rus
Issue #1/2014
I.Yaminsky, P.Gorelkin, A.Erofeev, O.Sinitsyna, G.Meshkov
Bionanoscopy in Biology and Medicine
Bionanoscopy in Biology and Medicine
Tools of nanoanalytics open new opportunities for observation of the wildlife. In the second part of the review the data concerning observation of the nucleic acids, proteins, bacteria, cells and tissues are generalized.
Теги: atomic balance bionanoscopy nanosansors scanning microscopy атомные весы бионаноскопия. наносенсоры сканирующая микроскопия
Invention of the scanning tunnel microscopy (SТМ) and atomic-force microscopy (АFМ) opened new opportunities for studying of DNA molecule. The initial observations done with STM were hardly reproducible and had serious artefacts. The tunnel microscopy had problems not only with the substrate – graphite, because of its poor adsorption ability, but also with tunneling of the electrons near a molecule surface.
DNA
When reliably fixed on a modified graphite surface, DNA is visible in the negative contrast not as an eminence, which is characteristic for most objects with a final electroconductivity, but as a deepening [1]. Well reproducible and informative results (figs.1 and 2) were obtained, when AFM was applied for DNA studies, as well as the methods of deposition on a mica surface borrowed from the electronic microscopy. In a water solution DNA and the surface of a freshly split off mica have a negative charge, therefore Coulomb repulsion appears between them.
For neutralization of the negative charge solutions of bivalent ions are necessary. Good results are achieved, when mica is modified by small concentrations of organic substances: aminopropiltrietoxisilan or silatran. Such a surface gives an insignificant background not interfering with obtaining of quality images of DNA.
АFМ allows us to study a conformational state of DNA molecules in solutions [3]. In order to estimate the geometry of a biomacromolecule, we should fix it on a substrate – then we will be able to observe conformation of a molecule not freely floating in a solution, but the one fixed on the interphase liquid-solid body border. At that, we must be sure in the minimum influence of a substrate.
RNA
RNA is a more complex object than DNA, because its molecule is less rigid and demands an especially delicate treatment. In one of the first observations [4] in order to eliminate possible artefacts, consistently examined were RNA-containing virus particles (tobacco mosaic virus), intermediate stages of a partial destruction of a protein shell (fig.3), loose ends of RNA and the moment of release of the molecules from a protein shell under the influence of urea. Helpful information for molecular biology was obtained due to research of the complex formation of RNA with various proteins, in particular, the transport ones.
Proteins. Single molecules and complexes
The sizes of separate protein molecules are comparable with the curve radius of the АFМ probe, therefore, a submolecular resolution can be obtained only in research of proteins of high molecular weight. So far the attempts to ensure packing of chains in small proteins, for example, lysozyme (molecular weight 14 kD) were unsuccessful. At that the works are known, in which the protein monomers were reliably distinguished from dimers and multimers (fig.4 [5]).
Probe microscopy (PМ) can be used for registration of a specific antigene-antibody binding. Measurements of the sizes of separate particles and the achieved accuracy allow us to separate single antibodies and antigenes from their complexes. PМ gives us a chance to obtain information about the character of aggregation of the proteins and their binding with the nucleic acids. In [6], for example, the character of binding of a transport protein with a virus RNA (fig.5) was determined.
protein Crystals
An advantage of PМ in observation of the protein crystals in saturated solutions is a possibility of registration of the processes’ dynamics in liquid environments, natural for many biological objects. Research works allowed us to register the kinetics of growth of the dislocation hills and two-dimensional germs of many protein crystals at the level of separate molecules [7, 8]. For example, for lysozyme the speed of movement of steps and breaks, probability of joining and detachment of building units, dependence of the kinetic parameters on such factors as temperature and supersaturation [9] were measured.
АFМ allows us to observe the structure of a surface of a growing crystal with a molecular resolution and to study the packing of the molecules near the dot defects, which is impossible with the other methods. In [10] reconstruction of the surface of a crystal was revealed, when the packing of the protein molecules on it differed from the volume structure (figs.6 and 7).
