rus
Issue #2/2014
E.Dubrovin, G.Meshkov, I.Yaminskiy
Observation of the Tobacco Mosaic Virus in the Laboratory Course of Scanning Probe Microscopy
Observation of the Tobacco Mosaic Virus in the Laboratory Course of Scanning Probe Microscopy
Successful development of the technologies was often followed the path of imitation of natural objects and processes, which called biomimicry. Undoubtedly, the biomimicry has a great potential also in the nanotechnology. The viruses are ones of the simplest (in terms of structure) natural objects, whose dimensions are in the nanometer range.
Теги: atomic-force microscopy research of the surface topography sample preparation атомно-силовая микроскопия исследование топографии поверхности пробоподготовка
Virus (from the Latin: Virus – poison) - a microscopic particle that can infect the cells of living organisms. The viruses are obligate parasites - they cannot reproduce themselves outside the cell. Today, the viruses reproduce themselves in the cells of plants, animals, fungi and bacteria (the latter usually called bacteriophages) are known. The virus particles (virions) represent proteinaceous capsule – capsid, containing the viral genome, presented by one or more molecules of DNA or RNA. The capsid is built of capsomeres - protein complexes, in turn consisting of the protomers. The nucleic acid in complex with proteins is denoted by the term nucleocapsid. Some viruses also have an outer lipid cover. The sizes of different viruses are ranged from 20 to 500 nm and more. The virions often have regular geometric shape (icosahedron, cylinder), more complicated structures also occurs (figs. 1-3).
The first known virus - TMV - was grown by Russian researcher Dmitry Ivanovsky in 1882 and studied in details by Dutch scientist Martinus Beyzherink in 1898. Currently, over 5,000 types viruses are described. The viruses are one of the most common forms of existence of organic matter in the world by the number: the ocean's contain a colossal amount of bacteriophages (about 1011 particles per milliliter of water).
Role of viruses in the biology
and its use by the human
The viruses have a genetic connection with the flora and fauna of the Earth. According to the recent studies, the human genome on more than 30% consists of the information encoded by the virus-like elements. The so-called horizontal gene transfer can occur with the help of viruses, i.e. the transfer of genetic information not from father to son and so on, but between two unrelated individuals (or even belong to the different species). Due to this property, the viruses significantly contribute to the biological evolution of the planet.
The viruses are used in molecular and cellular biology, as their participation can help to create the simple systems for studying of the cell’s functions. For example, the use of viruses has helped in understanding the basic mechanisms of molecular genetics and immunology. The viruses are commonly used as gene vectors in genetic engineering and, in particular, when creating the genetically engineered organisms (for example, cauliflower mosaic virus). Treatment of the infectious diseases using bacteriophages - phage therapy – is actively developed as an alternative to the antibiotics, which have side effects and low efficiency due to the adaptability of bacteria to them.
The viruses are a promising target for the development of nanotechnology (however, the applications described above, can also be attributed to this type of technology). From the point of view of materials science, the viruses are the organic nanoparticles, which are able to penetrate through the walls of the host cells. The size and shape of viruses, as well as the number and nature of functional groups on their surface, are exactly defined, whereby the viruses can be used as templates for organization of the material on the nanoscale level. For example, the particles of cowpea mosaic virus (CPMV) were used to amplify the signal of the biosensor based on the DNA matrix. In this study, the viral particles separated the fluorescent dyes in order to prevent occurrence of non-fluorescent dimers that act as quenchers [1]. An another example is the use of CPMV as nanomodel for molecular electronics [2]. The baculoviruses coated with metal nanoparticles or conducting polymers such as polyaniline, are used for creating of nanowires and new conductive materials.
