Method of nano and micro object study by probe microscopy

FIELD: biotechnologies.

SUBSTANCE: object is positioned on porous substrate, fixed to the substrate surface and scanned by probe microscopy method. Substrate with through pores of smaller size than the diameter of a study object is used, and an object is fixated by laminar flow of liquid or gas supplied to the substrate from the side of scanning, with clamping force exerted by the flow on an object within 10-12-10-3 N range.

EFFECT: possible study of structures and mechanical properties of organic and inorganic objects, enhanced information content of nano and micro object studies by probe microscopy.

7 ex

 

The invention relates to the field of probe microscopy and can be used to study the structure and mechanical properties of organic and inorganic nature.

The known method of the study of nano - and micro-objects by the method of scanning probe microscopy by placing the object on a substrate, its fixation on the surface of the substrate by using electrostatic force and scanning of the recorded object method probe microscopy (Rikke Louise Meyer, Xingfei Zhou, Lone Tang, Ayyoob Arpanaei, Peter Kingshott, Flemming Besenbacher. Immobilisation of living bacteria for AFM imaging underphysiological conditions // Ultramicroscopy (2010), p.1-8).

The known method of the study of nano - and micro-objects by the method of scanning probe microscopy by placing the object on a substrate, its chemical fixation on the substrate surface and scanning the recorded object method probe microscopy (Rikke Louise Meyer, Xingfei Zhou, Lone Tang, Ayyoob Arpanaei, Peter Kingshott, Flemming Besenbacher. Immobilisation of living bacteria for AFM imaging underphysiological conditions // Ultramicroscopy (2010), p.1-8).

The closest to the claimed is a method of study of nano - and micro-objects by the method of scanning probe microscopy by placing the object on a porous substrate, its fixation on the substrate surface and scanning the recorded object method probe microscopy (Kailas L., E. C. Ratcliffe, E. J. Hayhurst, M. G. Walker, S. J. Foster, J. K. Hobbs, Immobilizing live bacteria for AFM imaging of cellular processes // Ultramicroscopy 109 (2009), p775-780) - as a prototype. In this method, the object under study (bacterium) is placed in the environment of water on the surface of the porous substrate from silicon dioxide, containing of through pores, allow the water to evaporate, whereby the object is fixed on the surface of the substrate, then the substrate is placed in a measuring cell with a liquid and are scanning the recorded object by the method of scanning probe microscopy.

The disadvantage of this method is its complexity, which consists in a multi-stage preparation of the substrate with the sample before scanning probe microscope and a lack of data in ways that do not allow to study the properties change depending on the varying parameters of the environment in which measurement is carried out.

The technical problem of the invention is to reduce the complexity of the method, increasing the amount of information and expand the Arsenal of technical means that can be used to study nano - and micro-objects by the method of scanning probe microscopy.

Said technical result is achieved in that in the known method of the study of nano - and micro-objects by the method of scanning probe microscopy by placing the object on a porous substrate, its fixation on the substrate surface and scanning the recorded object method probe Mick�octopii, use a substrate with through pores larger than the size of the investigated object and the fixation of the object is carried out by laminar flow of liquid or gas supplied to the substrate from the side being scanned, the magnitude of the pressure force acting from the side of the stream to the object, is in the range 10-12-10-3the Newton (N).

The proposed method is new and not described in scientific literature.

This method can be used for studies using scanning probe microscopy nano - and micro-objects of organic and inorganic nature. Thus, the dimensions of the studied objects can be from 1 nanometer (nm) to 100 micrometers (μm). As such objects can be used, for example, viruses, bacteria, nano - and microparticles of inorganic materials, etc.

The proposed technical solution can be used to study various objects by scanning probe microscopy such as atomic force microscopy, near-field microscopy, scanning tunneling microscopy, etc.