Viruses
Although by its resolution PМ yields to the electronic microscopy, ease in preparation of samples justifies its application in examination of viruses for a qualitative analysis of their morphology. In virology the basic application of PМ is not for determination of the structure of viruses, but for studying of the processes occurring with them.
Crystallization processes were observed for various viruses [11, 12]. Stages of destruction of the protein shell of a tobacco mosaic virus with a release of RNA were registered in [4]. Since АFМ allows us to measure the mechanical properties of the nanostructures, Young modulus was determined for the virus particles during their cross-section compression by a probe: for the tobacco mosaic virus it is equal to 3·109 – 4·109 Pa, and for the less rigid potato X-virus – 8·108 Pa (fig.8). Good conformity to the experiments is ensured by calculations based on Hertz model of deformation (fig.9).
The tobacco mosaic virus can be used as a test structure [13] for calibration of Z-movement of АFМ and as a rigidity standard (fig.10). Research works show that viruses can serve as a system for delivery of nanoparticles in human tissues. Also they have found application in ecologically safe manufacture of nanoparticles with the use of plants [14].
Bacterial cells
Bacteria can be considered an ideal object for PM. They have micron sizes, therefore, we can watch separate molecules and their colonies, and also study fragments of cells [2]. Bacterial cells have a rigid polymer carcass and are not deformed by a microscope’s probe during scanning. For observation of bacteria in the air the cells grown up in a nutrient medium are transferred to a distilled water with a concentration up to about 109/ml (short-term stay in water does not lead to destruction of the bacteria). A drop of several microliters is placed on a freshly split mica. Water moistens it forming a thin film, after removal of which separate cells are deposited on a substrate or form monolayer coatings.
AFM makes it possible to obtain 3D images of the bacterial cells (fig.11). Such data can serve as additional criteria for preparation of the bacteria identifiers. In liquids the contrast of images is lower, which is due to the mobile polymer chains of the external membrane [15]. Figure 12 presents an image of E.coli bacterial cells in a buffer solution [15].
AFM allows us to reliably register the structural changes on the surface of the external membrane of a cell wall. Figure 13 presents a parent bacteria of Escherichia coli and a genetically engineered bacteria, into the initial DNA of which gene rfb-a3,4 was introduced, responsible for the synthesis of O-specified side chains of lipopolysaccharides. These chains create a lamellar structure on the surface of a cell, considerably different by its morphology from the surface of a parent cell [16].
Cells of higher organisms
Cells of higher organisms – animals and plants – are not very rigid and, therefore, an interaction with a probe leads to their deformation and an increase of the probe contact area with a cell surface, which reduces the spatial resolution [17]. On the other hand, PМ provides opportunity to measure not only the mechanical rigidity of separate cells, but also their adhesive and frictional properties.
PМ allows us to observe the dynamic processes occurring with the cells. For example, it helps to visualize the movement of the osteoblasts of mice on the surface of a glass substrate [18]; formation of units of these cells and aggregation of fibroblasts of chicken embryos; AFM-images were obtained of the primary osteoblasts of mice during cytocinesis; changes in the cells’ surface accompanying the apoptosis of the cells of an osteosarcoma were investigated. Scanning of the surface of cells with a probe edge allows us to visualize certain details of their internal structure. So, in the image of the surface of a cell it is possible to distinguish elements of cytoskeleton under the membrane, while in the picture of a dividing osteoblast a separating cellular nucleus is clearly visible.
Besides a morphological analysis with a nanometer resolution, we can determine the mechanical parameters of a membrane of cells and detect by means of optical labels the location of the cellular receptors. Also PМ can be applied for the diagnostic purposes, for example, in resuscitation [19].
