KR20030021091A - A Kit for Determination of Concentration, Type and Toxicity of Toxic Materials Employing Immobilized Recombinant Bioluminescent Bacteria - Google Patents

Abstract

PURPOSE: A kit for analysis of toxicity of toxic materials in a sample using fixed recombinant luminescent microorganisms is provided, thereby easily and rapidly analyzing the concentration, kinds and toxicity of toxic material. CONSTITUTION: The kit for analysis of toxicity of toxic materials in a sample contains recombinant luminescent microorganisms on a microplate using a fixing material, wherein the recombinant luminescent microorganisms increase the amount of luminescence when the concentration of the toxic materials is increased. The toxic material is oxidative-toxic material such as hydrogen peroxide, paraquat or heavy metals; cell membrane-toxic materials such as cresol, chlorophenol or nitrophenol; is DNA-toxic material such as methyl-nitro-nitrosoguanidine, ethidium bromide, bisphenol A, mitomycin C or gamma rays; or is protein-toxic material such as ethanol, PCP or phenol. The recombinant luminescent microorganism is Escherichia coli DPD2511, DPD2540, DPD2794 and TV1061, .the fixing material is agar, sodium alginate or polyacrylamide, and the sample is liquid pollutant collected from water, air or soil.

Description

A kit for Determination of Concentration, Type and Toxicity of Toxic Materials Employing Immobilized Recombinant Bioluminescent Bacteria}

The present invention relates to a toxicity analysis kit for analyzing the concentration, type and degree of toxicity of a toxic substance present in a sample by using a fixed recombinant recombinant microorganism. More specifically, the present invention injects a sample into the wells of the microplate of the toxicity assay kit and the electrotoxicity analysis kit fixed to the microplate as a immobilization material of a recombinant luminescent microorganism that increases the amount of light emission in proportion to the concentration of the toxic substance, The present invention relates to a method for analyzing the concentration, type, and degree of toxicity of a toxic substance present in a sample by measuring the amount of emitted light at regular intervals.

The most successful microbiological toxicity bioassay instrument to date has been Microtox developed by Azur Environmental of the United States. (Microtox ) Is a system that analyzes the toxicity of a sample based on a decrease in the amount of emitted light due to toxicity when the luminescent microorganism comes into contact with the toxic sample. The instrument consists of a luminescence detector, a temperature controller, and a lyophilized luminescent microorganism and a sample infusion unit, and the luminescent microorganism used in the instrument is a freeze-dried undersea microorganism Vibrio fisheri ( NRRL B-11177). . Although electrical equipment is widely used due to the simplicity of measurement, rapidity, and high accuracy within 20% of the error range, it is impossible to determine the type of toxic substance because it is based on the death of luminescent microorganisms and the emission of luminescence due to toxicity. It does not provide information on how the toxicity of the sample affects microbial cells.

As a method to solve the above-mentioned problems, there is a method using a recombinant light-emitting microorganism. Similar to the method used by electrical equipment, this method detects changes in the amount of emission of microorganisms due to toxicity, so detection is quick, and it is possible to determine the type of toxicity because it uses a recombined luminescent microorganism to emit light in response to a specific toxic substance. In addition, since the amount of light emission varies depending on the concentration of the toxic substance, the concentration of the toxic substance can be predicted. However, in order to use a recombinant microorganism, since the culture of the microorganism is essential (minimum incubation time: 2 days), there is a problem in that the time taken for detecting the toxicity is long.

Therefore, there is a need to develop a simple, quick and accurate method to analyze the concentration, type and degree of toxicity of the toxic substances present in the sample.

Accordingly, the present inventors have made intensive efforts to develop a method for analyzing the concentration, type, and degree of toxicity of toxic substances present in a sample. It is fixed to the plate, the sample is injected into the well of the electric microplate, and the data obtained by measuring the amount of emission at regular intervals is compared with the reference data, thereby simplifying the concentration, type and degree of toxicity of the toxic substances present in the sample. It was confirmed that it can be analyzed quickly and accurately, and completed the present invention.

