Method for detecting acute toxicity of water body by surface enhanced raman scattering technique

Abstract

The application relates to the technical field of water body detection, in particular to a method for detecting acute toxicity of a water body by using surface-enhanced Raman scattering technology, which comprises the following steps: configuring an indicator bacterial suspension; providing a water body sample to be detected, carrying out first mixed culture of the indicator bacterial suspension and the water body sample to be detected, and separating and collecting a precipitate to obtain a first mixture; providing a probe solution, carrying out second mixed culture of the probe solution and the first mixture, and separating and collecting supernatant to obtain a mixture to be detected; providing a surface-enhanced Raman scattering substrate, mixing the mixture to be detected and the surface-enhanced Raman scattering substrate, using a Raman instrument to detect the characteristic peak intensity of a probe reduction product in the mixture to be detected, and calculating through an inhibition rate formula to analyze the toxicity of the water body. The method for evaluating the acute toxicity of the water body by combining SERS and a biosensor has good sensitivity, can rapidly detect and evaluate the acute toxicity problem of the water body in a short time.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of water detection, and particularly relates to a method for detecting acute toxicity of water by surface-enhanced Raman scattering technology. BACKGROUND

[0002] Due to the increasingly serious water pollution, water safety has become a focus of widespread concern. Heavy metals, pesticides and antibiotics are common pollution sources in water, which pose a serious threat to the environment and even human beings. For example, excessive copper intake can cause damage to the cell membrane and symptoms such as abdominal pain and nausea. In order to protect the safety of human drinking water and protect the aquatic ecosystem, a series of water detection standards have been proposed, such as chemical oxygen demand, biochemical oxygen demand, total phosphorus and total nitrogen, etc. Among these standards, water acute toxicity is a special standard, which links the composition of water with natural organisms, and reflects the overall impact of water quality on organisms, rather than just providing the physical and chemical properties of water. Therefore, although traditional methods (such as high-performance liquid chromatography and inductively coupled plasma emission spectrometer) can accurately determine some specific pollutants, they cannot reflect the biological toxicity of toxic substances. Therefore, many unconventional methods combining biological response and chemical detection have been proposed to evaluate the toxicity of chemical substances.

[0003] Different trophic level organisms (such as algae, water fleas, fish, microorganisms, etc.) have been widely developed for the detection of water acute toxicity. However, the biological sensor method also has some unavoidable shortcomings. Among them, higher level organisms (algae, fish, etc.) have higher sensitivity to toxic substances, but due to the long cultivation time and complicated procedures, they are not suitable for water toxicity assessment in emergency situations. Compared with these relatively high-level organisms, ubiquitous and low-cost microorganisms are easy to cultivate and can respond quickly to toxic substances. Therefore, microorganism-based methods, such as bioluminescence inhibition method, biosensor combined with electrochemical method, colorimetric method, etc., have also been developed for the evaluation of water acute toxicity. However, the bioluminescent bacteria used in the bioluminescence method are almost all marine microorganisms, which must be maintained in a saline environment to maintain their activity. Therefore, when they are applied to the evaluation of acute toxicity of freshwater, there will inevitably be deviations. In addition, the bioluminescence method is directly added to the water body, which is greatly affected by the turbidity of the water body. Therefore, it is very important to establish a method with high sensitivity, rapid detection and comprehensive evaluation of water acute toxicity for environmental protection and human health. SUMMARY

[0004] The purpose of the present application is to provide a method for detecting acute toxicity of water by surface-enhanced Raman scattering technology, which aims to solve the problem of lack of methods with high sensitivity, rapid detection and comprehensive evaluation of water acute toxicity in the prior art.

[0005] To achieve the above application purposes, the technical scheme adopted by the present application is as follows:

[0006] In a first aspect, the present application provides a method for detecting acute toxicity of water body by surface-enhanced Raman scattering technology, comprising the following steps:

[0007] Providing an indicator bacterial suspension;

[0008] Providing a water body sample to be tested, mixing and culturing the indicator bacterial suspension with the water body sample to be tested for the first time, and separating and collecting the precipitate to obtain a first mixture;

[0009] Providing a probe solution, mixing and culturing the probe solution with the first mixture for the second time, and separating and collecting the supernatant to obtain a mixture to be tested;

[0010] Providing a surface-enhanced Raman scattering substrate, mixing the mixture to be tested with the surface-enhanced Raman scattering substrate, using a Raman instrument to detect the characteristic peak intensity of the probe reduction product in the mixture to be tested, and calculating through an inhibition rate formula to analyze the toxicity of the water body.

[0011] The method for detecting acute toxicity of water body by surface-enhanced Raman scattering technology provided in the first aspect of the present application is based on a bacterial-medium SERS method to comprehensively evaluate the acute toxicity of water body. The microorganism provided is ubiquitous, low in cost, easy to cultivate, and reacts quickly to toxic substances. In addition, the method of mediating microbial respiration through a medium avoids the influence of turbidity of the actual water body on the detection. The method of evaluating the acute toxicity of water body by combining SERS with a biosensor shows good sensitivity and can quickly detect and evaluate the acute toxicity of water body in a short time. The detection method is easy to operate and is conducive to widespread use. BRIEF DESCRIPTION OF DRAWINGS

[0012] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0013] Figure 1 is a flowchart of the method for detecting acute toxicity of water body by surface-enhanced Raman scattering technology provided in the embodiments of the present application.

[0014] Figure 2 is a schematic diagram of analysis of factors affecting bacterial toxicity inhibition rate provided in the embodiments of the present application.

