Publications by authors named "Yuri D Ivanov"

28 Publications

  • Page 1 of 1

Nanoribbon-Based Electronic Detection of a Glioma-Associated Circular miRNA.

Biosensors (Basel) 2021 Jul 13;11(7). Epub 2021 Jul 13.

Laboratory of Nanobiotechnology, Institute of Biomedical Chemistry, 119121 Moscow, Russia.

Nanoribbon chips, based on "silicon-on-insulator" structures (SOI-NR chips), have been fabricated. These SOI-NR chips, whose surface was sensitized with covalently immobilized oligonucleotide molecular probes (oDNA probes), have been employed for the nanoribbon biosensor-based detection of a circular ribonucleic acid (circRNA) molecular marker of glioma in humans. The nucleotide sequence of the oDNA probes was complimentary to the sequence of the target oDNA. The latter represents a synthetic analogue of a glioma marker-NFIX circular RNA. In this way, the detection of target oDNA molecules in a pure buffer has been performed. The lowest concentration of the target biomolecules, detectable in our experiments, was of the order of ~10 M. The SOI-NR sensor chips proposed herein have allowed us to reveal an elevated level of the NFIX circular RNA in the blood of a glioma patient.
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http://dx.doi.org/10.3390/bios11070237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8301916PMC
July 2021

Use of Silicon Nanowire Sensors for Early Cancer Diagnosis.

Molecules 2021 Jun 18;26(12). Epub 2021 Jun 18.

Institute of Biomedical Chemistry, 119121 Moscow, Russia.

The review covers some research conducted in the field of medical and biomedical application of devices based on silicon sensor elements (Si-NW-sensors). The use of Si-NW-sensors is one of the key methods used in a whole range of healthcare fields. Their biomedical use is among the most important ones as they offer opportunities for early diagnosis of oncological pathologies, for monitoring the prescribed therapy and for improving the people's quality of life.
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http://dx.doi.org/10.3390/molecules26123734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234636PMC
June 2021

Micro-Raman Characterization of Structural Features of High-k Stack Layer of SOI Nanowire Chip, Designed to Detect Circular RNA Associated with the Development of Glioma.

Molecules 2021 Jun 18;26(12). Epub 2021 Jun 18.

Laboratory of Nanobiotechnology, Institute of Biomedical Chemistry, 119121 Moscow, Russia.

The application of micro-Raman spectroscopy was used for characterization of structural features of the high-k stack (h-k) layer of "silicon-on-insulator" (SOI) nanowire (NW) chip (h-k-SOI-NW chip), including AlO and HfO in various combinations after heat treatment from 425 to 1000 °C. After that, the NW structures h-k-SOI-NW chip was created using gas plasma etching optical lithography. The stability of the signals from the monocrine phase of HfO was shown. Significant differences were found in the elastic stresses of the silicon layers for very thick (>200 nm) AlO layers. In the UV spectra of SOI layers of a silicon substrate with HfO, shoulders in the Raman spectrum were observed at 480-490 cm of single-phonon scattering. The h-k-SOI-NW chip created in this way has been used for the detection of DNA-oligonucleotide sequences (oDNA), that became a synthetic analog of circular RNA-circ-SHKBP1 associated with the development of glioma at a concentration of 1.1 × 10 M. The possibility of using such h-k-SOI NW chips for the detection of circ-SHKBP1 in blood plasma of patients diagnosed with neoplasm of uncertain nature of the brain and central nervous system was shown.
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http://dx.doi.org/10.3390/molecules26123715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234461PMC
June 2021

Effect of Spherical Elements of Biosensors and Bioreactors on the Physicochemical Properties of a Peroxidase Protein.

Polymers (Basel) 2021 May 15;13(10). Epub 2021 May 15.

Institute of Biomedical Chemistry, 119121 Moscow, Russia.

