Method for detecting synthetic toxicity in water

Technical Field

The invention relates to an environment-friendly in the method for detecting water Technical Field, in particular relates to a reaction system using the luciferase detection method for comprehensive toxicity in a water body.

Background Art

Detecting the current International major harmful chemical contaminants in drinking water is indeed natare process is a gas phase chromatography, high performance liquid chromatography, chromatography-mass spectrometry for; the organic contaminant in drinking water analysis method for the enrichment of the main with the top end of the distribution space, hydrophobic adsorption, gas-phase extraction, solvent extraction, solid phase extraction, macroporous resin adsorption process and reverse osmosis. Drinking water quality of the Chinese National standard GB5750-85 by detecting criterion of the in "the drinking water standard of life detection method" and construction industry standard CJ/T206-2005 of the in "city water supply water quality standards", in the drinking water, and the detection of harmful chemical pollution substance mainly substantially in order to instrument analysis method, large-scale water systems in our country and the major urban water pollution situation provides a very important data. But these authenticity disposable of the detection method of the detection range is narrow, and very time-consuming and laborious. Due to the wide variety of organic dye in drinking water, but the vast majority of the water content in the organic matter is extremely little, to all analyzed monitoring and study these pollution component, the workload is very great. These instrument long detection time of the method, the operation is complex, the need for costly to instrument, in a short time and the water sample is determined whether there is any biological toxicity; in addition, qualitative or quantitative detection of even single or more kinds of harmful substances, also the harmful substance to the comprehensive biological toxic effect, because different toxic substances or synergistic antagonism of the biological toxicity effect of these chemicals is not to calculate the specific amount. Therefore these authenticity method of the comprehensive toxicity of the detection quality with very large lacks limits the nature. Over the past ten years, some developed countries in Europe and the United States the comprehensive toxicity of very great importance to the detection technique of water quality, such as Germany, the United States, the Netherlands, Italy and Spain will water quality and environmental sample National standard test included in the comprehensive toxicity. The is a core technology of the bio-sensing technology-based detection system, in particular to a use of the light-emitting bacteria to the acute toxicity testing technology, have been many countries for detecting large cities used for drinking water supply system. The sensor has a convenient carrying, the detection time is short, wide detection range, and the like. China in 1995 year acute toxicity of the test method, standard number gigabyte/T15441-1995, the standards of toxic or harmful substances is utilized in the marine luminescent baumanni T3 biological luminescent has an inhibitory effect of the principle of the comprehensive toxicity of the water quality is judged, however, the method for the maintenance of T3 normal survival of the bacteria, is added to the test system to a relatively high concentration of NaCl (2%-3%). However, a large amount of chlorine ion or sodium ion in the presence of the affected to a considerable extent in a sample of biological availability of some pollutants and toxic sequence, at the same time in the sample to the test fresh water system there are certain limitations of toxic pollutants and contradictions.

Document retrieval is found on the prior art, Patent application number 200610150846.9 of China Patent, the name of "concentrated preconditioning is used to water the method of detection of the comprehensive toxicity", the patented method stadnicki ultrafiltration membrane, a reverse osmosis membrane is subjected to a concentration treatment to the sample, then utilize the harmful substance to the light-emitting bacteria inhibit the characteristic to determine the light-emitting of the comprehensive toxicity of the sample. The advantage of this method is the comprehensive toxicity of the sample can be detected, the detection time is short, the sensitivity is high; however, the method needs to be in the application of the light-emitting bacteria is cryopreserved reviviscence processing, require specific culture facilities and refrigeration equipment, so that this kind of method in the detection of the-site water sample by the comprehensive toxicity of certain restrictions; in addition, because the cryopreserved, reviviscence and in the process of training, the different batches of the light intensity of the blank control sample the large difference, make this kind of method is not good stability and repeatability.

Content of the invention

The purpose of this invention lies in to the deficiency of the prior art, provides a method for detecting synthetic toxicity in water, using luciferase reaction system to replace photobacteria for the detection of the comprehensive toxicity, has the advantages of high detection sensitivity, the operation is simple, the detection time is short, the stability and repeatability is good, the advantages of not needing valuable instrument, can realize on-the-spot detecting the comprehensive toxicity of the water sample, is also suitable for the large-scale social activities held emergency detection and anti-terrorism need.

