CN113390805A - Method for rapidly detecting and evaluating neurotoxicity of water sample in high throughput manner - Google Patents
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Abstract
The invention provides a method for quickly detecting and evaluating neurotoxicity of a water sample in a high-throughput manner, and belongs to the field of detection and analysis. The method comprises a toxicity detection method and a toxicity evaluation system, wherein the toxicity detection method is based on the principle of acetylcholinesterase (AChE) inhibition, adopts an electric eel AChE in-vitro exposure mode, and is combined with a microplate method to operate, so that the neurotoxicity of a tested water sample is rapidly detected; and the toxicity evaluation system adopts the equivalent conversion of methomyl and combines the emission limit value of methomyl to quantitatively represent the neurotoxicity level of the tested water sample in an equivalent interval. The method comprehensively inspects the neurotoxicity of the water sample from the perspective of in vitro biological tests, can quickly detect a plurality of water samples in batches in a single operation, and can quantitatively represent toxicity results, thereby quickly evaluating the neurotoxicity level of the water sample.
Description
Technical Field
The invention belongs to the field of detection and analysis, and particularly relates to a method for quickly detecting and evaluating neurotoxicity of a water sample in a high-throughput manner.
Background
Municipal sewage and industrial wastewater are complex in components, often contain various toxic and harmful pollutants, can produce certain toxic action on organisms after being discharged into the environment, and pose great threat to ecological systems and human health. At present, the water quality monitoring mainly focuses on the content of high-risk pollutants in chemical monitoring besides conventional indexes such as nitrogen, phosphorus, COD and the like. However, chemical monitoring cannot cover all chemicals present in water, and since the toxic action mechanisms of different chemicals on organisms are different, the comprehensive toxic effect of a large amount of pollutants in water cannot be evaluated by single-purification chemical monitoring. In recent years, the toxicity of wastewater is receiving more and more attention as a measure of the pollution level of wastewater, and the control of the toxicity of wastewater also becomes a hotspot in the fields of wastewater treatment and safe regeneration at present. With the development of wastewater toxicity monitoring towards the conventional water quality monitoring, the development of a high-throughput method for rapidly detecting and evaluating the toxicity of a water sample is of great significance, and particularly the method is directed at the batch treatment of the water sample.
Various pollutants with neurotoxicity, such as organophosphorus pesticides, benzene series and the like, exist in various waste water and polluted environmental water bodies, so that the neurotoxicity becomes one of important indexes for monitoring the water toxicity. At present, a mode of detecting and evaluating the neurotoxicity of a water body mainly adopts an in vivo exposure method of model organisms (in vivo), namely, after an aquatic model organism is exposed acutely or chronically by using a water sample to be detected, the change of nerve injury indexes (such as acetylcholinesterase) in the organism is detected, but the method has high cost and long period and cannot carry out batch detection on the water sample. In addition, the model biological method can only qualitatively evaluate the neurotoxicity of the water sample, but cannot quantitatively characterize the toxicity, which hinders the development of the toxicity evaluation and subsequent toxicity reduction of the water sample towards the direction of quantification and standardization to a certain extent. The aim needs to be achieved by developing a method suitable for detecting the neurotoxicity of the water sample in batches and establishing a quantitative evaluation system suitable for the neurotoxicity of the water sample.
In recent years, in vitro assay (in vitro) has attracted more and more attention because of its excellent correlation with the results of model in vivo exposure experiments, and it uses cells or enzymes to perform toxicity detection on the reaction of a sample in an in vitro environment (culture dish or culture plate), has advantages of short cycle, small reaction volume, low cost, and the like, and is suitable for batch detection when the number of samples is large.