External electromagnetic fields are known to be able to concentrate inside the construction elements of biosensors and bioreactors owing to reflection from their surface. This can lead to changes in the structure of biopolymers (such as proteins), incubated inside these elements, thus influencing their functional properties. Our present study concerned the revelation of the effect of spherical elements, commonly employed in biosensors and bioreactors, on the physicochemical properties of proteins with the example of the horseradish peroxidase (HRP) enzyme. In our experiments, a solution of HRP was incubated within a 30 cm-diameter titanium half-sphere, which was used as a model construction element. Atomic force microscopy (AFM) was employed for the single-molecule visualization of the HRP macromolecules, adsorbed from the test solution onto mica substrates in order to find out whether the incubation of the test HRP solution within the half-sphere influenced the HRP aggregation state. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was employed in order to reveal whether the incubation of HRP solution within the half-sphere led to any changes in its secondary structure. In parallel, spectrophotometry-based estimation of the HRP enzymatic activity was performed in order to find out if the HRP active site was affected by the electromagnetic field under the conditions of our experiments. We revealed an increased aggregation of HRP after the incubation of its solution within the half-sphere in comparison with the control sample incubated far outside the half-sphere. ATR-FTIR allowed us to reveal alterations in HRP's secondary structure. Such changes in the protein structure did not affect its active site, as was confirmed by spectrophotometry. The effect of spherical elements on a protein solution should be taken into account in the development of the optimized design of biosensors and bioreactors, intended for performing processes involving proteins in biomedicine and biotechnology, including highly sensitive biosensors intended for the diagnosis of socially significant diseases in humans (including oncology, cardiovascular diseases, etc.) at early stages.
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http://dx.doi.org/10.3390/polym13101601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8155990PMC
May 2021

AFM study of changes in properties of horseradish peroxidase after incubation of its solution near a pyramidal structure.

Sci Rep 2021 May 10;11(1):9907. Epub 2021 May 10.

Institute of Biomedical Chemistry, Pogodinskaya str., 10, Moscow, 119121, Russia.

In our present paper, the influence of a pyramidal structure on physicochemical properties of a protein in buffer solution has been studied. The pyramidal structure employed herein was similar to those produced industrially for anechoic chambers. Pyramidal structures are also used as elements of biosensors. Herein, horseradish peroxidase (HRP) enzyme was used as a model protein. HRP macromolecules were adsorbed from their solution onto an atomically smooth mica substrate, and then visualized by atomic force microscopy (AFM). In parallel, the enzymatic activity of HRP was estimated by conventional spectrophotometry. Additionally, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) has been employed in order to find out whether or not the protein secondary structure changes after the incubation of its solution either near the apex of a pyramid or in the center of its base. Using AFM, we have demonstrated that the incubation of the protein solution either in the vicinity of the pyramid's apex or in the center of its base influences the physicochemical properties of the protein macromolecules. Namely, the incubation of the HRP solution in the vicinity of the top of the pyramidal structure has been shown to lead to an increase in the efficiency of the HRP adsorption onto mica. Moreover, after the incubation of the HRP solution either near the top of the pyramid or in the center of its base, the HRP macromolecules adsorb onto the mica surface predominantly in monomeric form. At that, the enzymatic activity of HRP does not change. The results of our present study are useful to be taken into account in the development of novel biosensor devices (including those for the diagnosis of cancer in humans), in which pyramidal structures are employed as sensor, noise suppression or construction elements.
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http://dx.doi.org/10.1038/s41598-021-89377-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110588PMC
May 2021

Detection of Influenza Virus Using a SOI-Nanoribbon Chip, Based on an N-Type Field-Effect Transistor.

Biosensors (Basel) 2021 Apr 12;11(4). Epub 2021 Apr 12.

Laboratory of Nanobiotechnology, Institute of Biomedical Chemistry, 119121 Moscow, Russia.

The detection of influenza A virions with a nanoribbon detector (NR detector) has been demonstrated. Chips for the detector have been fabricated based on silicon-on-insulator nanoribbon structures (SOI nanoribbon chip), using a complementary metal-oxide-semiconductor (CMOS)-compatible technology-by means of gas-phase etching and standard optical photolithography. The surface of the SOI nanoribbon chip contains a matrix of 10 nanoribbon (NR) sensor elements. SOI nanoribbon chips of n-type conductance have been used for this study. For biospecific detection of target particles, antibodies against influenza virus have been covalently immobilized onto NRs. Influenza A virus detection was performed by real-time registration of the source-drain current through the NRs. The detection of the target viral particles was carried out in buffer solutions at the target particles concentration within the range from 10 to 10 viral particles per milliliter (VP/mL). The lowest detectable concentration of the target viral particles was 6 × 10 M (corresponding to 10 VP/mL). The use of solutions containing ~10 to 10 VP/mL resulted in saturation of the sensor surface with the target virions. In the saturation mode, detection was impossible.
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http://dx.doi.org/10.3390/bios11040119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8069153PMC
April 2021

Raman Spectroscopy-Based Quality Control of "Silicon-On-Insulator" Nanowire Chips for the Detection of Brain Cancer-Associated MicroRNA in Plasma.

Sensors (Basel) 2021 Feb 13;21(4). Epub 2021 Feb 13.

Laboratory of Nanobiotechnology, Institute of Biomedical Chemistry, 119121 Moscow, Russia.