After all, a main object of the present invention is to provide a toxicity assay kit in which a recombinant luminescent microorganism, which increases the amount of luminescence in proportion to the concentration of toxic substances, is immobilized on a microplate as an immobilization material.

Another object of the present invention is to provide a method for analyzing the concentration, type and degree of toxicity of a toxic substance present in a sample using an electrotoxicity analysis kit.

1A, 1B, 1C, and 1D are graphs showing changes in light emission at low and high concentrations of toxic substances with respect to absorbance of DPD2511, DPD2540, DPD2794 and TV1061 cultures measured at 600 nm, respectively.

Figure 2 is a graph showing the change in the amount of light emission with time of the DPD2511 strain for each concentration of hydrogen peroxide.

3A and 3B are graphs showing the change in the amount of luminescence over time of DPD2540 and TV1061 strains for various concentrations of phenol, respectively.

Figure 4 is a graph showing the change in the amount of light emission over time of the DPD2794 strain for each concentration of mitomycin C.

5A, 5B, 5C and 5D are graphs showing the amount of light emitted by DPD2511, DPD2540, DPD2794 and TV1061 according to the elapsed storage days.

According to the present invention, a sample is injected into a well of an toxicology kit and an electric microplate in which a recombinant luminescent microorganism which increases luminescence in proportion to the concentration of a toxic substance is fixed on a microplate, and the luminescence is measured at regular time intervals. Comparing the data with reference data includes analyzing the concentration, type, and degree of toxicity of the toxins present in the sample, wherein the toxins are oxidative-toxic substances of hydrogen peroxide, paraquat or heavy metals; Cell membrane-toxic substances of cresol, chlorophenol or nitrophenol; Methyl-nitro-nitrosoguanidine, ethidium bromide, bisphenolol A, mitomycin C or gamma-ray DNA-toxic substances; Or protein-toxic substances of ethanol, PCPs or phenols. Meanwhile, the immobilization material is agar, sodium alginate or polyacrylamide, and the sample contains liquid contaminants extracted from water quality, air or soil.

Genetically modified luminescent microorganisms are classified into microorganisms that increase the amount of luminescence in the presence of a specific toxic substance and microorganisms that reduce the amount of luminescent. E. coli DPD2511, which emits oxidative stress to toxic substances (oxidative-toxic substances), E. coli DPD2540, which emits toxic substances on cell membranes (cell membrane-toxic substances), toxic to cause DNA damage (DNA-toxic E. coli DPD2794 which emits light to a substance), or E. coli TV1061 which emits light to a toxic substance (protein-toxic substance) that causes protein damage. The genotypes of the plasmids included in the strains exemplified above were katG :: luxCDABE in the DPD2511 strain, fabA :: luxCDABE in the DPD2540 strain, recA :: luxCDABE in the DPD2794 strain and grpE :: luxCDABE in the TV1061 strain. KatG, fabA, recA and grpE, located upstream of the lux operon, are inducible promoters induced by oxidative-toxic, membrane-toxic, DNA-toxic and protein-toxic substances, respectively. Start operation to repair damage by toxic substances. Thus, for example, in the presence of cell membrane-toxic substances, the strain will damage the cell membrane and activate the fabA promoter to repair it, thereby inducing lux operon downstream of the promoter. As a result, the amount of light emitted is increased.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1 Preparation of Fixation and Toxicity Assay Kits for Luminescent Microorganisms

Using agar, E. coli DPD2511 (DuPont, USA), DPD2540 (DuPont, USA), DPD2794 (DuPont, USA) and TV1061 (ATCC 69315) recombinant luminescent microorganism strains were immobilized on 96-well microplates: Strains used for immobilization To determine the cell mass showing the maximum luminescence, each strain was cultured in LB medium containing ampicillin until OD ₆₀₀ became 0.4, 0.8, 1.5 or 2.3, and then the amount of luminescence for toxic substances was measured. 1A, 1B, 1C, and 1D are graphs showing changes in light emission at low and high concentrations of toxic substances with respect to absorbance of DPD2511, DPD2540, DPD2794 and TV1061 cultures measured at 600 nm, respectively. As shown in Figures 1a, 1b, 1c and 1d, the DPD2511 strain shows the largest difference in the amount of light emission at low and high concentrations when the absorbance is 1.5, the other strains of 0.8. As a result, in 100 mL LB medium containing 2 mg of ampicillin, the DPD2511 strain was cultured until the absorbance at 600 nm was 1.5, and the DPD2540, DPD2794 and TV1061 strains were cultured until the absorbance was 0.8, and high-speed centrifugation was performed. It was. Each strain precipitated by centrifugation was resuspended in 0.5 mL of a high-pressure, high-temperature sterilized agar solution by mixing 20 mL of distilled water, 0.5 g LB mixture, and 0.3 g agar. After dispensing three rows in -well microplate, and left at room temperature until the agar solution was solidified, a toxicity assay kit was prepared in which each strain was immobilized on the microplate.