For the first time the authors by a direct method demonstrated the character of damage (formation of nanopores) in the walls of erythrocytes during electroporation of various intensity. Research works were done on the human venous blood. The blood (1.2 ml) was placed in a quartz cuvette with titanium electrodes, with the distance between them of 15 mm. An electric field impulse was supplied to the electrodes. The source of the calibrated field was clinical defibrillator Lifepak 7 (USA). At the level of intensity of the field E in a solution of 1100 V/cm and duration of an impulse of 10 ms the induced transmembrane potential exceeded the threshold of a potential breakdown of the membrane (φt = 300÷500 mV), and its irreversible electroporation occurred.
Reduction of the number of erythrocytes as a result of haemolysis, and, hence, electroporation, was estimated by a kinetic curved line – dependence D (t), where D is an optical density of the suspension of blood. The kinetic curves of the haemolysis of erythrocytes were described by the exponential function:
D(t) = D0exp(–βt) + D1,
where β is a constant of the speed of reduction of the number of erythrocytes, D1 is the residual level of the cells, D0 is the initial number of erythrocytes. The research of the surface of the erythrocytes before the impact of the electric field was done on blood smears in accordance with a standard technique at a room temperature.
Images of the surface of the membranes (fig.14) were obtained at the Chair of Polymers and Crystals of the Physical Faculty of the Moscow State University named after M.V.Lomonosov by means of an atomic-force microscope FemtoScan, developed by NPP Center of Perspective Technologies. An erythrocyte in a smear on a glass substrate has the form of a discocyte. Its radius is 3600 nm, side height – 460 nm, difference between the highest point and the lowest point – 35 nm. This is shown on a profile in the points of installation of the first and second cursors.
Figure 15 demonstrates erythrocytes after an impulse breakdown with the energy of 112 J. In the enlarged fragment the red and green cursors mark the lines of measurement of the profile of pores, and on the profiles the red dotted line designates average values by the surface. The left profile refers to the left bottom pore, and the right profile – to the right top pore. On the first profile the cursors are fixed in the middle and bottom (deepest) point of the pore, allowing us to measure its depth. On the right profile the diameter of a pore is shown – 93 nm and its depth – 14 nm.
The surface of even a small fragment of an erythrocyte’s membrane is not flat. On it there are roughnesses, hollows and slopes of plane in different directions. In a membrane structure there are also pores which can be a result of heterogeneity of the structure or of a phase transition of the membrane from a liquid-crystal state into a gel, because the blood temperature in the experiments was about 20°С, which is below the phase transition point.
Other biological objects
PМ also allows us to study tissues of animals, surfaces of bones and nails, structure of the tooth enamel. Promising are the opportunities for observation of the surface of leaves and stalks of live plants. Figure 16 presents a three-dimensional image of a fragment of the surface of a human hair. Interesting, that a visually better looking hair at the microlevel has more perfect structure with a smaller number of defects.
All the above-stated concerned, first of all, applications of PМ for research of morphology of the surface of the biological objects, obtaining of three-dimensional images and measuring of the sizes. The main data for studying of the processes’ kinetics are the chains of the three-dimensional images varying in time, for example, pictures of the surface of a growing crystal.
Another prominent aspect of PМ is measurement of the mechanical properties of the biological objects. This information is new to the biological science, therefore, its comprehension demands time. PМ allows us to measure the mechanical rigidity of an object, to determine the force of adhesion, to register variations of the friction factor, receiving a three-dimensional card of the frictional properties of a surface.
Direct measurements of DNA (one of its ends is attached to a substrate, another is connected to the mobile probe of АFМ) allowed us to estimate the force of a chemical bond in a single biomacromolecule. A two-chain molecule of DNA brakes off with application of force of 10–10 N. АFМ ensures measurement of the dynamics of deployment of a single protein globule in a linear chain of aminoacid residues. Scanning of the bacterial monolayer films demonstrated that bacteria Arthrobacter Globophormis in a mummified form are more fragile, than in a vegetative state. If regular live bacteria withstood forces up to 10–6 N, which was by several orders more than the values traditional for scanning in the air, the mummified bacteria were destroyed by such forces completely.