Application of micro-Raman spectroscopy for the monitoring of quality of nanowire sensor chips fabrication has been demonstrated. Nanowire chips have been fabricated on the basis of «silicon-on-insulator» (SOI) structures (SOI-NW chips). The fabrication of SOI-NW chips was performed by optical litography with gas-phase etching. The so-fabricated SOI-NW chips are intended for highly sensitive detection of brain cancer biomarkers in humans. In our present study, two series of experiments have been conducted. In the first experimental series, detection of a synthetic DNA oligonucleotide (oDNA) analogue of brain cancer-associated microRNA miRNA-363 in purified buffer solution has been performed in order to demonstrate the high detection sensitivity. The second experimental series has been performed in order to reveal miRNA-363 itself in real human plasma samples. To provide detection biospecificity, the SOI-NW chip surface was modified by covalent immobilization of probe oligonucleotides (oDNA probes) complementary to the target biomolecules. Using the SOI-NW sensor chips proposed herein, the concentration detection limit of the target biomolecules at the level of 3.3 × 10 M has been demonstrated. Thus, the approach employing the SOI-NW chips proposed herein represents an attractive tool in biomedical practice, aimed at the early revelation of oncological diseases in humans.
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http://dx.doi.org/10.3390/s21041333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918486PMC
February 2021

Optical Monitoring of the Production Quality of Si-Nanoribbon Chips Intended for the Detection of ASD-Associated Oligonucleotides.

Micromachines (Basel) 2021 Feb 3;12(2). Epub 2021 Feb 3.

Laboratory of Nanobiotechnology, Institute of Biomedical Chemistry, 119121 Moscow, Russia.

Gas-phase etching and optical lithography were employed for the fabrication of a silicon nanoribbon chip (Si-NR chip). The quality of the so-fabricated silicon nanoribbons (Si-NRs) was monitored by optical Raman scattering spectroscopy. It was demonstrated that the structures of the Si-NRs were virtually defect-free, meaning they could be used for highly sensitive detection of biological macromolecules. The Si-NR chips were then used for the highly sensitive nanoelectronics detection of DNA oligonucleotides (oDNAs), which represent synthetic analogs of 106a-5p microRNA (miR-106a-5p), associated with the development of autism spectrum disorders in children. The specificity of the analysis was attained by the sensitization of the Si-NR chip sur-face by covalent immobilization of oDNA probes, whose nucleotide sequence was complementary to the known sequence of miR-106a-5p. The use of the Si-NR chip was demonstrated to al-low for the rapid label-free real-time detection of oDNA at ultra-low (~10 M) concentrations.
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http://dx.doi.org/10.3390/mi12020147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913754PMC
February 2021

AFM and FTIR Investigation of the Effect of Water Flow on Horseradish Peroxidase.

Molecules 2021 Jan 9;26(2). Epub 2021 Jan 9.

Institute of Biomedical Chemistry, Moscow 119121, Russia.

Atomic force microscopy (AFM)-based fishing is a promising method for the detection of low-abundant proteins. This method is based on the capturing of the target proteins from the analyzed solution onto a solid substrate, with subsequent counting of the captured protein molecules on the substrate surface by AFM. Protein adsorption onto the substrate surface represents one of the key factors determining the capturing efficiency. Accordingly, studying the factors influencing the protein adsorbability onto the substrate surface represents an actual direction in biomedical research. Herein, the influence of water motion in a flow-based system on the protein adsorbability and on its enzymatic activity has been studied with an example of horseradish peroxidase (HRP) enzyme by AFM, attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and conventional spectrophotometry. In the experiments, HRP solution was incubated in a setup modeling the flow section of a biosensor communication. The measuring cell with the protein solution was placed near a coiled silicone pipe, through which water was pumped. The adsorbability of the protein onto the surface of the mica substrate has been studied by AFM. It has been demonstrated that incubation of the HRP solution near the coiled silicone pipe with flowing water leads to an increase in its adsorbability onto mica. This is accompanied by a change in the enzyme's secondary structure, as has been revealed by ATR-FTIR. At the same time, its enzymatic activity remains unchanged. The results reported herein can be useful in the development of models describing the influence of liquid flow on the properties of enzymes and other proteins. The latter is particularly important for the development of biosensors for biomedical applications-particularly for serological analysis, which is intended for the early diagnosis of various types of cancer and infectious diseases. Our results should also be taken into account in studies of the effects of protein aggregation on hemodynamics, which plays a key role in human body functioning.
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http://dx.doi.org/10.3390/molecules26020306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826892PMC
January 2021

Highly Sensitive Detection of CA 125 Protein with the Use of an n-Type Nanowire Biosensor.

