RU2413772C2 - Bioluminescent biomodule for analysing toxicity of various media and preparation method thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: biomodule contains the following, immobilised into gel: nicotinamide adenine dinucleotide (NADN): flavine mononucleotide (FMN)-oxidoreductase, myristic aldehyde, NADN, enzyme stabiliser - dithiothreitol in concentration of 1·10^-4 M, a FMN solution and a substrate. The method of preparing the biomodule involves preparation of 3-5% gel by boiling a starch suspension in a phosphate buffer or heating a gelatin suspension in a phosphate buffer to 60-80°C, cooling the gel to 24°-30°C, mixing the buffer solution of a bienzymatic system of luminescent bacteria NADN:FMN-oxidoreductase - luciferase, solutions of myristic aldehyde, reduced nicotinamide adenine dinucleotide, enzyme stabiliser - dithiothreitol in concentration of 1·10^-4 M with gel, depositing onto the substrate and drying at 4-10°C. The biomodule is activated with a solution of flavin mononucleotide.

EFFECT: invention increases activity output and maximum luminous intensity of the biomodule by 1,3-3 times, increases the time for constant illumination level of the biomodule to 2-20 minutes, increases the storage time of the biomodule without loss of activity by 2 times, increases thermal stability and resistance of the biomodule to chemical factors of the environment.

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Description

The invention relates to biochemistry, in particular to methods for producing immobilized enzymes, and can be used in chemistry, biochemistry, medicine, microbiology, ecology and agriculture for the analysis of substances by the bioluminescent method.

Known biomodule for the analysis of environmental toxicity, including immobilized recombinant luminous bacteria Escherichia coli. A method of manufacturing a biomodule consists in preliminary obtaining isolated colonies by using a medium containing a specific antibiotic, growing the colonies for 12 hours, inoculating them in 5 ml of culture medium, culturing for 12 hours at a temperature of 30-37 ° C, adding 100 μl of the resulting solution to 15 ml of culture medium, followed by cultivation to achieve an optical density of 0.8 at 600 nm. Then, centrifugation of the obtained cell cultures at 6000 g for 15 minutes, the addition of 2 ml of a matrix for immobilization to the obtained cell pellet, thorough mixing and dosing of the resulting mixture of 2 μl in the wells are required. After the mixture has hardened, additional nutrient medium is added to the wells. As a matrix for immobilization, a mixture consisting of microagar (Duchefa, Netherlands) with a concentration of 15 g / l and Luria-Bertani nutrient medium (Difco, USA) at a concentration of 30 g / l is used. The resulting biomodule must be stored at 4 ° C [Lee J.H., Mitchell R.J., Kim B.C., Cullen D.C., Gu M.B. A cell array biosensor for environmental toxicity analysis // Biosensors and Bioelectronics, 2005. V.21. P.500-507].

The disadvantages of the known biomodule and the method of its preparation are the complexity of the procedure for its preparation, the need to provide special conditions for storage (4 ° C), a long analysis time, because preliminary incubation of the biomodule for 1 hour at a temperature of 30 ° C is necessary to restore the activity of luminous bacteria, as well as incubation of the biomodule with the analyzed solution for 30 minutes. In addition, during the analysis, it is additionally required to create specific conditions to ensure the normal operation of the biomodule: dark boxing, maintaining a constant temperature (30 ° C), and the biotesting process itself takes 2 hours. Thus, the resulting biomodule is of little use for analytical studies, since it significantly increases the analysis time and reduces the accuracy of measurements, in particular during long-term analyzes.

Closest to the claimed invention are a multicomponent reagent and a method for its preparation, comprising a bienzyme system of luminous bacteria NADH: FMN-oxidoreductase-luciferase, substrates (NADH, FMN, myristin aldehyde), starch gel and phosphate buffer [US Pat. No. 22252963 RF, IPC C12Q 1/66, C12N 11/04, publ. 05/27/2005 (prototype)].

It is known that the prototype contains in the composition of the used enzyme preparation a stabilizer of SH-groups of enzymes - DTT. However, its amount is extremely small (0.02 mM), which leads to a number of significant disadvantages of the prototype: low reagent activity (120 mV), a rapid decrease in the luminescence intensity of the NADH: FMN-oxidoreductase-luciferase luminescence system (the intensity drops to 0 mV in within 2 minutes), as well as low sensitivity of the reagent to toxic substances and a relatively short shelf life of the reagent in the active state (180 days at a storage temperature of 4-10 ° C).

