptamers.

In the present study, the possibility of hepatitis C virus core antigen (HCVcoreAg) detection in buffer solution, using atomic force microscope chip (AFM-chip) with immobilized aptamers, has been demonstrated. The target protein was detected in 1mL of solution at concentrations from 10-10М to 10-13М. The registration of aptamer/antigen complexes on the chip surface was carried out by atomic force microscopy (AFM). The further mass-spectrometric (MS) identification of AFM-registered objects on the chip surface allowed reliable identification of HCVcoreAg target protein in the complexes. Aptamers, which were designed for therapeutic purposes, have been shown to be effective in HCVcoreAg detection as probe molecules.

[Monitoring of brightness temperature fluctuation of water in SHF range].

The purpose of the research consisted in detection of fluctuation of brightness temperature (TSHF) of water in the area of the temperature Т = 42°Ð¡ (that is critical for human) during its evaporation by SHF radiometry. Methods: Monitoring of the changes in brightness temperature of water in superhigh frequency (SHF) range (3.8-4.2 GHz) near the phase transition temperature of water Т = 42°Ð¡ during its evaporation in the cone dielectric cell. The brightness temperature measurements were carried out using radiometer. Results: Fluctuation with maximum of brightness temperature was detected in 3.8-4.2 GHz frequency range near at the temperature of water Т = 42°Ð¡. It was characteristic for these TSHF fluctuations that brightness temperature rise time in this range of frequencies in ~4°Ð¡ temperature range with 0.05-15°Ð¡/min gradient and a sharp decrease during 10 s connected with measuring vapor conditions. Then nonintensive fluctuation series was observed. At that, the environment temperature remained constant. Conclusion: The significant increasing in brightness temperature of water during its evaporation in SHF range near the temperature of Т ~42°Ð¡ were detected. It was shown that for water, ТSHF pull with the amplitude DТSHF ~4°C are observed. At the same time, thermodynamic temperature virtually does not change. The observed effects can be used in the development of the systems for diadnostics of pathologies in human and analytical system.

[AFM fishing of proteins under impulse electric field]. / ASM-fishing belka v impul'snom élektricheskom pole.

A combination of (atomic force microscopy)-based fishing (AFM-fishing) and mass spectrometry allows to capture protein molecules from solutions, concentrate and visualize them on an atomically flat surface of the AFM chip and identify by subsequent mass spectrometric analysis. In order to increase the AFM-fishing efficiency we have applied pulsed voltage with the rise time of the front of about 1 ns to the AFM chip. The AFM-chip was made using a conductive material, highly oriented pyrolytic graphite (HOPG). The increased efficiency of AFM-fishing has been demonstrated using detection of cytochrome b5 protein. Selection of the stimulating pulse with a rise time of 1 ns, corresponding to the GHz frequency range, by the effect of intrinsic emission from water observed in this frequency range during water injection into the cell.

Mass spectrometric detection of the amino acid sequence polymorphism of the hepatitis C virus antigen.

A method for detection and identification of the hepatitis C virus antigen (HCVcoreAg) in human serum with consideration for possible amino acid substitutions is proposed. The method is based on a combination of biospecific capturing and concentrating of the target protein on the surface of the chip for atomic force microscope (AFM chip) with subsequent protein identification by tandem mass spectrometric (MS/MS) analysis. Biospecific AFM-capturing of viral particles containing HCVcoreAg from serum samples was performed by use of AFM chips with monoclonal antibodies (anti-HCVcore) covalently immobilized on the surface. Biospecific complexes were registered and counted by AFM. Further MS/MS analysis allowed to reliably identify the HCVcoreAg in the complexes formed on the AFM chip surface. Analysis of MS/MS spectra, with the account taken of the possible polymorphisms in the amino acid sequence of the HCVcoreAg, enabled us to increase the number of identified peptides.

SHF radiation from albumin solution upon external excitation.

The purpose of the research consisted in the measurement of nonequilibrium radiation in superhigh frequency (SHF) range from aqueous solution of albumin upon its mechanical stimulation. Methods: the monitoring of change in the ratio between brightness temperatures TSHF and TIR values after the mechanical stimulation of aqueous solution of albumin in the measuring cell at 35-39°Ð¡. The measurements of brightness temperatures were carried out with use of radiothermometer. SHF frequency range corresponded to 3.4-4.2 GHz, SHF frequency range corresponded to 8-13 mm. Results: It was found that mechanical stimulation of aqueous solution of albumin at the temperature ~39°Ð¡, the change in the ratio between TSHF and TIR occurs. This corresponds to emergence of nonequilibrium SHF radiation from the solution near the phase transition with ~39°Ð¡ temperature. Conclusion: The effect of emergence of nonequilibrium SHF radiation from protein solution near its temperature 39°Ð¡ was found. This temperature corresponds to the temperature of human organism upon a number of pathological states connected with inflammatory processes. The discovered effect can be used in the development of novel non-invasive methods of disease diagnostics.

Monitoring of microwave emission of HRP system during the enzyme functioning.