Biological sensors
From the very beginning AFM looked advantageous from the point of view of development of unique biological sensors. Weight of one bacterium – about 10–13 N – ensures an additional deflection of a cantilever with rigidity of 0.06 N/m by 1.6·10–12 m, which corresponds to the level of the limit resolution of a microscope working in a contact mode. However, if we use a mode when a cantilever fluctuates in the resonant frequency sensitive to its weight, it is possible to measure the weight of one bacterium. This is the principle on which the chemical and biological sensors are based.
Having joined a cantilever, the weight of a reagent or biological object changes the frequency of its resonant fluctuations [20]. Measurement of a frequency shift allows us to judge about the weight of the adsorbed substance. Measurements of the weight of nanoparticles are done, as a rule, on specialized atomic scales in which, just as in АFМ, a laser-optical system is used, but there is no necessity in a cantilever with a needle. A key part of most scales is an elastic element, and here the following principle works well – the tinier are the scales, the smaller weights can be weighed. Using this approach, it is possible to determine the weight of one bacterium [21], of a protein [22], and even of a separate atom [23].
High sensitivity is a feature of the cantilevers of a tuning type [24] and those made from piezomaterials. Resonant methods, as a rule, have higher sensitivity in comparison with the static ones. Therefore very interesting is the fact that exactly the static biosensors are very promising for the medical researches [25]. The method’s essence consists in the following:
Surface of one of the sides of a cantilever is covered by a monolayer film of an adsorbing substance with a certain surface tension;
If such a cantilever is placed in a biological liquid, a specific binding may appear on its surface;
Adsorption of a material on the surface of a cantilever leads to a change of the surface tension of the film and to its bending.
For development of chemical and biological sensors the cantilevers with a low mechanical rigidity are used (fig.17) [26]. If the physical principle for their development is clear, the biological aspects of a sensor’s structure are more difficult, because a biologically active coating with a biospecific binding is required. It would also be desirable to have a biosensor suitable for a repeated usage, that is, its ability of adsorption of a concrete substance should be restored by washing. Thus, the problems of creation of a biosensor are more connected with biology, than with the atom-force microscopy.
In general it should be pointed out that the scanning PМ does its first steps in the field of biomedical and biosensor applications. By combining it with the other analytical methods it is possible to make essential progress on the way to the personified medicine, which should take into account the specific features of each individual at a molecular level. ■
The work was done with the support of FTP “Scientific and Scientific-pedagogical Personnel for Innovative Russia for the period of 2009 – 2013” and FTP “R&D in the Priority Directions of Development of the Scientific-technological Complex of Russia for the period of 2007-2013”.
DNA
When reliably fixed on a modified graphite surface, DNA is visible in the negative contrast not as an eminence, which is characteristic for most objects with a final electroconductivity, but as a deepening [1]. Well reproducible and informative results (figs.1 and 2) were obtained, when AFM was applied for DNA studies, as well as the methods of deposition on a mica surface borrowed from the electronic microscopy. In a water solution DNA and the surface of a freshly split off mica have a negative charge, therefore Coulomb repulsion appears between them.
For neutralization of the negative charge solutions of bivalent ions are necessary. Good results are achieved, when mica is modified by small concentrations of organic substances: aminopropiltrietoxisilan or silatran. Such a surface gives an insignificant background not interfering with obtaining of quality images of DNA.
АFМ allows us to study a conformational state of DNA molecules in solutions [3]. In order to estimate the geometry of a biomacromolecule, we should fix it on a substrate – then we will be able to observe conformation of a molecule not freely floating in a solution, but the one fixed on the interphase liquid-solid body border. At that, we must be sure in the minimum influence of a substrate.
RNA
RNA is a more complex object than DNA, because its molecule is less rigid and demands an especially delicate treatment. In one of the first observations [4] in order to eliminate possible artefacts, consistently examined were RNA-containing virus particles (tobacco mosaic virus), intermediate stages of a partial destruction of a protein shell (fig.3), loose ends of RNA and the moment of release of the molecules from a protein shell under the influence of urea. Helpful information for molecular biology was obtained due to research of the complex formation of RNA with various proteins, in particular, the transport ones.