The technical result of the invention is to increase the activity output and maximum intensity of the biomodule glow from 30 to 300%, increase the time of a constant level of glow of the biomodule up to 2-20 minutes, increase the storage time of the biomodule without loss of activity by 2 times, increase the thermal stability and resistance of the biomodule to chemical factors environment, in ensuring high sensitivity to the action of toxic substances.

The technical result is achieved by the fact that in a bioluminescent biomodule for analyzing the toxicity of various media containing luciferase, NADH: FMN oxidoreductase, myristic aldehyde, NADH immobilized in a gel, as well as a substrate and solution of FMN, it is new that the enzyme stabilizer dithiothreitol in concentration of 1 · 10 ^-4 M.

The technical result is also achieved by the method for preparing the bioluminescent biomodule according to claim 1, which includes preparing the gel in phosphate buffer, cooling the gel to 24-30 ° C, mixing the buffer solution of the bienzyme system of luminous bacteria NADH: FMN oxidoreductase-luciferase, solutions of myristine aldehyde, reduced nicotinamide adenine dinucleotide, dithiothreitol enzyme stabilizer at a concentration of 1 x 10 ^-4 M to the gel, dosing on a substrate and drying at a temperature of from above 4 to 10 ° C and the activation biomodule sol rum flavin mononucleotide.

The addition of dithiothreitol to the composition of the biomodule at a concentration of 1 · 10 ^-4 M can significantly improve the characteristics of the biomodule (activity, storage time, resistance to changes in pH and temperature) while ensuring high sensitivity to the action of toxic substances.

When the content of DTT exceeds the specified concentration, the sensitivity of the reagent to the action of toxic substances is significantly reduced, which is unacceptable, since the biomodule is designed to analyze the toxicity of various environments and must respond to the presence of even small amounts of toxic substances. Apparently, in the case where excess concentrations of DTT are present, DTT molecules are able to restore FMN, and reduced FMN (FMN · H ₂ ) is a substrate for the light emission reaction catalyzed by luciferase. Thus, in the presence of excess concentrations of DTT, greater light emission is observed even in the presence of toxic substances, which leads to a decrease in the sensitivity of the biomodule to the action of toxicants.

In the case when the concentration of DTT in the biomodule is lower than the indicated concentration, a significant decrease in the activity of the biomodule, a rapid decrease in the intensity of its glow, a decrease in the storage time of the biomodule are observed.

Comparison of the claimed technical solutions with the prototype and other technical solutions from this technical field made it possible to establish their compliance with the criteria of "novelty" and "inventive step".

Figure 1 presents the obtained results of the activity of the biomodule based on starch gel depending on its composition and method of preparation (drying the biomodule directly in the wells of the tablet). Figure 2 gives the kinetics of the glow of the biomodule based on starch gel depending on its composition and method of preparation (drying the biomodule directly in the wells of the tablet). Figure 3 shows the yield of activity of a biomodule based on starch gel depending on its composition and method of preparation (drying the biomodule on lavsan followed by use in a tablet luminometer). Figure 4 shows the kinetics of the glow of the biomodule based on starch gel depending on its composition and method of preparation (drying the biomodule on lavsan followed by use in a tablet luminometer). Figure 5 shows the yield of activity of a biomodule based on starch gel depending on its composition and method of preparation (drying the biomodule on lavsan followed by use in a cuvette luminometer). Figure 6 presents the kinetics of the glow of the biomodule based on starch gel depending on its composition and method of preparation (drying the biomodule on lavsan followed by use in a cuvette luminometer). Figure 7 shows the dependence of the activity of a biomodule based on starch gel on the time of its storage at a temperature of + 25 ° C. On Fig presents the kinetics of the glow of the biomodule based on a gelatin gel when introduced into its composition as a stabilizer of dithiothreitol. Figure 9 shows the dependence of the maximum activity of the starch gel-based biomodule on the drying temperature. Figure 10 shows the time dependence of a constant level of luminescence of the biomodule on the drying temperature. 11 shows the activity of a biomodule prepared on the basis of starch gel, and its sensitivity to benzoquinone, depending on the content of DTT in the composition of the biomodule. On Fig presents the activity of the biomodule, prepared on the basis of starch gel, and its sensitivity to CuSO ₄ depending on the content of DTT in the composition of the biomodule.