Acetylcholinesterase (AChE) is a key enzyme in biological nerve conduction, and in cholinergic synapses, the enzyme can degrade acetylcholine, terminate the excitatory action of neurotransmitters on postsynaptic membranes, and ensure the normal transmission of nerve signals in organisms. The neurotoxicity of the tested chemical can be characterized to a certain extent by detecting the inhibition rate of the chemical on acetylcholinesterase in vitro. The theory provides a good theoretical basis for detecting the neurotoxicity of a water sample by using the inhibition of the acetylcholine esterase. However, in the prior art, acetylcholinesterase is mainly used for detecting the toxicity of pure chemicals, the operation method is not suitable for treating a water sample, and particularly when the water sample contains trace pollution, the sample is directly added to further dilute the sample, so that the toxicity result cannot be detected. Currently, there is little research on the detection of neurotoxicity in water samples. In addition, since acetylcholinesterase from different species has different sensitivity to pollutants, and the types of pollutants in water samples, especially in waste water and polluted water are various, the sensitive species need to be selected for evaluation by considering the possible difference between different species when evaluating the comprehensive neurotoxicity of the water samples. However, at present, no complete water sample neurotoxicity detection and evaluation method system exists.
Disclosure of Invention
1. Problems to be solved
In view of the fact that detection and evaluation of water sample neurotoxicity are more and more concerned in water quality safety evaluation, and the problems that a traditional neurotoxicity detection method is high in cost, long in period, incapable of batch operation, and lack of a toxicity evaluation system and the like exist, the invention provides a method for detecting and evaluating the neurotoxicity of a water sample in vitro through acetylcholinesterase, which can not only quickly detect the neurotoxicity of a plurality of water samples in a single test, but also can quantitatively represent the toxicity and the toxicity level of a tested water sample.
2. Technical scheme
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the invention provides a method for quickly detecting and evaluating neurotoxicity of a water sample in a high-throughput manner, which comprises the following steps:
(1) the method is characterized by concentrating organic matters in a water sample, wherein the concentration of the organic matters in the water sample comprises the following steps:
(a) organic matters in the enriched water sample can be separated from other component pollutants, so that the influence of other pollutants is reduced, and the accuracy of a detection result is improved;
(b) and concentrating the enriched organic matters, recording the concentration times, and increasing the concentration of the substances to be detected after concentration, thereby being beneficial to improving the accuracy of the detection result.
(2) The sample and the toxicity detection reference compound are subjected to gradient dilution, wherein the gradient dilution comprises the step of performing gradient dilution on the organic matters and the reference compound concentrated in the step (1) in proportion, so that the organic matters and the reference compound in the diluted water sample are kept at proper concentrations, and the accuracy of a detection result can be improved; the reference compound is a nonvolatile toxicity detection reference substance commonly used in the field; the dilution ratio and the dilution gradient described above can also be determined by preliminary experiments.
(3) And (3) acetylcholinesterase reaction, wherein the acetylcholinesterase reaction comprises the steps of reacting the sample, the solvent control and the reference compound under each dilution gradient in the step (2) with electric eel acetylcholinesterase (AChE), 5' -dithiobis (2-nitrobenzoic acid) (DTNB) and thioacetylcholine iodide (ATCH) respectively, detecting the absorbance values of the sample, the solvent control and the reference compound under the wavelength of 415nm, and taking the solvent control as a blank control without inhibiting the acetylcholinesterase.
(4) And (4) calculating the toxicity equivalent of the water sample, and respectively fitting the dose-effect curves of the reference compound and the sample according to the detection result in the step (3) to calculate the Toxicity Equivalent (TEQ) of the water sample.
(5) And (4) evaluating the toxicity grade of the water sample according to the toxicity equivalent of the water sample calculated in the step (4).
Preferably, the enrichment in step (1) is performed by solid phase extraction, liquid-liquid extraction, etc.
Preferably, the concentration in the step (1) is performed by nitrogen blowing, and the concentration multiple is 1000-5000 times.
Preferably, the sample concentrated in the above step (1) is dissolved in methanol.
Preferably, in the step (2), the reference compound is methomyl, which is diluted by 10-12 times of the reference compound according to a 2-3-fold proportion, wherein the diluted concentration range is 1000-0.001 mg/L.
Preferably, the sample gradient dilution in the step (2) includes 2-3 times of dilution to 4-6 concentrations.
Preferably, the solvent control described in step (3) above comprises methanol.
