RU2437937C1 - METHOD FOR Escherichia coli BACTERIA IDENTIFICATION BY DETECTING THEIR FRAGMENTS BY ATOMIC-FORCE MICROSCOPY - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves specific immobilisation of fragments of the specified bacteria from a solution on an affine surface consisting of a protein G layer coated with a layer of antibodies specific to the identified bacterial cells. Two-stage washing of a sample from a non-specifically bound material implies washing in a buffer pH 8.5-9.5 and washing in deionised water. The atomic-force microscopy is used as a visualisation tool of the bound bacterial fragments and enables direct count of a bound analyte amount per a surface unit.

EFFECT: offered invention allows identifying the minute cell concentration in the analysed solution.

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Description

The claimed method for determining the presence of Escherichia coli bacteria by detecting their fragments relates to the field of sensory technologies aimed at detecting and visualizing bacterial cells, as well as their fragments. A bacterial cell fragment is understood to mean any part thereof containing an antigenic determinant.

There are a number of technical solutions that are close in meaning to the proposed method: a diagnostic test system for detecting a disease (patent RU 2361215 C1), a nanobiochip used to register proteins and protein complexes, a method for its preparation, and a method for recording proteins and protein complexes using probe microscopy (patent RU 2007117787A), a method for registering specific macromolecules in a biological sample and a device for its implementation (patent RU 2006129865A), a method for registering macromolecules during proteomic studies nny and a biochip used in the registration (patent RU 2004119864).

However, the methods developed in all of the above patents have one significant drawback: they do not describe the registration or visualization of bacterial cells and their fragments, including Escherichia coli, but refer only to macromolecules (mainly proteins and protein complexes) and viruses.

There are methods for detecting bacteria that specifically bind to the affinity surface, based on the use of quartz microbalance, ellipsometry, and surface plasma resonance methods (Lazcka O., Del Campo FJ, Munoz FX, Biosens. Bioelectron, 2007, 22, 1205-1217; Ivnitski D., Abdel-Hamid I., Atanasov P., Wilkins E., Biosens. Bioelectron, 1999, 14, 599-624). The disadvantages of these methods are (1) a high signal level in control experiments, i.e. in case of nonspecific binding, (2) the inability to visualize the bound analyte, which for bacterial cells (and their fragments) means confirmation of the physical event of their binding to the affinity surface, (3) inability to detect a single bacterial cell or part of a bacterial cell, (4) risk of contamination premises and the possibility of infection of personnel during the detection of pathogenic bacteria.

The problem solved by the invention is the determination of the presence of bacterial cells of Escherichia coli and their fragments in solutions with low concentrations using atomic force microscopy. The proposed method makes it possible to determine the presence of ultra-low concentrations of cells in the analyzed solution, including the detection of fragments of a single bacterium bound to the affinity surface. The method is equally sensitive both to whole bacterial cells and to their fragments, since antibodies bind to an antigenic determinant. The detection time from the moment of exposure of the analyte to the affinity surface to obtain the result is from 20 minutes. This method is suitable for controlling the quality of drinking water and any other liquids for the content of Escherichia coli bacteria in them. Since Escherichia coli bacterial fragments can be detected using this method, this allows the use of a solution of destroyed bacterial cells for analysis, which reduces the risk of infection for maintenance personnel.

The technical result in the present invention is achieved by the following sequence of steps: preparing an affinity surface based on specific antibodies, exposing the analyte to this surface, two-stage washing of the substrate, obtaining a topography of the surface of the sample using atomic force microscopy. The exposure of the analyte to the affinity surface is carried out surrounded by a buffer solution with a pH value, and at temperatures that are optimal for specific binding of the antibody to the antigen. An essential feature is also a two-stage washing: first in a buffer that allows you to most effectively wash non-specifically bound material, and then with water, which allows you to wash the substrate from salts.

Consider the implementation of the invention by the example of detecting fragments of the bacterium Escherichia coli from a suspension with a concentration of 10 ⁷ CFU / ml in whole cells. As a control experiment, an analytical solution (analyte) containing fragments of the bacterium Bacillus subtilis of the same concentration is taken.

To prepare the affinity surface, a common method is used for applying a layer of protein G to the surface of the mica, on which specific antibodies to Escherichia coli are in turn applied. To increase the adhesive properties of mica, before applying protein G to it, it is treated in a glow discharge for 60 seconds at a current strength of 0.4 mA (voltage 1.5 kV, distance between electrodes 5 mm, electrode area 0.5 cm ² ). After applying the antibody layer to the surface of the protein G layer, the substrate is washed in deionized water and dried. The analyte is exposed to the biofunctional surface by placing it on top of a small drop (20 μl) of the analyzed suspension in a pH 8.5-9.5 buffer at 37 ° C for 10 minutes, after which it is washed by immersion of the substrate in a buffer solution and its swinging on a shaker , while the substrate is rigidly fixed in the swing system. After that, the substrate is washed with deionized water from salts, dried in a stream of compressed nitrogen and examined using atomic force microscopy. By analyzing the AFM image, the number of bacterial cells bound or the total volume of bound cell fragments per unit surface is counted.

Figure 1 presents a three-dimensional AFM image of fragments of Escherichia coli cells that bind to their affinity surface from a suspension of 10 ⁷ CFU / ml (left), as well as the same surface to which a suspension of fragments of Bacillus subtilis cell fragments of the same concentration was exposed (on right). The size of the scanning area of the images is 10 × 10 μm ² . Analysis of AFM images demonstrates that the total volume of the bound Escherichia coli fragments with a height of more than 50 nm is 2.47 × 10 ¹⁴ nm ³ in the observed field, while this value for Bacillus subtilis is zero, i.e. in the case of a non-specific antibody-antigen pair, the binding of objects with a height of more than 50 nm is not observed at all.

Thus, the present invention allows the diagnosis of liquids, including food liquids, for the content of microorganisms, and in the case it is possible to preliminarily carry out deliberate destruction of cells to reduce the risk of contamination of the premises and infection of maintenance personnel. In this case, the detection sensitivity will not decrease.

Claims (1)

A method for determining the presence of Escherichia coli bacteria by detecting their fragments in a solution, which includes specific immobilization of fragments of these bacteria from a solution on an affinity surface, consisting of a layer of protein G with a layer of antibodies specific for detectable bacterial cells, comprising a two-stage washing of the sample from non-specifically bound material, including washing in a buffer with a pH value of 8.5-9.5 and subsequent washing with deionized water, the use of atomic force micro scopy as a tool for visualizing bound bacterial fragments and the ability to directly count the volume of bound analyte per unit surface.

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