Biosensors (Basel) 2020 Dec 18;10(12). Epub 2020 Dec 18.

Laboratory of nanotechnology, Institute of Biomedical Chemistry, 119121 Moscow, Russia.

The detection of CA 125 protein in a solution using a silicon-on-insulator (SOI)-nanowire biosensor with n-type chip has been experimentally demonstrated. The surface of nanowires was modified by covalent immobilization of antibodies against CA 125 in order to provide the biospecificity of the target protein detection. We have demonstrated that the biosensor signal, which results from the biospecific interaction between CA 125 and the covalently immobilized antibodies, increases with the increase in the protein concentration. At that, the minimum concentration, at which the target protein was detectable with the SOI-nanowire biosensor, amounted to 1.5 × 10 M.
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http://dx.doi.org/10.3390/bios10120210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766891PMC
December 2020

AFM Imaging of Protein Aggregation in Studying the Impact of Knotted Electromagnetic Field on A Peroxidase.

Sci Rep 2020 06 2;10(1):9022. Epub 2020 Jun 2.

Institute of Biomedical Chemistry, Pogodinskaya str., 10, Moscow, 119121, Russia.

The phenomenon of knotted electromagnetic field (KEMF) is now actively studied, as such fields are characterized by a nontrivial topology. The research in this field is mainly aimed at technical applications - for instance, the development of efficient communication systems. Until present, however, the influence of KEMF on biological objects (including enzyme systems) was not considered. Herein, we have studied the influence of KEMF on the aggregation and enzymatic activity of a protein with the example of horseradish peroxidase (HRP). The test HRP solution was irradiated in KEMF (the radiation power density was 10 W/cm at 2.3 GHz frequency) for 40 min. After the irradiation, the aggregation of HRP was examined by atomic force microscopy (AFM) at the single-molecule level. The enzymatic activity was monitored by conventional spectrophotometry. It has been demonstrated that an increased aggregation of HRP, adsorbed on the AFM substrate surface, was observed after irradiation of the protein sample in KEMF with low (10 W/cm) radiation power density; at the same time, the enzymatic activity remained unchanged. The results obtained herein can be used in the development of models describing the interaction of enzymes with electromagnetic field. The obtained data can also be of importance considering possible pathological factors that can take place upon the influence of KEMF on biological objects- for instance, changes in hemodynamics due to increased protein aggregation are possible; the functionality of protein complexes can also be affected by aggregation of their protein subunits. These effects should also be taken into account in the development of novel highly sensitive systems for human serological diagnostics of breast cancer, prostate cancer, brain cancer and other oncological pathologies, and for diagnostics of diseases in animals, and crops.
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http://dx.doi.org/10.1038/s41598-020-65888-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265551PMC
June 2020

Pilot data of serum proteins from children with autism spectrum disorders.

Data Brief 2019 Dec 25;27:104558. Epub 2019 Sep 25.

Institute of Biomedical Chemistry, Russia.

Protein profiles of 13 serum samples from children with autism spectrum disorders (ASD) and 11 serum samples from healthy volunteers was obtained using panoramic ultra-high resolution mass spectrometry. The analysis of measurements was performed using the proteomics search engine. We identified a group of 74 proteins which we term a "protein fingerprint" specific for serum samples collected from children with autism. Components of the protein fingerprint are involved in hemostasis maintenance including biological regulation, the response to stimulus, regulation of metabolism, and proteins of the immune system.
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http://dx.doi.org/10.1016/j.dib.2019.104558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817683PMC
December 2019

Challenges of the Human Proteome Project: 10-Year Experience of the Russian Consortium.

J Proteome Res 2019 12 28;18(12):4206-4214. Epub 2019 Oct 28.

Institute of Biomedical Chemistry , Moscow 119435 , Russia.