Preferably, the step of loading the sample for acetylcholinesterase reaction in step (3) above comprises: firstly adding the sample, the solvent control and the reference compound under each dilution gradient, standing until the solvent is completely volatilized, then sequentially adding the electric eel AChE and the DTNB, standing for a certain time at 37 ℃, finally adding the ATCH, standing for 10-15 min at room temperature in a dark place, and then detecting the absorbance, wherein the detection effect in the sample adding step is better. Further, placing the mixture in a fume hood for 15-20 min until the solvent is completely volatilized, and/or standing the mixture for 10-20 min at 37 ℃.
Preferably, the acetylcholinesterase reaction in step (3) above is carried out in a 96-well plate.
Preferably, the addition amount of each substance for acetylcholinesterase reaction in step (3) is: the amount of the electric eel acetylcholinesterase is 0.005-0.015U relative to 10 mu L of a sample to be detected; the amount of the 5,5' -dithiobis (2-nitrobenzoic acid) is 0.02-0.03 mg; the amount of thioacetyl choline iodide is 0.02-0.03 mg.
Preferably, the addition amount of each substance for acetylcholinesterase reaction in step (3) is: the amount of the electric eel acetylcholinesterase is 0.01U relative to 10 mu L of the sample to be detected; the amount of 5,5' -dithiobis (2-nitrobenzoic acid) was 0.025 mg; the amount of thioacetylcholine iodide was 0.025 mg.
Preferably, in the step (3), the addition amount of each substance per well of the acetylcholinesterase reaction is as follows: the samples, solvent control and reference compound at each dilution gradient were 10. mu.L, electric eel AChE (dissolved in phosphate buffer) at a concentration of 0.067U/mL, DTNB (dissolved in phosphate buffer) at a concentration of 0.5g/L, and ATCH (dissolved in phosphate buffer) at a concentration of 0.5g/L, at a concentration of 150. mu.L.
Preferably, the calculation method of the water-like toxicity equivalent in the step (4) is as follows:
(i) calculating the inhibition rate EC of AChE by the sample and the reference compound at each concentration according to the formula (1);
(ii) plotting dose-response curves for the sample and reference compounds, respectively, to yield EC 50;
(iii) calculate sample TEQ according to equation (2).
Formula (1)
Formula (2)
Preferably, the water-like toxicity equivalent in the step (4) is characterized in a manner of mg methomyl/L water sample.
Preferably, the evaluation system of toxicity level in step (5) is:
according to the discharge standard of water pollutants for pesticide industry (survey prompter), the limit value of the methomyl discharge is 0.2 mg/L. Therefore, a water sample with the methomyl equivalent of more than 0.2mg/L is regarded as high-toxicity, and the toxicity grade is set as grade I; in addition, with reference to the acute toxicity threshold setting, with 1/10 the threshold being the limit, grade II and III toxicity levels are set, respectively.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) the method for quickly detecting and evaluating the neurotoxicity of the water sample with high flux provided by the invention utilizes the high-sensitivity in-vitro test method of the electric eel acetylcholinesterase to quickly detect the neurotoxicity of the water sample, can quickly detect a plurality of samples in batches in a single operation, and improves the detection efficiency.
(2) According to the method for rapidly detecting and evaluating the neurotoxicity of the water sample with high flux, provided by the invention, the sample adding step in the acetylcholinesterase reaction is optimized, as shown in figure 3, so that the inhibition rate of the water sample on the acetylcholinesterase is improved, and the detection limit of the neurotoxicity of the water sample is further improved.
(3) The method for rapidly detecting and evaluating the neurotoxicity of the water sample with high throughput provided by the invention provides a neurotoxicity evaluation system based on the toxicity equivalent of methomyl and the emission limit value, and can be used for quantitatively evaluating the neurotoxicity size and grade of the tested water sample.
(4) The method for rapidly detecting and evaluating the neurotoxicity of the water sample with high flux provided by the invention is used for comprehensively evaluating the neurotoxicity of the water sample from a biological perspective aiming at the characteristics of mixed pollutants in wastewater and polluted water, can provide a method and a technical support for quantitative and standardized development of neurotoxicity monitoring in water quality safety evaluation, and has wide application prospect and popularization value.
