Method for producing a biological receptor element

Abstract

A method for producing a biological receptor element based on microorganisms immobilized in a xerogel matrix includes successively mixing a microbial biomass with a phosphate buffer solution, tetraethoxysilane and polyvinyl alcohol to obtain a microorganism-containing suspension. A nitrocellulose-based substrate is used which is pre-soaked with a solution of NaF then passed through the obtained microorganism-containing suspension and, after immobilization of the microorganisms, the resulting matrix is dried and cut into fragments.

Description

[0001] METHOD FOR OBTAINING A BIOLOGICAL RECEPTOR ELEMENT

[0002] Field of technology

[0003] The invention relates to the field of biotechnology, namely to biocatalysts based on immobilized microorganism cells in a matrix of a synthetic carrier and can be used in combination with devices for determining biochemical oxygen demand (BOD), for example with the EXPERT-009 liquid analyzer.

[0004] State of the art

[0005] Developments in the field of bioreceptor elements over the past decade have focused on methods for forming hybrid structures in which living cells are surrounded by synthetic protective shells, particularly those made of inorganic materials such as silicon oxide. The advantages of silicon oxide as a cell-surrounding material include its inertness, porosity, and mild production conditions. These artificial structures mimic natural single-cell microorganisms—diatoms—which form a protective silicate exoskeleton.

[0006] One of the approaches to creating an artificial silicate shell around cells is the use of sol-gel technologies. For targeted regulation of the characteristics of silica materials, it has been proposed to use hydrophilic polymers, in particular, polyvinyl alcohol (PVA), which is capable of forming hydrogels, participating in sol-gel processes of formation of silicate and organosilicate materials and, as a result, influencing the architecture of the final material [Gribanov I. A., Lavrova D. G., Alferov V. A. Yeast encapsulated in organosilicate matrices as biocatalysts in the development of biofilters / / Bulletin of Tula State University. Natural Sciences. - 2022. - No. 1. - P. 3-8.].

[0007] A composition based on tetramethoxysilane and Escherichia coli cells is known, which is used to form bioreceptor elements in luminescent biosensors [E. Sagi, N. Never, R. Rosen, AJ Bartolome, JR Premkumar, R. Ulber, O. Lev, T. Scheper, S. Belkin. Fluorescence and bioluminescence reporter functions in genetically modified bacterial sensor strains. / / Sensors and Actuators 90, p. 2-8, 2003]. The activity of biomass immobilized in this way is significantly reduced compared to the activity of non-immobilized cells, which is due to the absence of a structure-forming agent in the process of obtaining such a matrix. Sensors based on cells immobilized in such a matrix do not have a stable analytical signal and have a narrow range of detectable concentrations.

[0008] The closest analogue of the claimed invention is a method for producing an organosilicon sol-gel matrix for immobilizing microorganisms in biosensor analyzers, disclosed in the Russian Federation patent for invention RU2492236, 10.09.2013. The matrix composition consists of a 20% solution of polyethylene glycol in a phosphate buffer solution, tetraethoxysilane and 0.2 mol / dm 3 a solution of the NaF catalyst, a hydrophobic additive - methyltriethoxysilane, while the components are taken in a volume ratio of PEG: TES: MTES: NaF 4: (18-3.4): (2-16.6): 1. To prepare such an organosilicon matrix and immobilize microorganisms (with subsequent use in biosensor analyzers), 0.1 ml of a 20% solution of polyethylene glycol in a phosphate buffer solution (pH = 6.8) is taken into a microtube and 0.25 ml of a suspension of Pichia angusta yeast cells in a phosphate buffer solution with a concentration of 150 mg / cm is added to it 3, stir the mixture for 3 minutes, then add 0-0.45 ml of tetraethoxysilane and 0.05-0.5 ml of methyltriethoxysilane, after which stir the mixture for 3 minutes. Then add 0.025 ml-0.2 mol / dm 3 NaF solution and stirred for 15 minutes. The resulting gel (10 μl) was transferred to a glass fiber filter and air-dried for 5 minutes at room temperature. The prepared bioreceptor element was placed under the cap of a Clark-type oxygen electrode and secured with nylon mesh.

[0009] The main drawback of this method for producing a bioreceptor element is the addition of the primary catalyst, NaF, to the microtube during gel production, which requires prolonged mixing of the components (15 minutes) and subsequently requires additional drying time for the gel applied from an automatic pipette. Such product compositions are fragile due to the use of a fiberglass filter as a substrate and the impossibility of storing the finished bioreceptor element separately from the sensor, making it impossible to transport, for example, for field research.

[0010] Thus, the technical problem that the claimed invention is aimed at solving is the increased time required to manufacture bioreceptor elements, as well as their low strength characteristics.

[0011] Disclosure of the essence of the invention

[0012] The technical result of the invention is a reduction in time costs and an increase in productivity in the manufacture of a biological receptor element, a reduction in the fragility of the element, as well as an increase in its long-term stability and shelf life separately from the sensor.

[0013] This technical result is achieved by a method for producing a biological receptor element based on microorganisms immobilized in a xerogel matrix. This method involves sequentially mixing the microbial biomass with a phosphate buffer solution, tetraethoxysilane, and polyvinyl alcohol to produce a suspension containing the microorganisms. A nitrocellulose-based substrate is used, which is pre-wetted with a NaF solution. The resulting suspension, containing the microorganisms, is then passed through the substrate. After immobilization of the microorganisms, the resulting matrix is ​​dried and cut into fragments.