The structure and composition of TMV
TMV is a classic object of virology. It causes tobacco mosaic disease, which manifests itself as spots on the leaves. The virus represents the rods with the diameter of 18 nm and the length of 300 nm (fig.1) consisting of RNA and protein [3]. The RNA molecule of the nucleotide with the length of 6390 bases has the form of a spiral coil with a length of 2.3 nm, while the shell is formed of 2130 protein subunits. The protein of the cover is self-organizing in a rod-helical structure (16.3 monomeric protein on one turn of the spiral) around the RNA, which is located in a cylindrical ring with the radius of about 6 nm, which could not be affected by the cellular enzymes, destroying it. The electron micrographs show the presence of virions in the internal channel with the diameter of about 4 nm. TMV is a thermostable virus: it can withstand the temperatures of up to 50°C for 30 minutes. The modern virology owes not only its birth to this virus, but also the basic concepts that add up to the present time, so its further study, especially with new techniques, is relevant to this day.
Which the atomic force microscopy of viral particles is required for?
At the AFM study of the viruses surface topography in continuous or intermittent contact, the following issues can be studied: the particle’s morphology, especially the adsorption of viral particles to the surface (adsorption kinetics, the degree of adhesion, the relative orientation of the particles on the surface [4-6]), dependence of the adsorption on the surface form (e.g., at modification of the substrate with the various chemical compounds [5]), crystallization processes at the surface of the viral particles [7], mechanical strength and individual virion protein subunits [8], localization of the genome inside of the virions [9]. Such processes can also be studied, as: dressing-undressing of viral particles [10], release of RNA (DNA) from the particles [4], the interaction with the host cell.
The examples of AFM images of six types of viruses are shown in fig. 2. Fig. 3 shows the AFM image, which shows the adsorption of the bacteriophages on the Escherichia coli cells’ surface. AFM can also manipulate the individual viral particles using cantilever and create a given architecture in the nanoscale level. In addition, the modes of AFM spectroscopy (removal of power curves, i.e. the dependence of the force, exerted from the side of the cantilever on the sample from the vertical position of the piezoscanner), the additional opportunities occur to quantitatively study the strength of interaction of the virion with the substrate, RNA (DNA) with a protein cover, the protein subunits between each other.
The AFM provides unique information about the sample. It is extremely important that the AFM helps to study the viral particles without additional contrasting by the atoms of heavy metals (as it is needed, for example, in electron microscopy). You can get a three-dimensional topography with high resolution (the ultimate solution for soft objects: the fractions of nanometers for lateral and the fractions of angstroms for vertical directions), to study the living objects on the surface, as in the air, and aquatic environments. The information obtained by AFM is required both for basic science and for development of the nanotechnology.
The TMV were repeatedly investigated by AFM [4,5,11-13]. In particular, a higher adsorption capacity of virus particles on the surface of highly oriented pyrolytic graphite (HOPG) as compared with the surface of mica [4] was noted, which was explained by hydrophobic interactions of the particles with the substrate. In this work, the "undressing" of the virus particle and extraction of the RNA molecule after treatment of the TMV virions with dimethyl sulfoxide or the urea were observed. A more detailed study of the adsorption of TMV particles on surfaces modified by various chemical compounds showed high sensitivity to the nature of the modifier of the adsorption process [5]. For example, modification of the mica by cetyltrimethylammonium bromide increased adhesion of TMV to the mica in more than 10 times. It should be noted that in the study of the TMV on such substrates as mica or graphite, we face with the physical adsorption of the virions on the surface, i.e. adsorption primarily due to van der Waals interactions (and the hydrophobic for graphite surface).
TMV samples preparation for AFM
A typical procedure of TMV sample preparation for the study by AFM is described in [5].
Before application to the substrate for AFM, the aliquot of virus solution was centrifuged at 13,000 rpm on the Eppendorf centrifuge 5415S, diluted with phosphate buffer (5 mM sodium phosphate, pH 7.3, 150 mM NaCl) to a concentration of 1.3 mg/ml for the mica or up to 0.325 mg/ml for the graphite. 5 mcl of the sample were applied to a fresh chip of the mica or HOPG and were incubated in a humid chamber for 30 min on mica or 15 min on the graphite. The sorbed virus samples were washed for 2-3 times in tridistilled water and dried in a vacuum desiccator.