The biomodule is an enzymatic active substance consisting of two components:

1. enzymes: (luciferase and NADH: FMN oxidoreductases), substrates: (NADH and myristic aldehyde), a stabilizer of the enzymes dithiothreitol and buffer, jointly immobilized in a gel;

2. FMN solution.

Example 1

A biomodule for bioluminescence analysis is prepared as follows: a solution of the NADH bienzyme system: FMN-oxidoreductase-luciferase is prepared on 0.05 M phosphate buffer pH 7.0. Solutions of NADH, FMN and dithiothreitol (DTT) are prepared in distilled water or 0.05 M phosphate buffer pH 7.0. A 0.02% myristic aldehyde solution is prepared from a 0.2% alcohol solution by dilution in distilled water or 0.05 M phosphate buffer pH 7.0. A portion of 0.630 g of starch is placed in 20 ml of 0.05 M phosphate buffer pH 7.0, stirred vigorously and boiled the mixture until a clear solution is obtained. To 10 ml of gel cooled to 25-28 ° C, 50 μl of a solution of the NADH: FMN-oxidoreductase-luciferase system containing 20 μg of luciferase and 0.018 units of NADH: FMN-oxidoreductase activity, 200 μl of a 5 · 10 ^-3 M DTT solution are added, 250 μl of a 0.02% solution of myristic aldehyde, 250 μl of a NADH solution of various concentrations (0.8 · 10 ⁻² M; 1.6 · 10 ⁻² M or 3.2 · 10 ⁻² M). Dose the resulting mixture of 50 μl into the wells of the tablet, dried at a temperature of 4 ° C for 24 hours. The biomodule is activated by adding 150 μl of 3.3 · 10 ^-5 M FMN solution. Luminescence was recorded using a Lumat TriStar flatbed bioluminometer (Berthold Technologies, Germany). The activity output of the biomodule Q is determined by the formula:

Q = (I ₁ / I ₂ ) × 100%, where

I ₁ - the maximum intensity of the glow of the immobilized biomodule,

I ₂ is the maximum luminescence intensity of the soluble bienzyme system (the number of enzymes, substrates and stabilizer in one biomodule corresponds to their amount in solution in a single measurement).

In particular, in the technology for creating a biomodule, which consists in introducing 1 · 10 ^-4 M DTT into it and dosing the biomodule into the wells of the tablet with subsequent drying, it is optimal to have 4 · 10 ^-4 M NADH and 1 · 10 ^-4 M DTT in the disk . In this case, the activity yield is 3% (Fig. 1).

Example 2

The immobilization of the NADH bienzyme system: FMN-oxidoreductase-luciferase and its substrates is carried out in a similar manner (see example 1). Solutions of the NADH bienzyme system: FMN-oxidoreductase-luciferase, DTT, myristic aldehyde and NADH are added to 10 ml of gel cooled to 25-28 ° C. The concentration of tetradecanal in one biomodule is 0.0002%, NADH - 1 · 10 ^-4 M, DTT - 1 · 10 ^-4 M, luciferase content - 0.2 μg. Dose the resulting mixture of 50 μl into the wells of the tablet, dried at a temperature of 4 ° C for 24 hours. The biomodule is activated by adding 150 μl of 3.3 · 10 ^-5 M FMN solution. Luminescence was recorded using a Lumat TriStar flatbed bioluminometer (Berthold Technologies, Germany). The kinetic characteristics of the biomodule are measured (shown in FIG. 2). The introduction of the DTT biomodule into the biomodule can significantly improve the biomodule characteristics: increase the maximum activity by 2.5 times, increase the time of a constant level of luminescence by 3 times (the maximum luminescence intensity decreases by 10% in 130 s for a biomodule containing an additional amount of DTT) ( figure 2).

Example 3

The immobilization of the NADH bienzyme system: FMN-oxidoreductase-luciferase and its substrates is carried out in a similar manner. Solutions of the NADH bienzyme system: FMN-oxidoreductase-luciferase, DTT, myristic aldehyde and NADH of various concentrations are added to 10 ml of gel cooled to 25-28 ° C.