This invention is realized through the following technical scheme, the invention utilizes luciferase to substitute photobacteria for the detection of the comprehensive toxicity, luciferase in vivo from firefly insect is extracted or obtained using gene engineering technology commercialization of the enzyme, the enzyme is in the water environment or the presence of a trace amount of toxic or harmful substances is very sensitive. The harmful substances exist in the water, comprises a heavy metal, inorganic chemical contaminants, such as organic pollutants, the activity of the luciferase can be changed, thereby allowing the luciferase in the presence of triphosadenine of catalytic D-luciferin reaction is inhibited, produced by the reaction with the intensity of the light and change the content of the toxic, according to the degree of decrease in the chemical luminous intensity of the comprehensive toxicity of the water sample is judged.

This invention adopts the luciferase enzyme by extracting firefly insect ; fluorescein on the extracted firefly insect D-fluorescein, through the commercial, such as can be purchased directly from the US Sigma. The through the detection of toxic or harmful substances in the water sample, the fluorescence rope enzyme institute catalytic chemical luminous intensity of whether the water sample is judged whether the contamination of drinking water or for determining the security can be drunk.

The invention comprises the following steps:

Section 1 step: preparation of the buffer solution of the reaction system: the reaction system buffer solution, including A liquid-enzymatic reaction buffer solution, B liquid-substrate diluent, and storing fluid C liquid-luciferase D liquid-substrate application liquid.

Wherein A liquid preparation method is as follows: 0.025mol/L N-Troxerutin methyl glycine of 20 milliliter -100 milliliter, pH7 . 5 the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution 5 milliliter -20 milliliter, the two buffer, with the pH of the mixed solution 0.1mol/L the HCl or 0.1mol/L   NaOH adjusted to 6.0-8.5.

Wherein B liquid preparation method is as follows: that the albumin takes the cow blood serum 5 milligram -20 milligram, MgCl₂. 6H₂ O   30 milligram -60 milligram, b pharmaceutical preparations 2 milligram -10 milligram, finally A liquid constant volume to 10 milliliters.

C liquid preparation method of which are as follows: the luciferase would buy for freeze-dried powder 0.01mol/L -0.05 mol/L   pH   7.4 Troxerutin of methyl amino methane-hydrochloric acid buffer solution prepared into concentration is 0.15 milligram/ml -0.55 milligram/ml enzyme storing fluid of, according to the 100 microliters each pipe, the packaged frozen-stored in the -80--20 the [...][...] in the refrigerator.

Wherein D liquid preparation method is as follows: the first for-fluorescein pH7.5 D the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution prepared into concentration is 0.2 milligram/ml -1.0 milligram/ml solution, the solution is then 5 the the L-15  L, B liquid 500 the the L-1500  L, C liquid 50 the the L-150  L, finally the three solution application liquid D a substrate.

Section 2 step: adenosine triphosphate disodium preparation of storing fluid and /: pH7.5 for the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution to prepare a concentration of 2.0 milligram/ml -10.0 milligram/ml of adenosine triphosphate disodium storing fluid ; application liquid prepared, pH7.5 for the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution dilution 5-50 times can be.

Section 3 step: detection of the comprehensive water-body toxicity: heating 6 is made of a polytetrafluoroethylene material of good light transmission of the reaction cup, or can be applied to ordinary 48 plastic hole raises the board. When a blank test, to three parallel sample each time, to the first in each cup or each hole respectively added liquid A the 250  L, liquid D the 50  L, secondary distilled water 100 the  L, incubating at room temperature 15 minutes to 25 minutes; then the above-mentioned each reaction cup or a culture plate into a light-emitting in the instrumentation, to the reaction cup or reaction to adenosine triphosphate disodium application liquid 100 the  L, mixing door with the pipette gun; 15 seconds to 30 seconds after-recording light-emitting intensity. When the test each water sample, also to three parallel sample, test method is basically the same with the blank-like test, different water sample to be measured for the alternative secondary distilled water. According to the following formula to calculate the light intensity of the water sample to be measured:

The relative light-emitting intensity of the three parallel sample = sample an average luminous value of three parallel sample blank ÷ an average luminous value × 100%

The light-emitting intensity according to the calculated, in reference to Chinese 1995 year acute toxicity test of the water quality of standard method-GB/T15441-1995 method for judging the results, table 1.