Preparation and processing
of AFM images of the TMV sample
An image of the TMV sample can be get in a resonant mode. The atomic force microscopes "FemtoScan" are used in the laboratory course of scanning probe microscopy in the Moscow State University. Data processing software "FemtoScan" is used [14, 15]. Software with the one month test period can be downloaded from websites www.nanoscopy.ru and www.nanoscopy.net [15].
The command “Distance” of the software’s context menu measures the lengths of viral particles and constructs the histogram of length distribution. The measured particle is convenient to tag with the markers using the context menu command “Marks”. The most probable value of the length of the particles virus can be found and the variance of the resulting distribution can be estimated at the image analysis.
The training of a student in the laboratory course of scanning probe microscopy helps him to learn the theoretical material and to adequately interpret the received results [16]. ■
The first known virus - TMV - was grown by Russian researcher Dmitry Ivanovsky in 1882 and studied in details by Dutch scientist Martinus Beyzherink in 1898. Currently, over 5,000 types viruses are described. The viruses are one of the most common forms of existence of organic matter in the world by the number: the ocean's contain a colossal amount of bacteriophages (about 1011 particles per milliliter of water).
Role of viruses in the biology
and its use by the human
The viruses have a genetic connection with the flora and fauna of the Earth. According to the recent studies, the human genome on more than 30% consists of the information encoded by the virus-like elements. The so-called horizontal gene transfer can occur with the help of viruses, i.e. the transfer of genetic information not from father to son and so on, but between two unrelated individuals (or even belong to the different species). Due to this property, the viruses significantly contribute to the biological evolution of the planet.
The viruses are used in molecular and cellular biology, as their participation can help to create the simple systems for studying of the cell’s functions. For example, the use of viruses has helped in understanding the basic mechanisms of molecular genetics and immunology. The viruses are commonly used as gene vectors in genetic engineering and, in particular, when creating the genetically engineered organisms (for example, cauliflower mosaic virus). Treatment of the infectious diseases using bacteriophages - phage therapy – is actively developed as an alternative to the antibiotics, which have side effects and low efficiency due to the adaptability of bacteria to them.
The viruses are a promising target for the development of nanotechnology (however, the applications described above, can also be attributed to this type of technology). From the point of view of materials science, the viruses are the organic nanoparticles, which are able to penetrate through the walls of the host cells. The size and shape of viruses, as well as the number and nature of functional groups on their surface, are exactly defined, whereby the viruses can be used as templates for organization of the material on the nanoscale level. For example, the particles of cowpea mosaic virus (CPMV) were used to amplify the signal of the biosensor based on the DNA matrix. In this study, the viral particles separated the fluorescent dyes in order to prevent occurrence of non-fluorescent dimers that act as quenchers [1]. An another example is the use of CPMV as nanomodel for molecular electronics [2]. The baculoviruses coated with metal nanoparticles or conducting polymers such as polyaniline, are used for creating of nanowires and new conductive materials.
The structure and composition of TMV
TMV is a classic object of virology. It causes tobacco mosaic disease, which manifests itself as spots on the leaves. The virus represents the rods with the diameter of 18 nm and the length of 300 nm (fig.1) consisting of RNA and protein [3]. The RNA molecule of the nucleotide with the length of 6390 bases has the form of a spiral coil with a length of 2.3 nm, while the shell is formed of 2130 protein subunits. The protein of the cover is self-organizing in a rod-helical structure (16.3 monomeric protein on one turn of the spiral) around the RNA, which is located in a cylindrical ring with the radius of about 6 nm, which could not be affected by the cellular enzymes, destroying it. The electron micrographs show the presence of virions in the internal channel with the diameter of about 4 nm. TMV is a thermostable virus: it can withstand the temperatures of up to 50°C for 30 minutes. The modern virology owes not only its birth to this virus, but also the basic concepts that add up to the present time, so its further study, especially with new techniques, is relevant to this day.
Which the atomic force microscopy of viral particles is required for?