The concentrations of all components correspond to the concentrations in example 1. Dose the resulting mixture of 50 μl per mylar film, dried at 4 ° C for 24 hours. The biomodule is activated by adding 150 μl of 3.3 · 10 ^-5 M FMN solution. Luminescence was recorded using a Lumat TriStar flatbed bioluminometer (Berthold Technologies, Germany). Using biomodule containing DTT and predried on Dacron, significantly increases the yield of activity when measured in a luminometer tablet: Q maximum yield is 18% activity observed at concentrations of NADH and DTT, equal respectively 4 × 10 ^-4 M and 1 · 10 ^{- 4} M (FIG. 3).

Example 4

The immobilization of the NADH: FMN-oxidoreductase-luciferase bienzyme system and its substrates is carried out in a manner analogous to Example 3. To 10 ml of gel cooled to 25-28 ° C, solutions of the NADH: FMN-oxidoreductase-luciferase, DTT, myristine N-aldehyde aldehyde are added. The concentration of tetradecanal in one biomodule is 0.0002%, NADH - 4 · 10 ^-4 M, DTT - 1 · 10 ^-4 M, luciferase content - 0.2 μg. The resulting mixture is dosed in 50 μl onto the mylar film, dried at 4 ° C for 24 hours. The biomodule is activated by adding 150 μl of 3.3 · 10 ^-5 M FMN solution. Luminescence was recorded using a Lumat TriStar flatbed bioluminometer (Berthold Technologies, Germany). The kinetic characteristics of the biomodule are presented in figure 4. The additional introduction of the DTT stabilizer into the biomodule allows increasing the maximum activity by 2.8 times and increasing the time of a constant level of luminescence by 3 times (the maximum luminescence intensity decreases by 10% in 30 and 90 s for a biomodule containing 0.2 · 10 ^-4 M DTT and 1 · 10 ^-4 M DTT, respectively) (Fig. 4).

Example 5

The immobilization of the NADH bienzyme system: FMN-oxidoreductase-luciferase and its substrates is carried out in a similar manner. Solutions of the NADH bienzyme system: FMN-oxidoreductase-luciferase, DTT, myristic aldehyde and NADH of various concentrations are added to 10 ml of gel cooled to 25-28 ° C. The concentrations of all components correspond to the concentrations in examples 1 and 3. The resulting mixture is dosed in 50 μl onto the mylar film, dried at 4 ° C for 24 hours. The biomodule is activated by adding 500 μl of 1 · 10 ^-5 M FMN solution. Luminescence was recorded using a Lumat LB 9507 cuvette bioluminometer (Berthold Technologies, Germany). The use of a biomodule preliminarily dried on Dacron and containing DTT significantly increases the activity yield during measurements in a cuvette luminometer: the maximum activity yield Q is 55% and is observed at NADH and DTT concentrations equal to 2 · 10 ^-4 and 1 · 10 ^-4 , respectively (figure 5).

Example 6

The immobilization of the NADH: enzyme system FMN-oxidoreductase-luciferase and its substrates is carried out in a manner analogous to Example 5. To 10 ml of gel cooled to 25-28 ° C, solutions of the NADH: FMN-oxidoreductase-luciferase, DTT, myristine N-aldehyde solution are introduced. The resulting mixture is dosed in 50 μl onto the mylar film, dried at 4 ° C for 24 hours. The concentration of tetradecanal in one biomodule is 0.0002%, NADH - 2 · 10 ^-4 M, DTT - 1 · 10 ^-4 M, luciferase content - 0.2 μg. The biomodule is activated by adding 500 μl of 1 · 10 ^-5 M FMN solution. Luminescence was recorded using a Lumat LB 9507 cuvette bioluminometer (Berthold Technologies, Germany). The kinetic characteristics of the biomodule are presented in Fig.6. The introduction of the DTT stabilizer into the biomodule allows one to increase the maximum activity by a factor of 2, reduce the rate of decrease in the luminescence intensity of the biomodule, increase the time of a constant level of luminescence by 2.6 times (the maximum luminescence intensity decreases by 10% for 50 and 130 s for a biomodule that does not contain DTT and containing 1 · 10 ^-4 M DTT, respectively) (Fig.6).