Example 2 Analysis of Toxic Substances

Example 2-1 Analysis of Oxidative-Toxic Substances

Each well of the microplates of the toxicity assay prepared in Example 1 was injected with hydrogen peroxide giving oxidative stress to the cells and the change in the amount of emission was measured: 0.4375%, 0.2188%, 0.1094%, by diluting the hydrogen peroxide successively. Prepared at concentrations of 0.0547%, 0.0274%, 0.0137% and 0.0069%, 100 μl or 150 μl were injected into each well of the second to eighth rows of the microplate. The first row of the microplate was injected with 100 μl or 150 μl of distilled water and used as a control and standard. The change in the light emission amount by the microorganisms of the microplates after the sample injection was completed was measured at 3 minutes intervals at 490 nm using a 96-well microplate optical measuring device (DYNEX Technologies, USA). The amount of emission due to toxicity is expressed as a relative light unit (RLU), which is a relative value calculated by comparing the amount of emission of a sample with a reference emission amount owned by the optical measuring device. Table 1 below shows the emission amount of each strain for each concentration of hydrogen peroxide giving oxidative stress. The values given in Table 1 are the average of the R-RLUs measured in three wells for each concentration (= RLU of the sample containing the toxic substance / RLU of the sample without the toxic substance (control)). It is the value of the point which shows the maximum light emission amount on the way. As shown in Table 1, as the concentration of hydrogen peroxide increases, only the DPD2511 strain selectively shows an increase in the amount of emitted light in proportion to the concentration of hydrogen peroxide, and it can be seen that other strains show little change in the amount of emitted light. Figure 2 is a graph showing the change in the amount of light emission with time of the DPD2511 strain for each concentration of hydrogen peroxide. As shown in FIG. 2, it can be seen that the amount of emitted light of the DPD2511 strain increases in proportion to the concentration of hydrogen peroxide and shows a marked difference within 4 hours.

Table 1: Analysis of oxidative-toxic substances

density(%) DPD2511 error DPD2540 DPD2794 TV1061 0 One 0.081634 One One One 0.0069 49.31373 1.791982 1.03 0.98 1.02 0.0137 194.1569 7.857935 1.12 1.03 1.11 0.0274 460.4118 28.73735 1.03 1.01 0.95 0.0547 788.0588 38.27967 0.99 1.12 1.01 0.1094 1560.588 28.37565 1.04 0.99 1.12 0.2188 1489.255 194.7596 1.11 1.03 1.17 0.4735 2239.941 9.176471 1.23 1.1 0.98