[0014] The specified technical result is also achieved in particular forms of implementation of the invention due to the fact that:

[0015] - use a buffer solution with pH=6.8, containing 3 mM KH2PO4 + 33 mM Na2HPC>4, and in an amount that provides a cell titer of 150 mg / cm 3 ;

[0016] - tetraethoxysilane is introduced into the suspension in a volume ratio of 1:1;

[0017] - polyvinyl alcohol is introduced into the suspension in a suspension / alcohol ratio of 75:25 vol.%;

[0018] - use a 5-10% NaF solution.

[0019] - a suspension containing microorganisms is passed through the substrate in an amount of 5-10 ml;

[0020] - use Paracoccus yeei microorganisms.

[0021] The nitrocellulose substrate used in the claimed invention eliminates the disadvantages of the fiberglass filter used in its closest analogue. It has improved strength characteristics (reduced brittleness) and allows for the simultaneous production of several ready-to-use or ready-to-storage bioreceptor elements, increasing the productivity of element production and their shelf life separate from the sensor. Furthermore, the nitrocellulose substrate, in combination with the matrix composition, ensures improved long-term stability of the elements. Furthermore, the use of a substrate pre-wetted with a catalyst (sodium fluoride, NaF) significantly reduces the mixing time of the composition.

[0022] Brief description of the drawings

[0023] The invention is illustrated by figures, where: Figure 1 shows a diagram of a bioreceptor element based on microorganisms immobilized in a xerogel matrix on a nitrocellulose substrate, Figure 2 shows a diagram of a method for producing a bioreceptor element

[0024] The elements are designated on the figures by the following positions:

[0025] 1 - nitrocellulose substrate,

[0026] 2 - immobilized microorganisms in a matrix obtained as a result of sol-gel synthesis.

[0027] Implementation of the invention

[0028] The method for producing a bioreceptor element is as follows. A potassium-sodium phosphate buffer solution with a pH of 6.8, containing 33 mmol / dm3, is added to the biomass of microorganisms, in particular Paracoccus yeei. 3 ) KH2PO4 and 33 mM Na2HPO4. The buffer is introduced so that the cell titer is 150 mg / dm3 3The resulting mixture is stirred using a magnetic stirrer for 5 minutes. An equal volume (1:1) of tetraethoxysilane (TEOS) is added to the resulting suspension, followed by polyvinyl alcohol (PVA) in a suspension / PVA ratio of 75:25 vol%.

[0029] The nitrocellulose-based substrate is pre-wetted in a 5-10% catalyst solution (NaF) and placed in a plastic filtration device. A plastic syringe with a capacity of 5-10 cm 3 The microorganisms are then filled with a suspension (sol) containing the microorganisms, which is passed through the substrate. After the gel has formed and the microorganisms have been immobilized, the element is removed, dried, and cut into fragments of the desired size to obtain the finished bioreceptor elements. The resulting matrices are stored in Eppendorf tubes at temperatures ranging from -20°C to +5°C.

[0030] Thus, the resulting product has two components: microorganisms (2) immobilized in a xerogel matrix, and a nitrocellulose substrate (1) (see Fig. 1). During processing, the nitrocellulose substrate does not become soggy, and the xerogel matrix effectively protects the microorganisms from the effects of heavy metals and ultraviolet radiation.

[0031] The table presents the characteristics of bioreceptor elements obtained according to the methods of the closest analogue and according to the claimed invention.

[0032] Table The relative standard deviation of a series of 15 consecutive analytical signals was improved from 0.6% to 0.4% by using microorganisms isolated from activated sludge. Long-term stability was increased from 27 days (the closest analogue) to 30 days. In addition, the claimed bioreceptor element allows for the measurement of a wide range of detectable BOD5 concentrations (from 0.5 to 35 mg Og / dm3). 3). The time required to manufacture such bioreceptor elements is reduced by 25-30 minutes by pre-wetting the substrate with a catalyst and passing the sol through it.

[0033] These bioreceptor elements can be used for rapid analysis of BOD sensors and are compatible with first-generation biosensors.

[0034] Although the invention is illustrated using Paracoccus yeei as an example, it can be successfully applied to other microorganisms, as their type does not significantly affect the manufacturing efficiency, mechanical properties, or stability of the bioreceptor elements. For example, Debaryomyces hansenii and Blastobotrys adeninivorans can be used.

[0035] The given concentrations and characteristics of the components that form the xerogel matrix may also vary, taking into account the knowledge of a specialist in this field of technology, which will not have a negative impact on the achievement of the technical result.

Claims

CLAUSES OF THE INVENTION 1. A method for producing a biological receptor element based on microorganisms immobilized in a xerogel matrix, comprising sequentially mixing the biomass of microorganisms with a phosphate buffer solution, tetraethoxysilane and polyvinyl alcohol to obtain a suspension containing microorganisms, characterized in that a nitrocellulose-based substrate is used, which is pre-wetted with a NaF solution, then the resulting suspension containing microorganisms is passed through the substrate, and after immobilization of the microorganisms, the resulting matrix is ​​dried and cut into fragments.

2. The method according to I.1, characterized in that a buffer solution with pH=6.8 is used, containing 3 mM KH2PO4 + 33 mM Na2HPO4, and in an amount providing a cell titer of 150 mg / cm 3 .

3. The method according to I.1, characterized in that tetraethoxysilane is introduced into the suspension in a volume ratio of 1:

1.

4. The method according to I.1, characterized in that polyvinyl alcohol is introduced into the suspension in a suspension / alcohol ratio of 75:25 vol.%.

5. The method according to paragraph 1, characterized in that a 5-10% NaF solution is used.

6. The method according to claim 1, characterized in that the suspension containing microorganisms is passed through the substrate in an amount of 5-10 ml.

7. The method according to claim 1, characterized in that Paracoccus yeei microorganisms are used.

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