This manuscript collects all the efforts of the Russian Consortium, bottlenecks revealed in the course of the C-HPP realization, and ways of their overcoming. One of the main bottlenecks in the C-HPP is the insufficient sensitivity of proteomic technologies, hampering the detection of low- and ultralow-copy number proteins forming the "dark part" of the human proteome. In the frame of MP-Challenge, to increase proteome coverage we suggest an experimental workflow based on a combination of shotgun technology and selected reaction monitoring with two-dimensional alkaline fractionation. Further, to detect proteins that cannot be identified by such technologies, nanotechnologies such as combined atomic force microscopy with molecular fishing and/or nanowire detection may be useful. These technologies provide a powerful tool for single molecule analysis, by analogy with nanopore sequencing during genome analysis. To systematically analyze the functional features of some proteins (CP50 Challenge), we created a mathematical model that predicts the number of proteins differing in amino acid sequence: proteoforms. According to our data, we should expect about 100 000 different proteoforms in the liver tissue and a little more in the HepG2 cell line. The variety of proteins forming the whole human proteome significantly exceeds these results due to post-translational modifications (PTMs). As PTMs determine the functional specificity of the protein, we propose using a combination of gene-centric transcriptome-proteomic analysis with preliminary fractionation by two-dimensional electrophoresis to identify chemically modified proteoforms. Despite the complexity of the proposed solutions, such integrative approaches could be fruitful for MP50 and CP50 Challenges in the framework of the C-HPP.
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http://dx.doi.org/10.1021/acs.jproteome.9b00358DOI Listing
December 2019

Detection of Hepatitis C Virus Core Protein in Serum Using Aptamer-Functionalized AFM Chips.

Micromachines (Basel) 2019 Feb 15;10(2). Epub 2019 Feb 15.

Institute of Biomedical Chemistry, Moscow 119121, Russia.

In the present study, we demonstrate atomic force microscopy (AFM)-based detection of hepatitis C virus (HCV) particles in serum samples using a chip with aptamer-functionalized surface (apta-based AFM chip). The target particles, containing core antigen of HCV (HCVcoreAg protein), were biospecifically captured onto the chip surface from 1 mL of test solution containing 10 µL of serum collected from a hepatitis C patient. The registration of aptamer/antigen complexes on the chip surface was performed by AFM. The aptamers used in the present study were initially developed for therapeutic purposes; herein, these aptamers have been successfully utilized as probe molecules for HCVcoreAg detection in the presence of a complex protein matrix (human serum). The results obtained herein can be used for the development of detection systems that employ affine enrichment for protein detection.
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http://dx.doi.org/10.3390/mi10020129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413090PMC
February 2019

Micro-Raman Spectroscopy for Monitoring of Deposition Quality of High-k Stack Protective Layer onto Nanowire FET Chips for Highly Sensitive miRNA Detection.

Biosensors (Basel) 2018 Jul 27;8(3). Epub 2018 Jul 27.

Institute of Biomedical Chemistry (IBMC), Moscow 119121, Russia.

Application of micro-Raman spectroscopy for the monitoring of quality of high-k (h-k) dielectric protective layer deposition onto the surface of a nanowire (NW) chip has been demonstrated. A NW chip based on silicon-on-insulator (SOI) structures, protected with a layer of high-k dielectric ((h-k)-SOI-NW chip), has been employed for highly sensitive detection of microRNA (miRNA) associated with oncological diseases. The protective dielectric included a 2-nm-thick Al₂O₃ surface layer and a 8-nm-thick HfO₂ layer, deposited onto a silicon SOI-NW chip. Such a chip had increased time stability upon operation in solution, as compared with an unprotected SOI-NW chip with native oxide. The (h-k)-SOI-NW biosensor has been employed for the detection of DNA oligonucleotide (oDNA), which is a synthetic analogue of miRNA-21 associated with oncological diseases. To provide biospecificity of the detection, the surface of (h-k)-SOI-NW chip was modified with oligonucleotide probe molecules (oDVA probes) complementary to the sequence of the target biomolecule. Concentration sensitivity of the (h-k)-SOI-NW biosensor at the level of ~10 M has been demonstrated.
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http://dx.doi.org/10.3390/bios8030072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6164057PMC
July 2018

Atomic Force Microscopy for Protein Detection and Their Physicoсhemical Characterization.

Int J Mol Sci 2018 Apr 10;19(4). Epub 2018 Apr 10.

Institute of Biomedical Chemistry, 10, Pogodinskaya St., 119121 Moscow, Russia.

This review is focused on the atomic force microscopy (AFM) capabilities to study the properties of protein biomolecules and to detect the proteins in solution. The possibilities of application of a wide range of measuring techniques and modes for visualization of proteins, determination of their stoichiometric characteristics and physicochemical properties, are analyzed. Particular attention is paid to the use of AFM as a molecular detector for detection of proteins in solutions at low concentrations, and also for determination of functional properties of single biomolecules, including the activity of individual molecules of enzymes. Prospects for the development of AFM in combination with other methods for studying biomacromolecules are discussed.
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http://dx.doi.org/10.3390/ijms19041142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5979402PMC
April 2018

Spontaneous Charge Generation in Flowing Albumin Solutions at 35 °C and 38 °C.

Biosensors (Basel) 2017 Dec 11;7(4). Epub 2017 Dec 11.