Example 2-2 Analysis of Cell Membrane-Toxins and Protein-Toxics

In each well of the microplate of the toxicity assay kit prepared in Example 1, cells were injected with phenol, which causes cell membrane damage and protein damage, and the change in the amount of luminescence was measured: Phenol from lines 2 to 8 of the microplate. Concentrations of 1000 ppm, 600 ppm, 360 ppm, 216 ppm, 130 ppm, 78 ppm and 47 ppm in the wells of each column to which the DPD2511, DPD2540 and DPD2794 strains were fixed, and 1000 ppm, 500 ppm, 250 ppm, 125 ppm, 62.5 ppm, 31.25 ppm in the wells of each column to which the TV1061 strain was fixed. And 100 μl or 150 μl each at a concentration of 15.625 ppm. The first row of the microplate was injected with 100 µl or 150 µl of distilled water and used as a control and reference. In the same manner as in Example 2-1, the change in the amount of light emitted by the light-emitting microorganisms of the microplates after sample injection was measured at intervals of 3 minutes using a 96-well microplate light measuring device. Table 2 below shows the amount of luminescence of each strain for each concentration of phenol giving cell membrane damage and protein damage. As shown in Table 2, only the DPD2540 and TV1061 strain showed an increase in light emission in proportion to the concentration of phenol, and the other strains showed little change in light emission as the concentration of phenol increased. 3A and 3B are graphs showing the change in the amount of luminescence over time of DPD2540 and TV1061 strains for various concentrations of phenol, respectively. 3A and 3B, it can be seen that the emission amount of the DPD2540 and TV1061 strains increased in proportion to the concentration of phenol and showed a marked difference within 3 hours and 4 hours (168 minutes and 201 minutes, respectively).

Table 2: Analysis of Cell Membrane-Toxins and Protein-Toxics

Concentration (ppm) DPD2511 DPD2540 error DPD2794 Concentration (ppm) TV1061 error 0 One One 0.368743 One 0 One 0.607692 47 1.03 3.660388 0.395068 1.06 15.625 3.057365 0.452065 78 1.01 3.706592 0.425623 0.97 31.25 2.361538 0.391628 130 0.97 6.277663 0.080783 0.91 62.5 2.761538 0.46942 216 One 14.51001 0.170353 0.84 125 4.887179 0.653182 360 1.05 26.39729 1.918376 0.91 250 9.948718 1.082741 600 1.01 38.71984 1.609034 0.97 500 14.82692 2.866502 1000 1.13 39.57563 3.251343 0.84 1000 26.43846 1.928316

Example 2-3 Analysis of DNA-Toxic Substances

Each well of the microplates of the toxicity assay kit prepared in Example 1 was injected with mitomycin C, which causes DNA damage to cells, and the change in the amount of luminescence was measured: 1250 ppb, 750 ppb, 420 ppb, 270 ppb, 162 ppb, and mitomycin C. 97ppb and 58ppb were prepared, injected into each well of the microplate, and the change in the amount of light emitted by the light-emitting microorganisms was measured at 3 minute intervals in the same manner as in Example 2-1. Table 3 below shows the amount of emission of each strain for each concentration of mitomycin C giving DNA damage. As shown in Table 3, as the concentration of mitomycin C increases, only the DPD2794 strain selectively shows an increase in the amount of light emitted in proportion to the concentration of mitomycin C, and it can be seen that other strains show little change in the amount of light emitted. Figure 4 is a graph showing the change in the amount of light emission over time of the DPD2794 strain for each concentration of mitomycin C. As shown in Figure 4, the amount of luminescence of the DPD2794 strain increases in proportion to the concentration of mitomycin C, it can be seen that the continuous increase over time shows a noticeable difference within 4 hours (238 minutes).

Table 3: Analysis of DNA-Toxic Substances

Concentration (ppb) DPD2511 DPD2540 DPD2794 error TV1061 0 One One One 0.100203 One 58.32 1.01 0.98 35.29476 2.007868 1.01 97.2 1.05 1.01 46.22758 5.991663 0.96 162 0.97 1.04 64.99644 2.903601 0.94 270 0.96 1.07 97.7077 2.887305 1.01 450 1.04 1.14 117.0502 8.546394 1.03 750 1.1 1.08 136.178 9.48204 1.21 1250 1.04 1.11 130.8549 6.463943 0.97

Example 3 Expiration Date of Fixed Luminescent Microorganisms

In order to evaluate the shelf life of the microplates of the toxicity assay kit prepared in Example 1, the amount of luminescence for toxic substances was measured in the microplates refrigerated for various time periods: the microplates prepared in Example 1 were capped. After covering with an aluminum foil (fiol) and stored for 4 hours at various temperatures, the amount of luminescence for toxic substances was measured. 5A, 5B, 5C and 5D are graphs showing the amount of light emitted by DPD2511, DPD2540, DPD2794 and TV1061 according to the elapsed storage days. As shown in Figures 5a, 5b, 5c and 5d, in most strains the amount of light emission decreases gradually with increasing storage days, but the proportional increase in the amount of light emission with respect to the concentration of the toxic substance is found to be unchanged up to 3 weeks. have. Therefore, it can be seen that the expiration date of the light-emitting microorganisms fixed on the microplate by the immobilization method is about 3 weeks at 4 ° C.