Monitoring of microwave emission from aqueous solution of horseradish peroxidase (HRP) in the process of the enzyme functioning was carried out. For the monitoring, a system containing HRP, luminol and Н2О2 was employed. Microwave emission measurements were carried out in the 3.4-4.2 GHz frequency range using the active and passive modes (active-mode and passive-mode measurements). In the active mode, excitation of the solution in the pulsed electromagnetic field was accomplished. In the passive mode, no excitation was induced. It appears that the passive-mode measurements taken in the course of the peroxidase reaction in the enzyme system have shown a 0.5 °Ð¡ increase of the microwave signal. Upon the active-mode measurements, taken in the same reaction conditions, the forced excitation of the solution has also led to the increase (by 2 °Ð¡) of the level of the microwave signal - i.e. to its 4-fold enhancement compared to the signal obtained in passive-mode measurements.

[Atomic force microscopy fishing of gp120 on immobilized aptamer and its mass spectrometry identification].

A method of atomic force microscopy-based fishing (AFM fishing) has been developed for protein detection in the analyte solution using a chip with an immobilized aptamer. This method is based on the biospecific fishing of a target protein from a bulk solution onto the small AFM chip area with the immobilized aptamer to this protein used as the molecular probe. Such aptamer-based approach allows to increase an AFM image contrast compared to the antibody-based approach. Mass spectrometry analysis used after the biospecific fishing to identify the target protein on the AFM chip has proved complex formation. Use of the AFM chip with the immobilized aptamer avoids interference of the antibody and target protein peaks in a mass spectrum.

[Microwave emission from water in bioanalytical systems].

Mechanical stimulation of aqueous protein solution was found to be accompanied by electromagnetic radiation in the superhigh frequency range. This radiation was observed at solution temperatures near the phase transitions of water. This effect may occur in various bioanalytical systems, associated with injection of aqueous solutions in these systems, and may have impact on the results of bioalytical measurements. The discovered effect of generation of nonequilibrium microwave emission of water medium (which is the main component of the organism) in the 38-39°C temperature range, i.e. in the range of elevated temperature of the organism (which accompanies pathological condition in humans upon inflammatory diseases, i.e., infactious etc.) may be used for development of new non-invasive methods of disease diagnostics.

[Irreversible chemical AFM-fishing for the detection of low-copied proteins].

The atomic-force microscopy-based method of irreversible chemical AFM-fishing (AFM-IF(Ch)) has been developed for the detection of proteins at ultra-low concentrations in solution. Using this method, a very low concentration of horseradish peroxidase (HRP) protein (10(-17) M) was detected in solution. A theoretical model that allows the description of obtained experimental data, is proposed. This model takes into consideration both the transport of the protein from the bulk solution onto the AFM-chip surface and its irreversible binding to the activated area.

[Atomic force microscopy visualization and measurement of the activity and physicochemical properties of single monomeric and oligomeric enzymes].

An approach to measure the activity of single oligomers of the heme-containing enzyme the cytochrome P450 CYP102A1 (CYP102A1) by atomic force microscopy (AFM) has been developed. It was found that the amplitude of fluctuations of the height of single CYP102A1 molecules performing the catalytic cycle is twice as great as the amplitude of fluctuations of the height of the same enzymes in the inactive state. It was shown that the amplitude of height fluctuations of a CYP102A1 protein globule depends on temperature, the maximum of this dependence being observed at 22 degrees C. The activity of a single CYP102A1 molecule in the unit amplitude of height fluctuations of a protein globule reduced to the unit time was 5+/-2 Ac. The elasticity of a single protein molecule was measured from the deformation of this molecule by the action of an AFM probe. The use of AFM probes of different geometry made i t possible t o determine t he integral andlocal Young's modulus for the monomers of the protein putidaredoxin reductase from the cytochrome P450 CYP101 (P450cam)-containing monooxigenase system, which were 37+/-117 and 1+/-3 MPa, respectively.

[Visualization and identification of hepatitis C viral particles by atomic force microscopy combined with MS/MS analysis].

Possibility of detection and identification of hepatitis C viral particles with mass spectrometry (MS) in combination with atomic force microscopy (AFM) had been investigated. AFM/MS approach is based on two technologies: (1) AFM-biospecific fishing that allows to detect, concentrate from solution and to count protein complexes on a surface of AFM-nanochip; (2) mass spectrometric identification of these complexes. AFM-biospecific fishing of HCVcoreAg from solution was carried onto surface of AFM-nanochips with immobilized anti-HCVcoreAg. It was shown that HCVcoreAg/anti-HCVcore(im) complexes were formed onto AFM-nanochips in quantity sufficient for mass spectrometric identification. Thus, AFM/MS approach allows to identify fragments of hepatitis C virus fished onto a surface of AFM-nanochip from serum.

[Atomic force microscopy detection of serological markers of viral hepatites B and C].

The aim of the study was to demonstrate the possibility of detection of serological markers, containing the hepatitis B surface antigen (HBsAg) and hepatitis C virus core-antigen (HCVcoreAg) in human serum, by use of a new atomic force microscopy (AFM)-based nanotechnological approach. In this study, the immobilization on AFM-chip of antibodies against the hepatitis B virus surface antigen (anti-HBsAg) as well as the antibodies against the hepatitis C virus core antigen (anti-HCVcoreAg) was performed. It was shown that such approach enables to detect: HBsAg, HCVcoreAg and the viral fragments containing these antigens in the serum. Comparative analysis of detection of HBsAg- and HCVcoreAg-containing particles by use of the AFM method vs. traditional methods (ELISA, PCR) has demonstrated the 75% coincidence of results between the AFM and the latter two methods.