Table 1

According to the table look-up result the comprehensive toxicity of the water sample to be detected: security, suspicious or not the security.

The invention utilizes luciferase reaction is very sensitive to the characteristics of toxins, the establishment of the detection in the water body of the new method for the comprehensive toxicity, compared with the existing detection method, the invention has the following characteristics and advantages:

1, fast. In 30 the detection result can be obtained within minutes. In the actual detection, only needs to the preparation and storage of the solution of the reaction system, adenosine triphosphate two sodium fluids field dilution, then adding more than ten minutes incubation the water sample to be detected can be carried out after the test. Is not necessary as Background Art, among to the poison sensitive to recovery and re-culture, from the light-emitting bacteria treated to a sample test is complete, the actual need for 2 hours or more. While the present invention from a preparation reagent to obtain detection result, only 20 to 30 minutes, thereby greatly save time. If the application of the current National standard assay for detection of toxic or harmful substances by the water pollution, will need to be a few days before a result, cannot realize on-the-spot quick warning river water, and city water supply system and the safety of the drinking water the market.

2, high detection sensitivity. The invention has the advantages of the different toxins firefly insect derived luciferase reaction is very sensitive, that is, a trace amount of toxic substances can be significantly inhibiting the activity of the luciferase. Furthermore, luminescence apparatus for used in the present invention is the use of modern photon technology to detect the luminescent signal, this technology can detect the weak chemical or biological luminescence, and can distinguish between small light intensity change. Therefore the invention has the light-emitting system established by the comprehensive toxicity of water than the sensitivity of the detection method of using other enzymes thousands of times higher, than the general biological or biological cell detection method is higher than several tens of times to several hundred times.

3, all in the water sample to be detected can be detected the comprehensive toxicity of the poison. The National standard a certain method can only be detected specifically detecting a certain material, which cannot be judged that the detection items of toxic or harmful substances, thus very easily lead to undetected. And the method of the invention is to utilize the luciferase detection result to determine all the toxic and harmful substances in the sample to be detected of the comprehensive toxicity of the, therefore, even if the human also not found from the toxic materials or new chemical synthetic toxins, will also be a significant impact on the activity of the luciferase, through the method of the invention can measure the strength of its toxicity.

4, can detect a wide range of the material, can reach hundreds of toxic harmful substance detection of the comprehensive toxicity in the: heavy metal (1) and its kind of ion compound, such as inorganic mercury, MeHg, sodium ethylmercurithiosalicylate, cadmium, lead, copper, silver, or the like ; (2) the inorganic compound, such as cyanide, arsenic compounds, selenium compounds, ammonia, and the like; and (3) an organic compound, such as phenol, P-amino phenol, aniline, benzidine, formaldehyde, five chlorophenol, phenyltheophylline, dioxin, four phenyl toluene, Carbonitride said device, four phenyl toluene, tNT , paraquat, diazinon, Malathion, other organophosphorous pesticides and organochlorine pesticide, and the like; biological toxin (4), such as strychnin, reddish brown aspergillin , botulinum toxin, such as aflatoxin.

5, wide application range. The invention can be applied to : (1) the detection of the surface water toxicity ; (2) because the leakage, environmental pollution, natural disasters, man-made destruction and drinking water pollution caused by the detection of the situation; water for processing food (3) detection of toxicity ; (4) giang, river, lake and aquaculture water in the detection of the comprehensive toxicity ; (5) major social activity safety monitoring quality of the water in the; safety detection city water supply system (6).