Example 7

The immobilization of the NADH: enzyme system FMN-oxidoreductase-luciferase and its substrates is carried out in a manner analogous to Example 5. To 10 ml of gel cooled to 25-28 ° C, solutions of the NADH: FMN-oxidoreductase-luciferase, DTT, myristine N-aldehyde solution are introduced. The resulting mixture is dosed in 50 μl onto the mylar film, dried at 4 ° C for 24 hours. The concentration of tetradecanal in one biomodule is 0.0002%, NADH - 4 · 10 ^-4 M, DTT - 1 · 10 ^-4 M, luciferase content - 0.2 μg. The biomodule is activated by adding 500 μl of 1 · 10 ^-5 M FMN solution. Luminescence was recorded using a Lumat LB 9507 cuvette bioluminometer (Berthold Technologies, Germany). Measure the activity of the biomodule over time at a storage temperature of + 25 ° C. The introduction of the stabilizer DTT into the biomodule allows 2 times to increase the shelf life of the biomodule without loss of activity (Fig.7).

Example 8

The immobilization of the NADH bienzyme system: FMN-oxidoreductase-luciferase and its substrates is carried out in a gelatin gel. To prepare the gel, 0.5 g of gelatin is placed in 10 ml of phosphate buffer and incubated for 30 minutes to swell the gelatin. The resulting suspension is heated to 60-80 ° C, then the resulting gel is cooled to 25-28 ° C and solutions of the NADH bienzyme system: FMN-oxidoreductase-luciferase, myristine aldehyde and NADH, and also DTT as a stabilizer are successively added. The resulting mixture is dosed in 50 μl onto the mylar film, dried at 4 ° C for 24 hours. The concentration of tetradecanal in one biomodule is 0.0002%, NADH - 1 · 10 ^-4 M, DTT - 3 · 10 ^-3 M, luciferase content - 0.2 μg. The biomodule is activated by adding 410 μl of a 1.25 · 10 ^-5 M FMN solution. Luminescence was recorded using a Lumat LB 9507 cuvette bioluminometer (Berthold Technologies, Germany). The introduction of the DTT stabilizer into the biomodule allows increasing the maximum activity by 70% and lowering the rate of decrease in the luminescence intensity of the biomodule: the maximum luminescence intensity of the biomodule containing DTT remains at a constant level for more than 20 minutes (Fig. 8).

Example 9

The immobilization of the NADH: enzyme system FMN-oxidoreductase-luciferase and its substrates is carried out in a manner analogous to Example 5. To 10 ml of gel cooled to 25-28 ° C, solutions of the NADH: FMN-oxidoreductase-luciferase, DTT, myristine N-aldehyde solution are introduced. The resulting mixture is dosed in 50 μl onto the mylar film, dried at different temperatures: 2 ° C, 4 ° C, 6 ° C, 8 ° C, 10 ° C, 12 ° C and 14 ° C for 24 hours. The biomodule is activated by adding 500 μl of 1 · 10 ^-5 M FMN solution. Luminescence was recorded using a Lumat LB 9507 cuvette bioluminometer (Berthold Technologies, Germany). When drying the biomodule in the temperature range from 4 ° C to 10 ° C, an increase in the maximum intensity of the glow of the biomodule is observed (Fig. 9).

Example 10

The immobilization of the NADH: enzyme system FMN-oxidoreductase-luciferase and its substrates is carried out in a manner analogous to Example 5. To 10 ml of gel cooled to 25-28 ° C, solutions of the NADH: FMN-oxidoreductase-luciferase, DTT, myristine N-aldehyde solution are introduced. The resulting mixture is dosed in 50 μl onto the mylar film, dried at different temperatures: 2 ° C, 4 ° C, 6 ° C, 8 ° C, 10 ° C, 12 ° C and 14 ° C for 24 hours. The biomodule is activated by adding 500 μl of 1 · 10 ^-5 M FMN solution. Luminescence was recorded using a Lumat LB 9507 cuvette bioluminometer (Berthold Technologies, Germany).

When drying the biomodule in the temperature range from 4 ° C to 10 ° C, an increase in the time values of the constant level of luminescence of the biomodule is observed (Fig. 10).

Example 11

The immobilization of the NADH: FMN-oxidoreductase-luciferase bienzyme system and its substrates is carried out in a manner analogous to Example 1. To 10 ml of starch gel cooled to 25-28 ° C, the NADH: FMN-oxidoreductase-luciferase, DTT aldehyde different concentration solutions are introduced. and NADN. The resulting mixture is dosed in 50 μl onto the mylar film, dried at 4 ° C for 24 hours. The concentration of tetradecanal in one biomodule is 0.0002%, NADH - 2 · 10 ^-4 M, DTT - from 0.02 mm to 0.8 mm, luciferase content - 0.2 μg.

