rus
Issue #1/2017
A.Akhmetova, I.Yaminsky
Early detection of viruses and bacteria using methods of nanotechnology
Early detection of viruses and bacteria using methods of nanotechnology
Modern nanotechnology opens up new effective ways of early detection of viral and bacterial infections. This paper presents two related methods based on the use of scanning probe microscopes and piezoelectric biochips.
Теги: biochip flow-through liquid cell scanning probe microscopy биочип проточная жидкостная ячейка сканирующий зондовый микроскоп
The detection of the causative agent of an infectious viral or bacterial diseases is a demanding task. Even more important is the detection of the virus in the environment until it struck the plant, animal or human. Our project "Development of sensor technologies for molecular diagnostics for personalized medicine" is dedicated to this purpose.
Modern personalized medicine is largely focused on disease prevention to avoid disease and its possible consequences. The development of science and technologies allows to create a new effective measures for prevention and treatment of infections. However, there are significant difficulties due to the return of the old and emergence of new infectious diseases that are a serious challenge to modern medicine. Many scientists are actively involved in solving these problems.
The Science journal has identified 10 of the most groundbreaking technologies of 2016. Among them is the work of David Baker, Professor of biochemistry at the University of Washington. Together with his team, he learned how to model proteins and predict the formation of their spatial structures. For example, he was able to determine how the synthesized proteins, changing their configuration, will form a spatial structure. This discovery gave impetus to the creation of flu vaccines of the new generation. Flu viruses mutate fairly quickly, which makes difficult the development of a drug that would be effective for all strains. But each strain of influenza contains the hemagglutinin protein that helps it invade a host cell. Part of the hemagglutinin molecule, consisting of α-helices, is identical in many strains. D.Baker has developed a new protein that binds to the unchanging part of hemagglutinin, thereby preventing the invasion of the virus into the cell. The results of the experiment with the mice with introduction of a new protein and a lethal dose of influenza virus were published in 2016. As a result of experiments, it was shown that rodents were protected from the virus [1].
Scanning probe microscopy allows to detect both bacterial cells (Fig.1) and individual viral particles (Fig.2). Fig.2 shows particles of tobacco mosaic virus, which is actively used for virological research and educational purposes in laboratory workshops [2].
To identify viruses, we investigate the ability of hemagglutinin to attach to the cells. In the framework of the project "Development of sensor technologies for molecular diagnostics for personalized medicine " we develop two devices for detection of influenza A virus and E.coli bacteria: an advanced scanning probe microscope "FemtoScan" and the biosensor based on piezoelectric cantilevers. Both devices use a special biochip on the basis of a piezoceramic disk, the electrodes of which are covered by a sensory layer. For detection of influenza virus sensory layer of the biochip contains polysaccharides with sialic acids, providing biospecifically binding to hemagglutinin of the virus. In case of detection of bacterial cells, the antibodies against surface antigenic determinants of the cells are placed on the surface of the sensory layer. Details of the design and the working principle of biosensor and microscope are described in previous publications [3–5]. Detection of biological agents in the biosensor is carried out by registration of the amplitude, phase, frequency and the quality factor of the mechanical vibrations of the biochip. In a probe microscope, in addition to the determination of these parameters, you can carry out the direct calculation of the number of pathogens on the surface of the biochip.
In 2016, to create a workable prototype of the biosensor we have developed the following key elements:
flow-through liquid cell of biosensor in which the biochip is placed and the sample is circulated;
control unit of the biosensor, which detects the vibrations of the biochip and provides the transfer of this information to the computer;
package for the biosensor with cell and biochip;
holder of a cantilever for flow-through liquid cell.
Electronic measuring system and software are essential. As the last, the multiuser management platform of the FemtoScan scanning probe microscope is chosen. The software allows to determine the resonant frequency of biochip. The most sensitive and noise-resistant method of determining the resonant frequency, as was shown earlier, is the method of "center of mass" [6].
In the experiments we use H3N6 and H4N6 influenza virus weakened by formaldehyde, which was provided by the Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of RAS. Images of H3N6 and H4N6 influenza virus strains are shown in Fig.3.
The models of the sealed flow-through liquid cell (Fig.4) and of the head for resonance atomic force microscopy were created for the advanced scanning probe microscope. The design of the cell allows to install it in the microscope, and remove from the microscope in the assembled state without seal failure. This design is applied for the patents [7, 8]. Flow-through liquid cell can be designed for single use only. In any case, the biological entities –viruses and bacteria are in a closed isolated space during the measurement in scanning probe microscope.