6, practically is strong. Detecting by the invention method is simple comprehensive water-body toxicity, only the test buffer solution and substrate of the reaction mix, then is placed in a chemical light emitting instrument the result of detection can be obtained. When detecting in the field, in addition to utilizing portable sends study the luminescence apparatus for, without other special apparatus and the device, the detection time is short, the operation is convenient, comprehensive toxicity of the water sample can realize on-the-spot detection.

Description of drawings

Figure 1 for different concentrations of mercury to the luciferase inhibition schematic;

Figure 2 for different concentration of the inhibiting effect of the luciferase the cadmium is right schematic diagram.

Mode of execution

In conjuction with the embodiment of the present invention in detail: in the embodiment to the technical proposal of the present invention is implemented under the precondition, provides detailed and specific embodiment of the process of operation, the scope of protection of the present invention, however, is not limited to the following embodiments.

Example 1 detection of heavy metal pollution the comprehensive toxicity of the water sample

1, heavy metal pollution of the water sample

In this embodiment, first of all artificial preparing heavy metal mercury, lead, cadmium, copper contaminated water sample. In artificial preparation mercury pollution of the water sample, for double-distilled water is added to the standard mercury chloride, mercury content in the water sample are 0.006 milligram/L, 0 . 03 milligram/L, 0 . 15 milligram/L, 0 . 75 milligram/L, 3 . 75 milligram/L, 18 . 75 milligram/L, 93 . 75 milligram/L. According to the above-mentioned method using nitric acid respectively lead, cadmium chloride, copper sulfate preparation of heavy metal lead, cadmium, artificial polluted water samples of copper. Single or mixed heavy metal pollution in the preparation of the water sample are as follows : (1) concentration 0.03 milligram/L of the water sample mercury pollution ; (2) the concentration of 0.03 milligram/L of the water sample of lead pollution ; (3) concentration is 0.75 milligram/L of the water sample cadmium ; (4) the concentration of 0.75 milligram/L of copper contamination of the sample water ; (5) the concentration of 0.03 milligram/L of the water sample mercury pollution + 0.15 milligram/L of the water sample of lead pollution ; (6) the concentration of 0.03 milligram/L of the water sample mercury pollution + 0.15 milligram/L of the water sample of lead pollution + 0.75 milligram/L copper contamination of the sample water ; (7) 0.006 milligram/L + 0.03 milligram/L mercury pollution water sample water sample of lead pollution + 0.15 milligram/L cadmium water sample ; (8) the concentration of 0.15 milligram/L of the water sample of lead pollution + 0.75 milligram/L + 3.75 milligram/L of the water sample cadmium copper polluted water samples of; water tap (9).

2, the reaction system for the preparation of buffer solution

Preparation of buffer solution of the reaction system with the above-mentioned technical scheme step 1:

When in the preparation of liquid A, using 0.025mol/L N-Troxerutin of methyl glycine 40 milliliter, pH7 . 5 the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution 10 milliliter, with other pieces of stating;

When in the preparation of liquid B, said albumin takes the cow blood serum 10 milligram, MgCl₂. 6H₂ O   40 milligram, b pharmaceutical preparations 5 milligram, with other pieces of stating;

C liquid when in the preparation of, freeze-dried powder would buy for luciferase 0.03mol/L   pH   7.4 Troxerutin of methyl amino methane-hydrochloric acid buffer solution to prepare a concentration of 0.35 milligram/ml enzyme storing fluid of, the other pieces of stating;

When in the preparation of liquid D, the for-fluorescein pH7.5 D the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution into 0.5 milligram/ml solution, the solution is then the 10  L, liquid B the 1000  L, C liquid 100 the application liquid D   L into the substrate.

Adenosine triphosphate disodium storing fluid and/the preparation of technical scheme of the same in the step 2, the specific preparation, pH7.5 for the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution to prepare a concentration of 5.0 milligram/ml of adenosine triphosphate disodium storing fluid ; application liquid prepared, pH7.5 for the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution dilution 20 times can be.

3, the comprehensive toxicity of heavy metal pollution in the detection of the water sample

The comprehensive toxicity of heavy metal pollution in the detection of the water sample with the aforesaid technical proposal in the step 3, the blank and each of the control sample to three parallel examination type , in the course of the reaction is incubated at room temperature for 20 minutes, recording the luminous intensity of the time is 20 seconds.