As described and demonstrated in detail above, the present invention provides a sample in the well of the microplate of the toxicity assay kit and the electrotoxicity analysis kit, which is immobilized on a microplate with a recombinant luminescent microorganism which increases the amount of emission in proportion to the concentration of the toxic substance. The method provides a method of analyzing the concentration, type, and degree of toxicity of a toxic substance present in a sample by injecting the light emission amount at regular time intervals. According to the analytical method of the present invention, an additional process such as culturing microorganisms is not required, and an error and an error analysis can be performed at the same time by simply injecting a sample and measuring the amount of emitted light. And toxic levels can be analyzed simply, quickly and accurately, and all samples that can be extracted in the liquid phase can be used as samples, so that contamination of the pre- and post-treatment stages of wastewater treatment plants, water pollution of chemical plants, air pollution, or It can be applied to various fields such as soil pollution.

Claims (13)

Toxicity analysis kit that analyzes the concentration, type, and degree of toxicity of the toxic substances present in the sample by fixing the recombinant luminescent microorganisms that increase the amount of luminescence in proportion to the concentration of the toxic substances on the microplate as an immobilization material. The method of claim 1, Toxic substances are oxidative-toxic substances of hydrogen peroxide, paraquat or heavy metals. Toxicity Assay Kit. The method of claim 1, Toxic substances are cell membrane-toxic substances of cresol, chlorophenol or nitrophenol. Toxicity Assay Kit. The method of claim 1, Toxic substances are methyl-nitro-nitrosoguani dine, ethidium bromide, bisphenol-A, mitomycin C or gamma-ray DNA-toxic substances Toxicity Assay Kit. The method of claim 1, Toxic substance is characterized in that the protein-toxic substance of ethanol, PCP or phenols Toxicity Assay Kit. The method of claim 1, Recombinant luminescent microorganisms are Escherichia coli DPD2511, DPD2540, DPD2794 and TV1061, characterized in that Toxicity Assay Kit. The method of claim 1, Immobilization material is characterized in that the agar, sodium alginate or polyacrylamide Toxicity Assay Kit. The method of claim 1, The sample is a liquid pollutant extracted from water, air or soil. Toxicity Assay Kit. Injecting a sample into the wells of the microplate of the toxicity analysis kit of claim 1, and comparing the data obtained by measuring the amount of light emission at regular intervals with a microplate light measuring device and the reference data, the toxicity present in the sample How to analyze the concentration, type and degree of toxicity of a substance. The data obtained by injecting a sample into the well of the microplate of the Escherichia coli DPD2511 fixed with the oxidative-toxic substance, and measuring the amount of luminescence at regular time intervals using a microplate optical measuring device. A method of analyzing the concentration of oxidative-toxic substances present in a sample and the degree of toxicity, comprising comparing with reference data. Based on the data obtained by injecting a sample into the well of the microplate of the Escherichia coli DPD2540 fixed to the cell membrane-toxic substance, and measuring the amount of luminescence at regular time intervals using a microplate optical measuring device. A method for analyzing the concentration and toxicity of cell membrane-toxic substances present in a sample, comprising comparing the data. Based on the data obtained by injecting a sample into the well of the microplate of the Escherichia coli DPD2794 fixed with DNA-toxic substances, and measuring the amount of luminescence at regular intervals with a microplate light measuring device. A method of analyzing the concentration and degree of toxicity of DNA-toxic substances present in a sample, comprising comparing the data. Based on the data obtained by injecting a sample into the well of the microplate of the Escherichia coli TV1061 fixed to the protein-toxic substance, and measuring the amount of luminescence at regular time intervals using a microplate light measuring device. A method for analyzing the concentration and degree of toxicity of protein-toxic substances present in a sample, comprising comparing the data.