At the AFM study of the viruses surface topography in continuous or intermittent contact, the following issues can be studied: the particle’s morphology, especially the adsorption of viral particles to the surface (adsorption kinetics, the degree of adhesion, the relative orientation of the particles on the surface [4-6]), dependence of the adsorption on the surface form (e.g., at modification of the substrate with the various chemical compounds [5]), crystallization processes at the surface of the viral particles [7], mechanical strength and individual virion protein subunits [8], localization of the genome inside of the virions [9]. Such processes can also be studied, as: dressing-undressing of viral particles [10], release of RNA (DNA) from the particles [4], the interaction with the host cell.
The examples of AFM images of six types of viruses are shown in fig. 2. Fig. 3 shows the AFM image, which shows the adsorption of the bacteriophages on the Escherichia coli cells’ surface. AFM can also manipulate the individual viral particles using cantilever and create a given architecture in the nanoscale level. In addition, the modes of AFM spectroscopy (removal of power curves, i.e. the dependence of the force, exerted from the side of the cantilever on the sample from the vertical position of the piezoscanner), the additional opportunities occur to quantitatively study the strength of interaction of the virion with the substrate, RNA (DNA) with a protein cover, the protein subunits between each other.
The AFM provides unique information about the sample. It is extremely important that the AFM helps to study the viral particles without additional contrasting by the atoms of heavy metals (as it is needed, for example, in electron microscopy). You can get a three-dimensional topography with high resolution (the ultimate solution for soft objects: the fractions of nanometers for lateral and the fractions of angstroms for vertical directions), to study the living objects on the surface, as in the air, and aquatic environments. The information obtained by AFM is required both for basic science and for development of the nanotechnology.
The TMV were repeatedly investigated by AFM [4,5,11-13]. In particular, a higher adsorption capacity of virus particles on the surface of highly oriented pyrolytic graphite (HOPG) as compared with the surface of mica [4] was noted, which was explained by hydrophobic interactions of the particles with the substrate. In this work, the "undressing" of the virus particle and extraction of the RNA molecule after treatment of the TMV virions with dimethyl sulfoxide or the urea were observed. A more detailed study of the adsorption of TMV particles on surfaces modified by various chemical compounds showed high sensitivity to the nature of the modifier of the adsorption process [5]. For example, modification of the mica by cetyltrimethylammonium bromide increased adhesion of TMV to the mica in more than 10 times. It should be noted that in the study of the TMV on such substrates as mica or graphite, we face with the physical adsorption of the virions on the surface, i.e. adsorption primarily due to van der Waals interactions (and the hydrophobic for graphite surface).
TMV samples preparation for AFM
A typical procedure of TMV sample preparation for the study by AFM is described in [5].
Before application to the substrate for AFM, the aliquot of virus solution was centrifuged at 13,000 rpm on the Eppendorf centrifuge 5415S, diluted with phosphate buffer (5 mM sodium phosphate, pH 7.3, 150 mM NaCl) to a concentration of 1.3 mg/ml for the mica or up to 0.325 mg/ml for the graphite. 5 mcl of the sample were applied to a fresh chip of the mica or HOPG and were incubated in a humid chamber for 30 min on mica or 15 min on the graphite. The sorbed virus samples were washed for 2-3 times in tridistilled water and dried in a vacuum desiccator.
Preparation and processing
of AFM images of the TMV sample
An image of the TMV sample can be get in a resonant mode. The atomic force microscopes "FemtoScan" are used in the laboratory course of scanning probe microscopy in the Moscow State University. Data processing software "FemtoScan" is used [14, 15]. Software with the one month test period can be downloaded from websites www.nanoscopy.ru and www.nanoscopy.net [15].
The command “Distance” of the software’s context menu measures the lengths of viral particles and constructs the histogram of length distribution. The measured particle is convenient to tag with the markers using the context menu command “Marks”. The most probable value of the length of the particles virus can be found and the variance of the resulting distribution can be estimated at the image analysis.
The training of a student in the laboratory course of scanning probe microscopy helps him to learn the theoretical material and to adequately interpret the received results [16]. ■
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