Drawings
FIG. 1 is a schematic diagram of the method for rapid detection and evaluation of neurotoxicity of water samples with high throughput according to the present invention;
FIG. 2 is a dose-response curve of the reference compound methomyl of the present invention;
FIG. 3 shows the inhibition ratio of acetylcholinesterase at different loading steps in the acetylcholinesterase reaction;
FIG. 4 is a water sample dose-effect curve and toxicity test result of the method of the present invention.
Wherein: (A) adding no standard water sample, dose-effect curve and toxicity detection result; (B) adding a standard water sample dose-effect curve and a toxicity detection result.
Detailed Description
The invention is further described with reference to specific examples.
The terms used in the present invention have meanings commonly understood by those skilled in the art unless otherwise specified. The invention is described in further detail below with reference to specific embodiments and with reference to the attached drawings. It should be noted that these examples are only intended to illustrate the present invention, and do not limit the scope of the present invention in any way.
The invention utilizes acetylcholinesterase from electric eel to comprehensively detect the neurotoxicity of a water sample and quantitatively evaluate the toxicity level, and the principle and the steps of the method are shown in figure 1.
Example 1
This example demonstrates the sensitivity of the method of the invention.
The methomyl solution was diluted with methanol in 3-fold (3 ×) gradients at concentrations of 0.01, 0.033, 0.1, 0.33, 1, 3.3, 10, 33, 100, 330, 1000mg/L, respectively. The pH 6.8 phosphate buffer was used to prepare 0.067U/mL electric eel AChE, 0.5g/L DTNB and 0.5g/L ATCH solutions.
Taking a 96-well plate, sequentially adding 10 mu L of methomyl solution with each dilution concentration into the plate, wherein each concentration is 3 in parallel, additionally arranging 3 wells, adding 10 mu L of methanol as a solvent contrast, placing the 96-well plate in a fume hood for 15min until the methanol is completely volatilized to a liquid substance without macroscopic view, sequentially adding 150 mu L of the prepared electric eel AChE solution and 50 mu L of the prepared DTNB solution into each well, standing at 37 ℃ for 15min, then adding 50 mu L of the prepared ATCH solution into each well, and standing in a dark place for 15min, and then measuring the absorbance at the 415nm wavelength by using an enzyme labeling instrument.
The final concentrations of methomyl in the reaction system are respectively as follows: 0.0004, 0.00132, 0.004, 0.0132, 0.04, 0.132, 0.4, 1.32, 4, 13.2, 40 mg/L. The inhibition rate of electric eel AChE under the methomyl exposure of each concentration is calculated according to the formula (1), and a dose-effect curve is drawn as shown in fig. 2. ResultsShows that the in-vitro test mode of the 96-well plate used in the method has good sensitivity to nerve toxicants, and the methomyl has good sensitivity to EC of electric eel AChE inhibition50It was 0.065 mg/L.
Formula (1)
Example 2
This example compares different loading steps.
Gradient methomyl solutions at concentrations of 0.0132mg/L, 0.04mg/L, 0.132mg/L, 0.4mg/L, 1.32mg/L, and 4mg/L, and AChE, DTNB, and ATCH solutions, respectively, were prepared as in example 1.
Two 96-well plates were used as plate 1 and plate 2, respectively. Sequentially adding 10 mu L of methomyl solution with each dilution concentration into the plate 1, wherein each concentration is 3 in parallel, additionally arranging 3 holes, adding 10 mu L of methanol as a solvent control, placing a 96-hole plate in a fume hood for 15min until the methanol is completely volatilized to a liquid substance without macroscopic view, then sequentially adding 50 mu L of prepared DTNB solution, 50 mu L of prepared ATCH solution and 150 mu L of prepared conger AChE solution into each hole, standing at room temperature in a dark place for 15min, and then measuring the absorbance at the 415nm wavelength by using an enzyme labeling instrument; the plate 2 loading and detection procedure was the same as in example 1.