Proteins. Single molecules and complexes
The sizes of separate protein molecules are comparable with the curve radius of the АFМ probe, therefore, a submolecular resolution can be obtained only in research of proteins of high molecular weight. So far the attempts to ensure packing of chains in small proteins, for example, lysozyme (molecular weight 14 kD) were unsuccessful. At that the works are known, in which the protein monomers were reliably distinguished from dimers and multimers (fig.4 [5]).
Probe microscopy (PМ) can be used for registration of a specific antigene-antibody binding. Measurements of the sizes of separate particles and the achieved accuracy allow us to separate single antibodies and antigenes from their complexes. PМ gives us a chance to obtain information about the character of aggregation of the proteins and their binding with the nucleic acids. In [6], for example, the character of binding of a transport protein with a virus RNA (fig.5) was determined.
protein Crystals
An advantage of PМ in observation of the protein crystals in saturated solutions is a possibility of registration of the processes’ dynamics in liquid environments, natural for many biological objects. Research works allowed us to register the kinetics of growth of the dislocation hills and two-dimensional germs of many protein crystals at the level of separate molecules [7, 8]. For example, for lysozyme the speed of movement of steps and breaks, probability of joining and detachment of building units, dependence of the kinetic parameters on such factors as temperature and supersaturation [9] were measured.
АFМ allows us to observe the structure of a surface of a growing crystal with a molecular resolution and to study the packing of the molecules near the dot defects, which is impossible with the other methods. In [10] reconstruction of the surface of a crystal was revealed, when the packing of the protein molecules on it differed from the volume structure (figs.6 and 7).
Viruses
Although by its resolution PМ yields to the electronic microscopy, ease in preparation of samples justifies its application in examination of viruses for a qualitative analysis of their morphology. In virology the basic application of PМ is not for determination of the structure of viruses, but for studying of the processes occurring with them.
Crystallization processes were observed for various viruses [11, 12]. Stages of destruction of the protein shell of a tobacco mosaic virus with a release of RNA were registered in [4]. Since АFМ allows us to measure the mechanical properties of the nanostructures, Young modulus was determined for the virus particles during their cross-section compression by a probe: for the tobacco mosaic virus it is equal to 3·109 – 4·109 Pa, and for the less rigid potato X-virus – 8·108 Pa (fig.8). Good conformity to the experiments is ensured by calculations based on Hertz model of deformation (fig.9).
The tobacco mosaic virus can be used as a test structure [13] for calibration of Z-movement of АFМ and as a rigidity standard (fig.10). Research works show that viruses can serve as a system for delivery of nanoparticles in human tissues. Also they have found application in ecologically safe manufacture of nanoparticles with the use of plants [14].
Bacterial cells
Bacteria can be considered an ideal object for PM. They have micron sizes, therefore, we can watch separate molecules and their colonies, and also study fragments of cells [2]. Bacterial cells have a rigid polymer carcass and are not deformed by a microscope’s probe during scanning. For observation of bacteria in the air the cells grown up in a nutrient medium are transferred to a distilled water with a concentration up to about 109/ml (short-term stay in water does not lead to destruction of the bacteria). A drop of several microliters is placed on a freshly split mica. Water moistens it forming a thin film, after removal of which separate cells are deposited on a substrate or form monolayer coatings.
AFM makes it possible to obtain 3D images of the bacterial cells (fig.11). Such data can serve as additional criteria for preparation of the bacteria identifiers. In liquids the contrast of images is lower, which is due to the mobile polymer chains of the external membrane [15]. Figure 12 presents an image of E.coli bacterial cells in a buffer solution [15].
AFM allows us to reliably register the structural changes on the surface of the external membrane of a cell wall. Figure 13 presents a parent bacteria of Escherichia coli and a genetically engineered bacteria, into the initial DNA of which gene rfb-a3,4 was introduced, responsible for the synthesis of O-specified side chains of lipopolysaccharides. These chains create a lamellar structure on the surface of a cell, considerably different by its morphology from the surface of a parent cell [16].