Institute of Biomedical Chemistry, 119121 Moscow, Russia.

The time dependence of a charge accumulation in a 10 M albumin solution, flowing through a measuring cell of an analytical flow system injector, had a nonlinear character under certain conditions, within a human physiological temperature range. Sharp charge increases depended on albumin concentration. This effect must be taken into consideration when generating models that describe electrokinetic phenomena in flowing protein solutions and when developing analytical flow systems for the registration of biomolecules in low concentration ranges.
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http://dx.doi.org/10.3390/bios7040060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746783PMC
December 2017

Highly sensitive protein detection by biospecific AFM-based fishing with pulsed electrical stimulation.

FEBS Open Bio 2017 08 10;7(8):1186-1195. Epub 2017 Jul 10.

Institute of Biomedical Chemistry Moscow Russia.

We report here the highly sensitive detection of protein in solution at concentrations from 10 to 10 m using the combination of atomic force microscopy (AFM) and mass spectrometry. Biospecific detection of biotinylated bovine serum albumin was carried out by fishing out the protein onto the surface of AFM chips with immobilized avidin, which determined the specificity of the analysis. Electrical stimulation was applied to enhance the fishing efficiency. A high sensitivity of detection was achieved by application of nanosecond electric pulses to highly oriented pyrolytic graphite placed under the AFM chip. A peristaltic pump-based flow system, which is widely used in routine bioanalytical assays, was employed throughout the analysis. These results hold promise for the development of highly sensitive protein detection methods using nanosensor devices.
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http://dx.doi.org/10.1002/2211-5463.12253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5537060PMC
August 2017

Detection of hepatitis C virus core protein in serum by atomic force microscopy combined with mass spectrometry.

Int J Nanomedicine 2015 25;10:1597-608. Epub 2015 Feb 25.

Institute of Biomedical Chemistry, Moscow, Russia.

A method for detection and identification of core antigen of hepatitis C virus (HCVcoreAg)-containing particles in the serum was proposed, with due account taken of the interactions of proteotypic peptides with Na(+), K(+), and Cl(-) ions. The method is based on a combination of reversible biospecific atomic force microscopy (AFM)-fishing and mass spectrometry (MS). AFM-fishing enables concentration, detection, and counting of protein complexes captured on the AFM chip surface, with their subsequent MS identification. Biospecific AFM-fishing of HCVcoreAg-containing particles from serum samples was carried out using AFM chips with immobilized antibodies against HCVcoreAg (HCVcoreAgim). Formation of complexes between anti-HCVcoreAgim and HCVcoreAg-containing particles on the AFM chip surface during the fishing process was demonstrated. These complexes were registered and counted by AFM. Further MS analysis allowed reliable identification of HCVcoreAg within the complexes formed on the AFM chip surface. It was shown that MS data processing, with account taken of the interactions between HCVcoreAg peptides and Na(+), K(+) cations, and Cl(-) anions, allows an increase in the number of peptides identified.
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http://dx.doi.org/10.2147/IJN.S71776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4346358PMC
August 2016

Atomic force microscopy fishing and mass spectrometry identification of gp120 on immobilized aptamers.

Int J Nanomedicine 2014 3;9:4659-70. Epub 2014 Oct 3.

Department of Personalized Medicine, Orekhovich Institute of Biomedical Chemistry of the Russian Academy of Medical Sciences, Moscow, Russia.

Atomic force microscopy (AFM) was applied to carry out direct and label-free detection of gp120 human immunodeficiency virus type 1 envelope glycoprotein as a target protein. This approach was based on the AFM fishing of gp120 from the analyte solution using anti-gp120 aptamers immobilized on the AFM chip to count gp120/aptamer complexes that were formed on the chip surface. The comparison of image contrasts of fished gp120 against the background of immobilized aptamers and anti-gp120 antibodies on the AFM images was conducted. It was shown that an image contrast of the protein/aptamer complexes was two-fold higher than the contrast of the protein/antibody complexes. Mass spectrometry identification provided an additional confirmation of the target protein presence on the AFM chips after biospecific fishing to avoid any artifacts.
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http://dx.doi.org/10.2147/IJN.S66946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200055PMC
June 2015

Highly sensitive protein detection by combination of atomic force microscopy fishing with charge generation and mass spectrometry analysis.

FEBS J 2014 Oct 4;281(20):4705-17. Epub 2014 Oct 4.

Institute of Biomedical Chemistry RAMS, Moscow, Russia.