Respectively according to the above-mentioned method for testing the above-mentioned 8 artificial heavy metal contaminated water sample and tap water sample, the chemical luminous intensity corresponding to the, buckle 1, judging the results are as follows : (1) is not safety; suspicious (2) ; (3) not security; suspicious (4); security not (5) ; (6) is not safety; suspicious (7) ; (8) not security ; (9) the security. At the same time, the method also can be sensitively reflect different different cadmium concentration and the mercury concentration of the inhibiting effect of the luciferase, are respectively in Figure 1 and Figure 2, Figure 1 in digital arab League uncle 1, 2, 3, 4, 5, 6, 7 that the mercury concentration of 0.00 milligram/L, 0 . 03 milligram/L, 0 . 15 milligram/L, 0 . 75 milligram/L, 3 . 75 milligram/L, 18 . 75 milligram/L, 93 . 75 milligram/L. Figure 2 in the digital arab League uncle 1, 2, 3, 4, 5, 6, 7, 8, 9 are respectively the concentration of the expresses the cadmium 0.00 milligram/L, 0 . 03 milligram/L, 0 . 15 milligram/L, 0 . 75 milligram/L, 3 . 75 milligram/L, 18 . 75 milligram/L, 93 . 75 milligram/L, 200.0 milligram/L, 400.0 milligram/L. Adopts the National standard gigabyte/T15441-1995 the water quality by the acute toxicity of the above-mentioned inspection method for testing a water sample, the results are as follows: is not safe (1) ; (2) safety ; (3) not security; suspicious (4) ; (5) is not safety; security not (6) ; (7) security; suspicious (8) ; (9) the security. The method basically with the National standard method with detection results, but at the same time shows that the method with regard to the detection of trace toxicity than existing National standard method is sensitive.

Example 2 detecting river water, industrial waste water and surface water of the comprehensive toxicity of the

1, the obtained sample: river water 3 from a downstream huangpu river water of Shanghai Minhang district section; water of the earth's surface 3 respectively from Shanghai Minhang district, Xuhui district and putuo district park terrian water sample; industrial waste water sample 3 respectively from Shanghai three food processing factory waste water have been processed.

2, the reaction system for the preparation of buffer solution

Preparation of buffer solution of the reaction system with the above-mentioned technical scheme step 1:

When in the preparation of liquid A, using 0.025mol/L N-Troxerutin methyl glycine of 20 milliliter, pH7 . 5 the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution 5 milliliter, with other pieces of stating;

When in the preparation of liquid B, said albumin takes the cow blood serum 5 milligram, MgCl₂. 6H₂ O   30 milligram, b pharmaceutical preparations 2 milligram, with other pieces of stating;

C liquid when in the preparation of, freeze-dried powder would buy for luciferase 0.01mol/L   pH   7.4 Troxerutin of methyl amino methane-hydrochloric acid buffer solution prepared into concentration is 0.15 milligram/ml enzyme storing fluid of, the other pieces of stating;

When in the preparation of liquid D, the for-fluorescein pH7.5 D the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution prepared into concentration is 0.2 milligram/ml solution, the solution is then the 5  L, liquid B the 500  L, C liquid 50 the application liquid D   L into the substrate.

Adenosine triphosphate disodium storing fluid and/the preparation of technical scheme of the same in the step 2, the specific preparation, pH7.5 for the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution to prepare a concentration of 2.0 milligram/ml of adenosine triphosphate disodium storing fluid ; application liquid prepared, pH7.5 for the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution diluted 5 times can be.

3, the detection of the comprehensive toxicity in the water sample to be tested

The detection of the comprehensive toxicity in the water sample to be tested with the above-mentioned technical scheme in the step 3, the blank and each of the control sample to three parallel examination type , in the course of the reaction is incubated at room temperature for 15 minutes, recording the luminous intensity of the time is 15 seconds.