The inhibition rate of electric eel AChE under the exposure of methomyl of each concentration is calculated according to the formula (1), and the result is shown in fig. 3, the AChE inhibition rate generated by methomyl of each concentration in the plate 2 is obviously higher than that of the plate 1, which indicates that the sample adding detection method has higher sensitivity.
Example 3
The method of the invention is used for detecting and evaluating the neurotoxicity of a certain municipal sewage sample.
Collecting 2L of a certain municipal sewage sample subjected to biochemical and advanced treatment, firstly adjusting the pH of the water sample to 3 by using hydrochloric acid, then filtering the water sample by using a glass fiber membrane with the aperture of 1.6 mu m, and filtering the filtered water sample by using 6cc
Subjecting to solid phase extraction with HLB column, eluting with 10mL methanol and 10mL acetic acidThe ethyl ester was dried by nitrogen blowing and redissolved in 0.8mL of methanol to give a sample with a concentration of 2500.
And (3) carrying out gradient dilution on the concentrated sample by 2-3 times by using methanol to obtain samples with concentration times of 2500, 1000, 500, 250 and 100 respectively. The methomyl gradient dilution solution was prepared as in example 1. The pH 6.8 phosphate buffer was used to prepare 0.067U/mL electric eel AChE, 0.5g/L DTNB and 0.5g/L ATCH solutions.
A96-well plate is taken, 10 mu L of methomyl solution and samples with each diluted concentration are respectively added into the plate, each concentration is 3 in parallel, and 3 wells are additionally provided and 10 mu L of methanol is added to serve as a solvent control. And (3) placing the 96-well plate in a fume hood for 15min until methanol is completely volatilized to a liquid substance without macroscopic view, then sequentially adding 150 mu L of the prepared electric eel AChE solution and 50 mu L of the prepared DTNB solution into each well, standing for 15min at 37 ℃, then adding 50 mu L of the prepared ATCH solution into each well, standing for 15min in a dark place, and then measuring the absorbance at the 415nm wavelength by using a microplate reader. The relative concentration coefficients of the sample in the reaction system are respectively as follows: 100. 40, 20, 10 and 4.
The inhibition rate of sample exposure to electric eel AChE at each concentration factor was calculated according to equation (1), and a dose-effect curve was plotted as shown in fig. 4 (a). The results show the EC of the samples5039.67, that is, the sample concentrated 39.67 times, which inhibited the electric eel AChE by 50%.
The toxicity equivalent TEQ of the sample was calculated according to the formula (2), and the result showed that the TEQ of the sample was 1.64X 10-3mg/L methomyl, toxicity grade III.
Formula (1)
Formula (2)
Example 4
The accuracy of the method for quantitatively detecting the neurotoxicity of the water sample is verified in a labeling mode.
The concentrated water of example 3 was takenAdding a certain amount of methomyl to make its concentration be 2.5mg/L, i.e. its concentration converted into original water sample is 1X 10-3mg/L, the reference value of the water sample neurotoxicity is 2.64 multiplied by 10-3mg/L methomyl. And (3) carrying out 2-3 multiplied gradient dilution on the concentrated sample by using methanol to obtain samples with concentration multiples of 2500, 1000, 500, 250 and 100 respectively.
The sample was subjected to sample application detection and data analysis by the procedure of example 2, and the dose-effect curve of the sample was obtained as shown in FIG. 4 (B). The results show the EC of the spiked samples5026.91 and TEQ of 2.42X 10-3mg/L methomyl. Compared with the reference value of the water sample neurotoxicity, the error of the water sample measured value is smaller.