An approach combining atomic force microscopy (AFM) fishing and mass spectrometry (MS) analysis to detect proteins at ultra-low concentrations is proposed. Fishing out protein molecules onto a highly oriented pyrolytic graphite surface coated with polytetrafluoroethylene film was carried out with and without application of an external electric field. After that they were visualized by AFM and identified by MS. It was found that injection of solution leads to charge generation in the solution, and an electric potential within the measuring cell is induced. It was demonstrated that without an external electric field in the rapid injection input of diluted protein solution the fishing is efficient, as opposed to slow fluid input. The high sensitivity of this method was demonstrated by detection of human serum albumin and human cytochrome b5 in 10(-17) -10(-18) m water solutions. It was shown that an external negative voltage applied to highly oriented pyrolytic graphite hinders the protein fishing. The efficiency of fishing with an external positive voltage was similar to that obtained without applying any voltage.
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http://dx.doi.org/10.1111/febs.13011DOI Listing
October 2014

SOI nanowire for the high-sensitive detection of HBsAg and α-fetoprotein.

Lab Chip 2012 Dec;12(23):5104-11

Institute of Biomedical Chemistry RAMS, Russia, Moscow.

Silicon-on-isolator-nanowires (SOI-NWs) were used for the label-free, real-time biospecific detection of the hepatitis B marker HBsAg and cancer marker α-fetoprotein (AFP). Specific protein-protein recognition was carried out using individual NWs that were functionalized with antibodies. To solve the problem of non-specific binding of target protein molecules to the sensor element the use of a reference NW with immobilized antibodies against non-target proteins was proposed. Using individual SOI-NW surface functionalization allowed the fabrication of a NW array, containing working NWs and reference NWs within one chip. It was shown that this approach allows us to reach a detection limit up to 10(-14) and 10(-15) M for HBsAg and AFP, respectively. Our investigations also allowed us to reveal the influence of the charged state of the target protein molecules and antibodies in solutions with various pH values on the target protein detection limit. A high sensitivity NW-detector is of interest for the creation of diagnosticums for hepatitis B and for the early stages of cancer diseases.
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http://dx.doi.org/10.1039/c2lc40555eDOI Listing
December 2012

Nanotechnologies in proteomics.

Proteomics 2006 Mar;6(5):1399-414

Institute of Biomedical Chemistry, Moscow, Russia.

Progress in proteomic researches is largely determined by development and implementation of new methods for the revelation and identification of proteins in biological material in a wide concentration range (from 10(-3) M to single molecules). The most perspective approaches to address this problem involve (i) nanotechnological physicochemical procedures for the separation of multicomponent protein mixtures; among these of particular interest are biospecific nanotechnological procedures for selection of proteins from multicomponent protein mixtures with their subsequent concentration on solid support; (ii) identification and counting of single molecules by use of molecular detectors. The prototypes of biospecific nanotechnological procedures, based on the capture of ligand biomolecules by biomolecules of immobilized ligate and the concentration of the captured ligands on appropriate surfaces, are well known; these are affinity chromatography, magnetic biobeads technology, different biosensor methods, etc. Here, we review the most promising nanotechnological approaches for selection of proteins and kinetic characterization of their complexes based on these biospecific methods with subsequent MS/MS identification of proteins and protein complexes. Two major groups of methods for the analysis and identification of individual molecules and their complexes by use of molecular detectors will be reviewed: scanning probe microscopy (SPM) (including atomic-force microscopy) and cryomassdetector technology.
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http://dx.doi.org/10.1002/pmic.200402087DOI Listing
March 2006

Direct electron transfer of cytochrome P450 2B4 at electrodes modified with nonionic detergent and colloidal clay nanoparticles.

Anal Chem 2004 Oct;76(20):6046-52

Department of Analytical Biochemistry, University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Golm, Germany.

A method for construction of biosensors with membranous cytochrome P450 isoenzymes was developed based on clay/detergent/protein mixed films. Thin films of sodium montmorillonite colloid with incorporated cytochrome P450 2B4 (CYP2B4) with nonionic detergent were prepared on glassy carbon electrodes. The modified electrodes were electrochemically characterized, and bioelectrocatalytic reactions were followed. CYP2B4 can be reduced fast on clay-modified glassy carbon electrodes in the presence of the nonionic detergent Tween 80. In anaerobic solutions, reversible oxidation and reduction is obtained with a formal potential between -0.292 and -0.305 V vs Ag/AgCl 1 M KCl depending on the preparation of the biosensor. In air-saturated solution, bioelectrocatalytic reduction currents can be obtained with the CYP2B4-modified electrode on addition of typical substrates such as aminopyrine and benzphetamine. This reaction was suppressed when methyrapone, an inhibitor of P450 reactions, was present. Measurement of product formation also indicates the bioelectrocatalysis by CYP2B4.
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http://dx.doi.org/10.1021/ac049927yDOI Listing
October 2004

Atomic force microscopy revelation of molecular complexes in the multiprotein cytochrome P450 2B4-containing system.