In this technical solution can be used various organic and/or inorganic liquids, such as alcohol, chloroform, acetone, water, aqueous saline solutions, physiological fluids, etc., and various gas�, for example, such as air, carbon dioxide, nitrogen, oxygen, etc. Can be used as individual liquids or gases and mixtures of liquids, or gases.

In the proposed method can be used on porous substrates made of various organic or inorganic materials, such as polymers, silica, silicon, alumina, etc. it Should be noted that the substrate used must have through pores whose size must be less than the size of the investigated object, and pore shape may be almost any. The pores can be either the same size or different. Such substrates are described in scientific literature. [Shashishekar P. Adiga, Chunmin Jin, Larry A. Curtiss, Nancy A. Monteiro-Riviere and Roger J. Narayan. Nanoporous membranes for medical and biological applications WIREs Nanomedicine and nanobiotechnology is, V. No. 1, 2009, p.568-581]. If the proposed technical solution, use a porous substrate not with through with through pores or pores, the size of which will match or exceed the size of the studied objects, the present invention becomes inoperative.

It should be noted that the fixation of the investigated object on the surface of the porous substrate have to be performed before microscopic investigations if the object is not secure, it is possible to study the object by scanning probe Mick�octopii fails.

In the proposed technical solution fixation test object on the surface of the porous substrate is carried out by laminar flow of the medium supplied to the substrate from the side being scanned. It should be noted that the flow must be laminar. When using a turbulent flow environment the proposed technical solution will not work. If the laminar flow of the fluid will be supplied to the substrate from the side opposite to the scanning, the technical solution will not be healthy.

It was established experimentally that the proposed invention the magnitude of the pressure force acting from the side of the flow object shall lie within the range of 10-12-10-3N. If the stream will exert a clamping force greater than 10-3N, it will cause critical bending of a cantilever probe microscope, which will allow for the scanning of the investigated object with the needle of the cantilever. The amount of downforce can be determined by a known method [J. Zlatanova, S. M. Lindsay, S. H. Leuba. "Single Molecule Force Spectroscopy in Biology Using the Atomic Force Microscope", Prog. Biophys. Mol. Biol, 74, 37, (2000)] by chemical attachment silatrane groups of the linker [molecular fragment, covalently linked to a solid substrate that contains reactive functional groups] to needle cream�avago cantilever probe microscope. However, other functional groups of a linker capable of covalently to contact the surface of the investigated object. As the linker can be used such chemical compounds, such as 1-ω-mercaptopropionate, 1-ω-aminoalkylsilane, etc. this is followed by scanning of the investigated object using a modified cantilever and carry out the removal of the force curves for the same 10 of the studied objects, which define the average effect of separation from the object surface in the presence and in the absence of fluid flow. The difference of certain forces of separation is equal to the clamping force of the stream.

In the present invention to fix the investigated objects on the surface of the porous substrate in two ways, the first of which is to place the object in its pure form or in the form of a slurry on a porous substrate followed by submission of a laminar flow of liquid or gas. The second way lies in making the test object directly in laminar flow of liquid or gas.

In the proposed technical solution is possible to vary the flow rate of liquid or gas, as well as other parameters of a liquid or gas, such as the concentration of dissolved substances, temperature, etc., in the course of the experiment. In the latter case, you can examine treason�their properties depending on the changes of flow parameters, what increases the information content of the method.

After the end of the experiments used membrane can be released from the studied objects by filing a counter-flow of liquid or gas and re-used in other experiments.

The advantages of the proposed technical solution is illustrated by the following examples.

Example 1

The experiment is performed using a liquid cell of the atomic force microscope containing a membrane of silicon oxide, having an area of 1×1 cm2. Used membrane has a thickness of 1 μm and contains through the pores of cylindrical shape with a diameter of 150 nm with an average distance between the pores of 200 nm. Using a syringe pump to create a laminar flow of buffer brand Tris-HCl (pH of 7.5, 0.01 M) through a porous membrane. Then in laminar flow add studied the tobacco mosaic virus with a size of 300 nm, taken at a concentration of 1 microgram/ml in buffer Tris-HCl. As a result of passing a laminar flow of the buffer containing the tobacco mosaic virus, through the porous membrane on its surface are fixed in one position viruses due to the downforce of the laminar flow of the buffer equal to 10-3N. Then produce a scan of tobacco mosaic virus, fixed onto the membrane surface using atomic force microscope brand Nanoscope and study the surface morphology of V�USA with a spatial resolution of 4 nm.