Cells of higher organisms
Cells of higher organisms – animals and plants – are not very rigid and, therefore, an interaction with a probe leads to their deformation and an increase of the probe contact area with a cell surface, which reduces the spatial resolution [17]. On the other hand, PМ provides opportunity to measure not only the mechanical rigidity of separate cells, but also their adhesive and frictional properties.
PМ allows us to observe the dynamic processes occurring with the cells. For example, it helps to visualize the movement of the osteoblasts of mice on the surface of a glass substrate [18]; formation of units of these cells and aggregation of fibroblasts of chicken embryos; AFM-images were obtained of the primary osteoblasts of mice during cytocinesis; changes in the cells’ surface accompanying the apoptosis of the cells of an osteosarcoma were investigated. Scanning of the surface of cells with a probe edge allows us to visualize certain details of their internal structure. So, in the image of the surface of a cell it is possible to distinguish elements of cytoskeleton under the membrane, while in the picture of a dividing osteoblast a separating cellular nucleus is clearly visible.
Besides a morphological analysis with a nanometer resolution, we can determine the mechanical parameters of a membrane of cells and detect by means of optical labels the location of the cellular receptors. Also PМ can be applied for the diagnostic purposes, for example, in resuscitation [19].
For the first time the authors by a direct method demonstrated the character of damage (formation of nanopores) in the walls of erythrocytes during electroporation of various intensity. Research works were done on the human venous blood. The blood (1.2 ml) was placed in a quartz cuvette with titanium electrodes, with the distance between them of 15 mm. An electric field impulse was supplied to the electrodes. The source of the calibrated field was clinical defibrillator Lifepak 7 (USA). At the level of intensity of the field E in a solution of 1100 V/cm and duration of an impulse of 10 ms the induced transmembrane potential exceeded the threshold of a potential breakdown of the membrane (φt = 300÷500 mV), and its irreversible electroporation occurred.
Reduction of the number of erythrocytes as a result of haemolysis, and, hence, electroporation, was estimated by a kinetic curved line – dependence D (t), where D is an optical density of the suspension of blood. The kinetic curves of the haemolysis of erythrocytes were described by the exponential function:
D(t) = D0exp(–βt) + D1,
where β is a constant of the speed of reduction of the number of erythrocytes, D1 is the residual level of the cells, D0 is the initial number of erythrocytes. The research of the surface of the erythrocytes before the impact of the electric field was done on blood smears in accordance with a standard technique at a room temperature.
Images of the surface of the membranes (fig.14) were obtained at the Chair of Polymers and Crystals of the Physical Faculty of the Moscow State University named after M.V.Lomonosov by means of an atomic-force microscope FemtoScan, developed by NPP Center of Perspective Technologies. An erythrocyte in a smear on a glass substrate has the form of a discocyte. Its radius is 3600 nm, side height – 460 nm, difference between the highest point and the lowest point – 35 nm. This is shown on a profile in the points of installation of the first and second cursors.
Figure 15 demonstrates erythrocytes after an impulse breakdown with the energy of 112 J. In the enlarged fragment the red and green cursors mark the lines of measurement of the profile of pores, and on the profiles the red dotted line designates average values by the surface. The left profile refers to the left bottom pore, and the right profile – to the right top pore. On the first profile the cursors are fixed in the middle and bottom (deepest) point of the pore, allowing us to measure its depth. On the right profile the diameter of a pore is shown – 93 nm and its depth – 14 nm.
The surface of even a small fragment of an erythrocyte’s membrane is not flat. On it there are roughnesses, hollows and slopes of plane in different directions. In a membrane structure there are also pores which can be a result of heterogeneity of the structure or of a phase transition of the membrane from a liquid-crystal state into a gel, because the blood temperature in the experiments was about 20°С, which is below the phase transition point.
Other biological objects
PМ also allows us to study tissues of animals, surfaces of bones and nails, structure of the tooth enamel. Promising are the opportunities for observation of the surface of leaves and stalks of live plants. Figure 16 presents a three-dimensional image of a fragment of the surface of a human hair. Interesting, that a visually better looking hair at the microlevel has more perfect structure with a smaller number of defects.