Proteomics 2004 Aug;4(8):2390-6

V.N.Orekhovich Institute of Biomedical Chemistry RAMS, Moscow, Russia.

The application of atomic force microscopy (AFM) to the identification and visualization of individual molecules and their complexes in a reconstituted monooxygenase P450 2B4 system without the phospholipid was demonstrated. The method employed in this study distinguishes the monomeric proteins from their binary complexes and, also, the binary from the ternary complexes. The AFM images of the full-length P450 2B4 system's constituent components - cytochrome P450 2B4 (2B4), NADPH-cytochrome P450 reductase and cytochrome b5 (b5), were obtained on highly-oriented pyrolitic graphite. The typical heights of the d-2B4, d-flavoprotein (Fp) and d-b5 molecules were measured and found to be 2.2 +/- 0.2, 2.3 +/- 0.2 and 1.8 +/- 0.1 nm, respectively. The measured heights of the binary d-Fp/d-2B4 and d-2B4/d-b5 complexes were estimated to be 3.4 +/- 0.2 and 2.8 +/- 0.2 nm, respectively. No formation of d-Fp/d-b5 complexes was registered. The ternary d-Fp/d-2B4/d-b5 complexes were visualized and their heights were found to be roughly equal to 4.3 +/- 0.3 nm and 6.2 +/- 0.3 nm.
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http://dx.doi.org/10.1002/pmic.200300751DOI Listing
August 2004

Protein-protein interactions as a target for drugs in proteomics.

Proteomics 2003 Apr;3(4):380-91

V N Orekhovich Institute of Biomedical Chemistry, Moscow, Russia.

Protein-protein interactions play a central role in numerous processes in the cell and are one of the main fields of functional proteomics. This review highlights the methods of bioinformatics and functional proteomics of protein-protein interaction investigation. The structures and properties of contact surfaces, forces involved in protein-protein interactions, kinetic and thermodynamic parameters of these reactions were considered. The properties of protein contact surfaces depend on their functions. The contact surfaces of permanent complexes resemble domain contacts or the protein core and it is reasonable to consider such complex formation as a continuation of protein folding. Characteristics of contact surfaces of temporary protein complexes share some similarities with active sites of enzymes. The contact surfaces of the temporary protein complexes have unique structure and properties and they are more conservative in comparison with active site of enzymes. So they represent prospective targets for a new generation of drugs. During the last decade, numerous investigations were undertaken to find or design small molecules that block protein dimerization or protein(peptide)-receptor interaction, or, on the contrary, to induce protein dimerization.
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http://dx.doi.org/10.1002/pmic.200390053DOI Listing
April 2003

Atomic force microscopy detection of molecular complexes in multiprotein P450cam containing monooxygenase system.

Proteomics 2002 Dec;2(12):1699-705

Institute of Biomedical Chemistry, Moscow, Russia.

The application of atomic force microscopy (AFM) technique in proteomic research, identification and visualization of individual molecules and molecular complexes within the P450cam containing monooxygenase system was demonstrated. The method distinguishes between the binary protein complexes and appropriate monomeric proteins and, also, between the binary and ternary complexes. The AFM images of the components of a cytochrome P450cam containing monooxygenase system - cytochrome P450cam (P450cam), putidaredoxin (Pd) and putidaredoxin reductase (PdR) - were obtained on a mica support. The molecules of P450cam, Pd and PdR were found to have typical heights of 2.6 +/- 0.3 nm, 2.0 +/- 0.3 and 2.8 +/- 0.3 nm, respectively. The measured heights of the binary Pd/PdR and P450cam/PdR complexes were 4.9 +/- 0.3 nm and 5.1 +/- 0.3 nm, respectively. The binary P450cam/Pd complexes were found to have a typical height of about (3.9 / 5.7 nm) and the ternary PdR/Pd/P450cam complexes, a typical height of about 9.1 +/- 0.3 nm.
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http://dx.doi.org/10.1002/1615-9861(200212)2:12<1699::AID-PROT1699>3.0.CO;2-RDOI Listing
December 2002

Optical biosensor and scanning probe microscopy studies of cytochrome P450 interactions with redox partners and phospholipid layers.

Methods Enzymol 2002 ;357:94-103

Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow 119992, Russia.

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http://dx.doi.org/10.1016/s0076-6879(02)57669-2DOI Listing
January 2003
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