The sensitivity of the biomodule to the action of benzoquinone is determined. To measure the luminescence parameters in a control sample, a biomodule, 500 μl of 0.1 M potassium phosphate buffer pH 7.0, 10 μl of 5 · 10 ^-4 M FMN solution is sequentially introduced into a cuvette of a Lumat LB 9507 bioluminometer (Berthold Technologies, Germany) and the maximum intensity is measured glow I _k . After reaching the maximum level of luminescence, add 50 μl of the analyzed solution of benzoquinone and again measure the maximum luminous intensity I _about . Calculate the residual glow in the presence of benzoquinone: I _o / I _k × 100%. The lower the level of residual glow, the higher the sensitivity of the biomodule to the action of a toxic substance.

The dependences of the activity of the biomodule and its sensitivity to the action of benzoquinone on the content of DTT in the composition of the biomodule are presented in Fig. 11. Best result i.e. the high sensitivity of the biomodule to the action of benzoquinone in combination with high activity is observed when DTT is included in the biomodule at a concentration of 1 · 10 ^-4 M (Fig. 11). A further increase in the amount of DTT in the biomodule leads to a decrease in its sensitivity to the action of toxic substances, as well as to a decrease in its activity. When lower concentrations of DTT are introduced into the biomodule, the activity of the biomodule is significantly reduced.

Example 12

The immobilization of the NADH: FMN-oxidoreductase-luciferase bienzyme system and its substrates is carried out in a manner analogous to Example 1. To 10 ml of starch gel cooled to 25-28 ° C, the NADH: FMN-oxidoreductase-luciferase, DTT aldehyde different concentration solutions are introduced. and NADN. The resulting mixture is dosed in 50 μl onto the mylar film, dried at 4 ° C for 24 hours. The concentration of tetradecanal in one biomodule is 0.0002%, NADH - 2 · 10 ^-4 M, DTT - from 0.02 mm to 0.8 mm, luciferase content - 0.2 μg.

Determine the sensitivity of the biomodule to the action of CuSO ₄ in a manner analogous to example 11. The dependences of the activity of the biomodule and its sensitivity to the action of CuSO ₄ on the content of DTT in the composition of the biomodule are shown in Fig. 12. Best result i.e. the high sensitivity of the biomodule to the action of CuSO ₄ in combination with high activity is observed when DTT is included in the biomodule at a concentration of 1 · 10 ^-4 M (Fig. 12). A further increase in the amount of DTT in the biomodule leads to a decrease in its sensitivity to the action of toxic substances, as well as to a decrease in its activity. When lower concentrations of DTT are introduced into the biomodule, the activity of the biomodule is significantly reduced.

The inventive biomodule is distinguished by long-term stable luminescence of high intensity, resistance to physical and chemical environmental factors (thermal stability, expansion of the ranges of pH optimal for the biomodule and ionic strength of solutions), storage stability without special conditions (remains active for 1 year at a storage temperature of 4 to 25 ° C), high sensitivity to the action of toxic substances. The biomodule is a dosed product intended for one analysis, and can be used in laboratory and field conditions.

Bioluminescent biomodule is designed to analyze the toxicity of various environments, including determining the quality of natural, waste water and aqueous solutions; determination of the toxicity of industrial and household waste, water extracts from the soil and bottom sediments; air toxicity assessments; determination of the presence of harmful substances in products of the food industry and agriculture; determining the degree of endotoxicosis of surgical, therapeutic and oncological patients.

Claims (2)

1. Bioluminescent biomodule for analysis of the toxicity of various media, containing luciferase, NADH: FMN oxidoreductase, myristic aldehyde, NADH immobilized in a gel, as well as a substrate and an FMN solution, characterized in that it contains 1 × 10 ^-4 dithiothreitol enzyme stabilizer M. 2. The method of preparation of the bioluminescent biomodule according to claim 1, comprising preparing the gel in phosphate buffer, cooling the gel to 24 ° -30 ° C, mixing the buffer solution of the bienzyme system of luminous bacteria NADH: FMN-oxidoreductase-luciferase, solutions of myristic aldehyde, reduced nicotinamidinidedendenine enzyme stabilizer dithiothreitol at a concentration of 1 · 10 ^-4 M with gel, dosing on a substrate and drying at a temperature of above 4 to 10 ° C and activation of the biomodule with a solution of flavin mononucleotide.

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