According to the above-mentioned method of the above-mentioned water sample are tested respectively, according to their chemical luminous intensity corresponding to the, buckle 1, judging the results are as follows : (1) 3 mined downstream huangpu river a water sample in Shanghai Minhang district section, 2 not security, 1 a suspicious ; (2) 3 in the water sample Shanghai park the earth's surface a, 2 a water sample security, 1 a suspicious ; (3) 3 a three food processing plant in the waste water samples are not safe. Adopts the National standard gigabyte/T15441-1995 the water quality by the acute toxicity of the above-mentioned inspection method for testing a water sample, the results are as follows : (1) 3 mined downstream huangpu river a water sample in Shanghai Minhang district section, 1 not security, 2 a suspicious ; (2) 3 in the water sample Shanghai park the earth's surface a, 2 a water sample security, 1 a suspicious ; (3) 3 a Shanghai three food processing plant in the waste water sample, 2 a suspicious, 1 not safe.

Example 3 detection of commercial bottled water and the tap water of the comprehensive toxicity

1, the obtained sample: from the Shanghai 6 a supermarket acquisition 6 a sample of commercial bottled water, agricultural market collected from the Shanghai 6 a water sample barrel; 3 a different tap water from the tap water sample of the three zones.

2, the reaction system for the preparation of buffer solution

Preparation of buffer solution of the reaction system with the above-mentioned technical scheme step 1:

When in the preparation of liquid A, using 0.025mol/L N-Troxerutin methyl glycine of 100 milliliter, pH7 . 5 the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution 20 milliliter, with other pieces of stating;

When in the preparation of liquid B, said albumin takes the cow blood serum 20 milligram, MgCl₂. 6H₂ O   60 milligram, b pharmaceutical preparations 10 milligram, with other pieces of stating;

C liquid when in the preparation of, freeze-dried powder would buy for luciferase 0.01mol/L   pH   7.4 Troxerutin of methyl amino methane-hydrochloric acid buffer solution prepared into concentration is 0.55 milligram/ml enzyme storing fluid of, the other pieces of stating;

When in the preparation of liquid D, the for-fluorescein pH7.5 D the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution prepared into concentration is 1.0 milligram/ml solution, the solution is then the 15  L, liquid B the 1500  L, C liquid 150 the application liquid D   L into the substrate.

Adenosine triphosphate disodium storing fluid and/the preparation of technical scheme of the same in the step 2, the specific preparation, pH7.5 for the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution to prepare a concentration of 10.0 milligram/ml of adenosine triphosphate disodium storing fluid ; application liquid prepared, pH7.5 for the 0.01mol/L Troxerutin methyl amino methane-hydrochloric acid buffer solution diluted 50 times can be.

3, the detection of the comprehensive toxicity in the water sample to be tested

The detection of the comprehensive toxicity in the water sample to be tested with the above-mentioned technical scheme in the step 3, the blank and each of the control sample to three parallel treats examines the type , in the course of the reaction is incubated at room temperature for 20 minutes, recording the luminous intensity of the time is 30 seconds.

According to the above-mentioned method of the above-mentioned water sample are tested respectively, according to their chemical luminous intensity corresponding to the, buckle 1, judging the results are as follows : (1) 6 a commercially available in supermarkets barreled sample all for safety; on the agricultural market 2 water washing is not safe, 1 a suspicious, 3 safety. (2) the sample of 3 one of the three zones of Shanghai tap water sample are for safety. Adopts the National standard gigabyte/T15441-1995 the water quality by the acute toxicity of the above-mentioned inspection method for testing a water sample, the results are as follows : (1) 6 a commercially available in supermarkets barreled sample all for safety; on the agricultural market 3 water washing is not safe, 2 a suspicious, 1 security. (2) the sample of 3 one of the three zones of Shanghai tap water sample are for safety.

The above-mentioned embodiment, the detection of luciferase and D-luciferase Company Sigma freeze-dried powder from the United States. As can be seen from the above embodiment, the present invention has the advantages of high detection sensitivity, the operation is simple, the detection time is short, the stability and repeatability is good, the advantages of not needing valuable instrument, can realize on-the-spot detecting the comprehensive toxicity of the water sample.