Method for escherichia coli bacteria identification by detecting their fragments by atomic-force microscopy

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.

1 dwg

 

The claimed method of determining the presence of E. coli bacteria no detection of their fragments relates to the field of sensor technologies for the detection and visualization of bacterial cells and their fragments. Under the fragment of bacterial cell refers to any part that contains antigenic determinant.

There are a number of technical solutions that are close in meaning to the proposed method: a diagnostic test system for detecting diseases (patent RU 2361215 C1), nanobiosym used for registration of proteins and protein complexes, process for its production and method for detecting proteins and protein complexes using probe microscopy (patent RU 2007117787A), a method of registering a specific macromolecules in a biological sample and device for its implementation (patent RU 2006129865A), the method of registration of macromolecules when conducting proteomic of research and biochip used in their registration (patent RU 2004119864).

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

There are ways you can check specifically contacting affine the surface of bacteria, based on the application methods of the quartz microbalance, ellipsometry, surface plasma resonance (Lazcka O., F.J. Del Campo, Munoz F.X., 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 level signal in the control experiments, i.e. when non-specific binding, (2) lack of visualization of bound peroxidase analyte, for which bacterial cells (and their fragments) means the confirmation of the physical events of their binding affinity surface, (3) the inability to detect a single bacterial cell or part of a bacterial cell, (4) the risk of contamination of the premises and the possibility of contamination of the personnel in the detection of pathogenic bacteria.

The problem solved by the invention is the detection of bacterial cells of Escherichia coli and their fragments in solutions with low concentrations using atomic force microscopy. The proposed method allows to determine the presence of ultra-low concentrations of cells in the sample solution, including to detect contacting affine surface fragments only bacteria. The method is equally sensitive to both bacterial whole cells and their fragments, because the antibodies bind to the antigenic determinant. Time de is the design from the moment of exposure of the analyte to the affinity surface until the result is 20 minutes. This method is suitable for quality control of drinking water and any other liquids on the concentration of E. coli bacteria. Because this method can be used to detect fragments of the bacteria Escherichia coli, it allows for analysis of the solution is destroyed bacterial cells, which helps to reduce the risk of infection to staff.

The technical result in the present invention achieves the following steps: preparation of the affine surface on the basis of specific antibodies, the exposure of the analyte to the surface, a two-stage rinsing the substrate receiving surface topography of the sample using atomic force microscopy. While the exposure of the analyte to the affinity surface is performed surrounded by a buffer solution with pH value, and at temperatures that are optimal for specific binding of the antibody to the antigen. The essential feature is dvuhetapnogo washing: a first buffer that allows the most efficient wash not specifically bound peroxidase material, and then with water, allowing you to wash the substrate from the salts.

Consider an implementation of the invention on the example of the detection of fragments of the bacteria Escherichia coli from suspension with a concentration of whole cells 107CFU/ml as control experience is Imanta undertakes analytical solution (analyte), containing fragments of the bacteria Bacillus subtilis the same concentration.

For the preparation of affine surface is used common method of application to the surface of the mica layer of protein G, which in turn are applied specific antibodies to Escherichia coli. To increase the adhesive properties of mica before application of a G protein treat her in a glow discharge within 60 seconds with an electric current of 0.4 mA (voltage 1.5 kV, the distance between the electrodes is 5 mm, the area of the electrodes 0.5 cm2). After applying a layer of antibodies to the surface layer protein G substrate was washed with deionized water and dried. The exposure of the analyte to the biofunctional surface conduct its location on top of a small drop (20 ál) of the analyzed suspension in buffer pH 8.5 to 9.5 at 37°C for 10 minutes, then washed by immersing the substrate in a buffer solution and swinging on the shaker, while the substrate is rigidly fixed in the shaken system. After that, the substrate was washed with deionized water from salts, dried in a stream of compressed nitrogen and examined using atomic force microscopy. Analyzing AFM image, calculate the amount of bound peroxidase bacterial cells or the total amount of bound peroxidase cellular fragments per unit area.

Figure 1 presents a three-dimensional view of the AFM image f is of Ahmetov of Escherichia coli cells, contacting affine to him by the surface of the slurry concentration of 107CFU/ml (left)and the same surface, which exhibited suspension fragments of cells of Bacillus subtilis the same concentration (right). The size of the scanning area of the given image is 10×10 μm2. Analysis of the AFM images shows that the total amount of bound peroxidase fragments of Escherichia coli height of more than 50 nm is observed field 2,47×1014nm3while this value for Bacillus subtilis equal to zero, i.e. in case of nonspecific pair antelo-antigen binding objects to a height of more than 50 nm is not observed at all.

Thus, the present invention allows testing of liquids, including food, microbial content, and if it is possible to carry out the intentional destruction of the cells to reduce the risk of contamination of the premises and Contracting staff. Thus the sensitivity of detection will not fall.

The method of determining the presence of E. coli bacteria for the detection of fragments in solution, which includes specific immobilization of fragments of these bacteria from solution on an affine surface consisting of a layer of protein G coated with a layer of antibodies specific to identify bacterial glue the Cam, providing a two-stage rinsing of the sample from the non-specific bound peroxidase material comprising flushing the buffer with a pH value of 8.5-9.5 and subsequent washing with deionized water, using atomic force microscopy as a tool for visualization of bound peroxidase bacterial fragments and the possibility of a direct count of the amount of bound peroxidase analyte per unit surface area.



 

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