Example 2

The experiment is performed using a liquid cell of a near-field microscope brand Certus NSOM containing the porous membrane of aluminum oxide having a size of 2×2 cm2. Used membrane has a thickness of 1 μm and contains through the pores of square shape (square side 500 nm) with an average distance between the pores of 600 nm. The membrane is applied to a suspension of the bacteria Acinetobacter baumannii in bidistilled water with a concentration of 2 micrograms/ml through syringe pumps create circulating laminar flow bidistilled water through the membrane from the side being scanned. As a result of passing a laminar flow of water through a porous membrane on its surface are fixed in one position bacteria at the expense of downforce flux of 10-6N. After that make scan of bacteria fixed on the membrane surface. To study the effect of the antibiotic colistin on the surface structure of bacteria in laminar flow circulating through the membrane type in a given antibiotic, wherein the antibiotic concentration range from 0.5 to 32 micrograms/ml. In the result of the experiment was studied the surface morphology of the bacteria with a spatial resolution of 2 nm depending on the concentration of the antibiotic colistin, which indicates a high informative way.

Example 3

The experience carried out analogously to example 1, but the substrate using a polycarbonate membrane with an average diameter of through pores of 20 nm and an average distance between the pores is 30 nm, and instead of the fluid flow using a laminar flow of nitrogen gas, as a scanning probe microscope using tunneling microscope brand FemtoScan, the Russian Federation, and the quality of the object using spherical gold particles, whose diameter is 30 nm. The magnitude of the pressure force acting from the side of the gas flow on an object that is 10-12N. In the course of the experiment to study the surface morphology of the gold particles with a spatial resolution of 1 nm.

Example 4

The experience carried out analogously to example 3, however, instead of gaseous nitrogen of the air is used laminar flow which creates downforce equal to 10-10N. In the course of the experiment to study the surface morphology of the gold particles with a spatial resolution of 1 nm.

Example 5

The experience carried out analogously to example 3, however, instead of the nitrogen gas used argon, laminar flow which creates downforce equal to 10-11N. In the course of the experiment to study the surface morphology of the gold particles with a spatial resolution of 1 nm.

Example 6

The experience carried out analogously to example 3, but instead of laminar photokeratoscope nitrogen use laminar flow of chloroform, which creates downforce equal to 10-8N. However, instead of tunneling microscope using an atomic force microscope brand Nanoscope. In the course of the experiment to study the surface morphology of the gold particles with a spatial resolution of 2 nm.

Example 7

The experience carried out analogously to example 6, however, instead of the laminar flow of chloroform use laminar flow 70% aqueous solution of ethanol, which creates downforce equal to 10-9N. In the course of the experiment to study the surface morphology of the gold particles with a spatial resolution of 2 nm.

Thus, the given examples show that the proposed method is quite simple and really significantly reduces the complexity of the known method of the study of nano - and micro-objects by the method of scanning probe microscopy, increases the information content and expands the Arsenal of technical tools that can be used to study nano - and micro-objects by the method of scanning probe microscopy.

Method of the study of nano - and micro-objects by the method of scanning probe microscopy by placing the object on a porous substrate, its fixation on the substrate surface and scanning the recorded object method probe microscopy, characterized in that the substrate with through pores larger than the size of the investigated volume�KTA, and the documentation of the object is carried out by laminar flow of liquid or gas supplied to the substrate from the side being scanned, the magnitude of the pressure force acting from the side of the stream to the object, is in the range 10-12-10-3Newton.



 

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