Claims (10)
1. A method for rapidly detecting and evaluating the neurotoxicity of a water sample in a high-throughput manner is characterized by comprising the following steps:
(1) concentrating organic matters in the water sample, including the organic matters in the enriched water sample and the concentrated and enriched organic matters, and recording the concentration times;
(2) gradient dilution of the sample and the toxicity detection reference compound, which comprises the step of gradient dilution of the organic matter and the toxicity detection reference compound concentrated in the step (1) according to a proportion;
(3) performing acetylcholinesterase reaction, namely reacting the samples, the solvent contrast and the reference compound under each dilution gradient in the step (2) with the electric eel acetylcholinesterase, the 5,5' -dithiobis (2-nitrobenzoic acid) and the iodothioacetylcholine iodide respectively, and detecting the absorbance values of the samples, the solvent contrast and the reference compound at the wavelength of 415 nm; the solvent control is used as a blank control and has no inhibitory effect on acetylcholinesterase;
(4) calculating the toxicity equivalent of the water sample, respectively fitting the dose-effect curves of the reference compound and the sample according to the detection result in the step (3), and calculating the toxicity equivalent of the water sample;
(5) and (4) evaluating the toxicity grade of the water sample according to the toxicity equivalent of the water sample calculated in the step (4).
2. The method for high-throughput rapid detection and assessment of neurotoxicity of water sample according to claim 1, wherein the toxicity equivalent of water sample is calculated by:
(i) calculating the inhibition rate EC of the sample and the reference compound on the electric eel acetylcholinesterase under each concentration according to the formula (1);
(ii) dose-response curves were drawn for the sample and reference compounds, respectively, to give EC 50;
(iii) and (4) calculating the toxicity equivalent TEQ of the sample water sample according to the formula (2).
Formula (1)
Formula (2)
3. The method for the high-throughput rapid detection and evaluation of neurotoxicity of water samples according to claim 1 or 2, wherein the reference compound is methomyl, and the toxicity grade evaluation system is as follows:
(i) TEQ is more than 0.2mg/L, and the toxicity grade is grade I;
(ii) TEQ is more than or equal to 0.2mg/L and less than 0.02mg/L, and the toxicity grade is II grade;
(iii) TEQ is less than or equal to 0.02mg/L, and the toxicity grade is grade III.
4. The method for rapid high-throughput detection and assessment of neurotoxicity of aqueous sample according to claim 3, wherein the acetylcholinesterase reaction loading step in step (3) comprises: respectively adding the sample, the solvent control and the reference compound under each dilution gradient, and standing until the solvent is completely volatilized; sequentially adding electric eel acetylcholinesterase and 5,5' -dithiobis (2-nitrobenzoic acid), and standing at 37 ℃; finally adding iodized thioacetylcholine; standing for 10-15 min in dark at room temperature, and detecting the absorbance.
5. The method for high-throughput rapid detection and evaluation of neurotoxicity of water sample according to claim 4, wherein the acetylcholinesterase reaction substances added in step (3) are: the amount of the electric eel acetylcholinesterase is 0.005-0.015U relative to 10 mu L of a sample to be detected; the amount of the 5,5' -dithiobis (2-nitrobenzoic acid) is 0.02-0.03 mg; the amount of thioacetyl choline iodide is 0.02-0.03 mg.
6. The method for high-throughput rapid detection and evaluation of neurotoxicity of water sample according to claim 4 or 5, wherein the step (2) of gradient dilution of the concentrated organic substance comprises diluting the concentrated organic substance to 4-6 concentrations by a 2-3-fold ratio.
7. The method for high-throughput rapid detection and assessment of neurotoxicity of water sample according to claim 6, wherein the step (2) of gradient dilution of the reference compound comprises 2-3 times of dilution of the reference compound by 10-12 concentrations, wherein the dilution is in the range of 1000-0.001 mg/L.
8. The method for rapid detection and evaluation of high throughput neurotoxicity of water sample according to claim 7, wherein the solvent control in step (3) is methanol; and/or the sample in step (1) is dissolved in methanol after concentration.
9. The method for rapid high-throughput detection and assessment of neurotoxicity of water samples according to claim 8, wherein the enrichment in step (1) is performed by solid-phase extraction or liquid-liquid extraction.
10. The method for high-throughput rapid detection and evaluation of neurotoxicity of water sample according to claim 9, wherein the concentration in step (1) is 1000-5000 times, and/or nitrogen-blowing is used for concentration.
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