All the above-stated concerned, first of all, applications of PМ for research of morphology of the surface of the biological objects, obtaining of three-dimensional images and measuring of the sizes. The main data for studying of the processes’ kinetics are the chains of the three-dimensional images varying in time, for example, pictures of the surface of a growing crystal.
Another prominent aspect of PМ is measurement of the mechanical properties of the biological objects. This information is new to the biological science, therefore, its comprehension demands time. PМ allows us to measure the mechanical rigidity of an object, to determine the force of adhesion, to register variations of the friction factor, receiving a three-dimensional card of the frictional properties of a surface.
Direct measurements of DNA (one of its ends is attached to a substrate, another is connected to the mobile probe of АFМ) allowed us to estimate the force of a chemical bond in a single biomacromolecule. A two-chain molecule of DNA brakes off with application of force of 10–10 N. АFМ ensures measurement of the dynamics of deployment of a single protein globule in a linear chain of aminoacid residues. Scanning of the bacterial monolayer films demonstrated that bacteria Arthrobacter Globophormis in a mummified form are more fragile, than in a vegetative state. If regular live bacteria withstood forces up to 10–6 N, which was by several orders more than the values traditional for scanning in the air, the mummified bacteria were destroyed by such forces completely.
Biological sensors
From the very beginning AFM looked advantageous from the point of view of development of unique biological sensors. Weight of one bacterium – about 10–13 N – ensures an additional deflection of a cantilever with rigidity of 0.06 N/m by 1.6·10–12 m, which corresponds to the level of the limit resolution of a microscope working in a contact mode. However, if we use a mode when a cantilever fluctuates in the resonant frequency sensitive to its weight, it is possible to measure the weight of one bacterium. This is the principle on which the chemical and biological sensors are based.
Having joined a cantilever, the weight of a reagent or biological object changes the frequency of its resonant fluctuations [20]. Measurement of a frequency shift allows us to judge about the weight of the adsorbed substance. Measurements of the weight of nanoparticles are done, as a rule, on specialized atomic scales in which, just as in АFМ, a laser-optical system is used, but there is no necessity in a cantilever with a needle. A key part of most scales is an elastic element, and here the following principle works well – the tinier are the scales, the smaller weights can be weighed. Using this approach, it is possible to determine the weight of one bacterium [21], of a protein [22], and even of a separate atom [23].
High sensitivity is a feature of the cantilevers of a tuning type [24] and those made from piezomaterials. Resonant methods, as a rule, have higher sensitivity in comparison with the static ones. Therefore very interesting is the fact that exactly the static biosensors are very promising for the medical researches [25]. The method’s essence consists in the following:
Surface of one of the sides of a cantilever is covered by a monolayer film of an adsorbing substance with a certain surface tension;
If such a cantilever is placed in a biological liquid, a specific binding may appear on its surface;
Adsorption of a material on the surface of a cantilever leads to a change of the surface tension of the film and to its bending.
For development of chemical and biological sensors the cantilevers with a low mechanical rigidity are used (fig.17) [26]. If the physical principle for their development is clear, the biological aspects of a sensor’s structure are more difficult, because a biologically active coating with a biospecific binding is required. It would also be desirable to have a biosensor suitable for a repeated usage, that is, its ability of adsorption of a concrete substance should be restored by washing. Thus, the problems of creation of a biosensor are more connected with biology, than with the atom-force microscopy.
In general it should be pointed out that the scanning PМ does its first steps in the field of biomedical and biosensor applications. By combining it with the other analytical methods it is possible to make essential progress on the way to the personified medicine, which should take into account the specific features of each individual at a molecular level. ■
The work was done with the support of FTP “Scientific and Scientific-pedagogical Personnel for Innovative Russia for the period of 2009 – 2013” and FTP “R&D in the Priority Directions of Development of the Scientific-technological Complex of Russia for the period of 2007-2013”.
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