The results of the first year of joint development of Lomonosov MSU and Advanced Technologies Center were presented at the VUZPROMEXPO exhibition, which was held on 14–15 December 2016. We have demonstrated the model of the biosensor including control unit and a package in which a flow-through liquid cell with the biochip are located, and the model of the advanced scanning probe microscope with a sealed flow-through liquid cell (Fig.5). ■
The study was performed with financial support of RFBR (research project No. 15-04-07678) and Ministry of education and science under contract No. 02.G25.31.0135. The authors are grateful to D.Sc., head of the laboratory of molecular biology of influenza viruses at Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of RAS, Alexandra S. Gambaryan, for discussion and help.
Modern personalized medicine is largely focused on disease prevention to avoid disease and its possible consequences. The development of science and technologies allows to create a new effective measures for prevention and treatment of infections. However, there are significant difficulties due to the return of the old and emergence of new infectious diseases that are a serious challenge to modern medicine. Many scientists are actively involved in solving these problems.
The Science journal has identified 10 of the most groundbreaking technologies of 2016. Among them is the work of David Baker, Professor of biochemistry at the University of Washington. Together with his team, he learned how to model proteins and predict the formation of their spatial structures. For example, he was able to determine how the synthesized proteins, changing their configuration, will form a spatial structure. This discovery gave impetus to the creation of flu vaccines of the new generation. Flu viruses mutate fairly quickly, which makes difficult the development of a drug that would be effective for all strains. But each strain of influenza contains the hemagglutinin protein that helps it invade a host cell. Part of the hemagglutinin molecule, consisting of α-helices, is identical in many strains. D.Baker has developed a new protein that binds to the unchanging part of hemagglutinin, thereby preventing the invasion of the virus into the cell. The results of the experiment with the mice with introduction of a new protein and a lethal dose of influenza virus were published in 2016. As a result of experiments, it was shown that rodents were protected from the virus [1].
Scanning probe microscopy allows to detect both bacterial cells (Fig.1) and individual viral particles (Fig.2). Fig.2 shows particles of tobacco mosaic virus, which is actively used for virological research and educational purposes in laboratory workshops [2].
To identify viruses, we investigate the ability of hemagglutinin to attach to the cells. In the framework of the project "Development of sensor technologies for molecular diagnostics for personalized medicine " we develop two devices for detection of influenza A virus and E.coli bacteria: an advanced scanning probe microscope "FemtoScan" and the biosensor based on piezoelectric cantilevers. Both devices use a special biochip on the basis of a piezoceramic disk, the electrodes of which are covered by a sensory layer. For detection of influenza virus sensory layer of the biochip contains polysaccharides with sialic acids, providing biospecifically binding to hemagglutinin of the virus. In case of detection of bacterial cells, the antibodies against surface antigenic determinants of the cells are placed on the surface of the sensory layer. Details of the design and the working principle of biosensor and microscope are described in previous publications [3–5]. Detection of biological agents in the biosensor is carried out by registration of the amplitude, phase, frequency and the quality factor of the mechanical vibrations of the biochip. In a probe microscope, in addition to the determination of these parameters, you can carry out the direct calculation of the number of pathogens on the surface of the biochip.
In 2016, to create a workable prototype of the biosensor we have developed the following key elements:
flow-through liquid cell of biosensor in which the biochip is placed and the sample is circulated;
control unit of the biosensor, which detects the vibrations of the biochip and provides the transfer of this information to the computer;
package for the biosensor with cell and biochip;
holder of a cantilever for flow-through liquid cell.
Electronic measuring system and software are essential. As the last, the multiuser management platform of the FemtoScan scanning probe microscope is chosen. The software allows to determine the resonant frequency of biochip. The most sensitive and noise-resistant method of determining the resonant frequency, as was shown earlier, is the method of "center of mass" [6].
In the experiments we use H3N6 and H4N6 influenza virus weakened by formaldehyde, which was provided by the Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of RAS. Images of H3N6 and H4N6 influenza virus strains are shown in Fig.3.
The models of the sealed flow-through liquid cell (Fig.4) and of the head for resonance atomic force microscopy were created for the advanced scanning probe microscope. The design of the cell allows to install it in the microscope, and remove from the microscope in the assembled state without seal failure. This design is applied for the patents [7, 8]. Flow-through liquid cell can be designed for single use only. In any case, the biological entities –viruses and bacteria are in a closed isolated space during the measurement in scanning probe microscope.
The results of the first year of joint development of Lomonosov MSU and Advanced Technologies Center were presented at the VUZPROMEXPO exhibition, which was held on 14–15 December 2016. We have demonstrated the model of the biosensor including control unit and a package in which a flow-through liquid cell with the biochip are located, and the model of the advanced scanning probe microscope with a sealed flow-through liquid cell (Fig.5). ■
The study was performed with financial support of RFBR (research project No. 15-04-07678) and Ministry of education and science under contract No. 02.G25.31.0135. The authors are grateful to D.Sc., head of the laboratory of molecular biology of influenza viruses at Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of RAS, Alexandra S. Gambaryan, for discussion and help.
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