[0015] Figure 3 is a schematic diagram of linear curve analysis of the probe reduction product hydroquinone provided in the embodiments of the present application.

[0016] Figure 4 FIG. 5 is a schematic diagram of spectral analysis of a reaction of a water sample to be tested inhibiting bacteria from reducing BQ to produce HQ, according to an embodiment of the present application.

[0017] Figure 5 FIG. 6 is a curve analysis diagram of inhibition rate and concentration, according to an embodiment of the present application. DETAILED DESCRIPTION

[0018] In order to make the technical problems to be solved by the present application, technical solutions and beneficial effects clearer, the present application will be further described in detail below with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0019] In the present application, the term "and / or" describes the association relationship of the associated objects, which means that there can be three kinds of relationships, for example, A and / or B can mean that A exists alone, A and B exist together, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after it.

[0020] In the present application, "at least one" means one or more, and "multiple" means two or more. "At least one of the following" or similar expressions means any combination of these items, including any combination of single item or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can mean a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, wherein a, b, and c can be single or multiple.

[0021] It should be understood that in various embodiments of the present application, the size of the sequence number of the above-mentioned processes does not mean the order of execution, and part or all of the steps can be executed in parallel or in sequence. The execution order of each process should be determined according to its function and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0022] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a" and "the" used in the embodiments of the present application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0023] The weight of the related components mentioned in the embodiment specification of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of each component. Therefore, as long as the content of the related components in the embodiment specification of the present application is scaled up or down in proportion, it is within the scope disclosed in the embodiment specification of the present application. Specifically, the mass in the embodiment specification of the present application can be μg, mg, g, kg, etc. mass units commonly known in the chemical industry.

[0024] The terms "first", "second" are only for descriptive purposes, to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. For example, without departing from the scope of the present application, the first XX can also be referred to as the second XX, and similarly, the second XX can also be referred to as the first XX. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features.

[0025] The first aspect of the embodiment of the present application provides a method for detecting acute toxicity of water body by surface enhanced Raman scattering technology, as shown in the formula: Figure 1 The method comprises the following steps:

[0026] S01. Configure an indicator bacterial suspension;

[0027] S02. Provide a water body sample to be tested, mix the indicator bacterial suspension with the water body sample to be tested for a first time, and separate and collect the precipitate to obtain a first mixture;

[0028] S03. Provide a probe solution, mix the probe solution with the first mixture for a second time, and separate and collect the supernatant to obtain a test mixture;

[0029] S04. Provide a surface enhanced Raman scattering substrate, mix the test mixture and the surface enhanced Raman scattering substrate, use a Raman instrument to detect the characteristic peak intensity of the probe reduction product in the test mixture, and calculate through an inhibition rate formula to analyze the toxicity of the water body.

[0030] The method for detecting acute toxicity of water body by surface enhanced Raman scattering technology provided in the first aspect of the embodiment of the present application is based on a bacterial-medium SERS method to comprehensively evaluate the acute toxicity of the water body. The microorganism provided is ubiquitous and low in cost, easy to cultivate, and reacts quickly to toxic substances. In addition, the method of mediating microbial respiration through a medium avoids the influence of the turbidity of the actual water body on the detection. The method of evaluating the acute toxicity of the water body by combining SERS with a biosensor shows good sensitivity and can quickly detect and evaluate the acute toxicity of the water body in a short time. The detection method is easy to operate and is conducive to widespread use.

[0031] In step S01, the indicator bacteria suspension is prepared. The microorganism is provided as a detection indicator, and the main purpose is that the basic principle of toxicity detection is based on the entry of external toxic substances into the microorganism growth environment and the inhibition of the respiration of the microorganism. In this process, the toxic substances in the water body can intervene in the respiration of the microorganism, accept electrons to form a reducing mediator, and in the presence of toxic chemicals, the respiration and metabolism of the microorganism will be inhibited to different degrees due to the toxicity of the toxic substances, and the amount of the reducing mediator will also be reduced accordingly, and this change can be sensitively monitored by SERS technology. The spectral information can be used to determine the acute toxicity of the chemical substance. When SERS is used to detect the reducing mediator in the reaction mixture, it can be expected that the intensity of the characteristic peak corresponding to the reducing mediator in the spectrum will gradually decrease with the increase of the concentration of the toxic chemical substance, and by predicting the concentration of the metabolic product of the toxic chemical substance, the content of the toxic substance in the water body can be further analyzed and determined.

[0032] In some embodiments, the indicator bacteria include at least one of Escherichia coli, Actinomyces, Pseudomonas fluorescens, and Pseudomonas aeruginosa. The selected indicator bacteria are conventional and readily available microorganisms, and are inexpensive, easy to cultivate, and can be used for rapid experiments. In specific embodiments, the indicator bacteria are selected from Escherichia coli.

[0033] Since the optimal growth conditions of different indicator bacteria are different, during the cultivation of the indicator bacteria, the bacteria can be cultured to the logarithmic growth phase level according to the optimal culture temperature and the optimal culture time of different indicator bacteria.

[0034] In some embodiments, in the step of preparing the indicator bacteria suspension, the indicator bacteria are cultured at the optimal growth temperature of the indicator bacteria for 22-24 hours, a sodium chloride solution is provided for washing and resuspension treatment, and the indicator bacteria suspension is prepared.

[0035] In some embodiments, the sodium chloride solution is provided for washing, and the purpose is to remove the culture medium and bacterial metabolites in the collected bacterial bodies to ensure that there is no interference with the subsequent spectrum. In some specific embodiments, the concentration of the sodium chloride solution is 0.5%-1.0%.

[0036] In some embodiments, the OD 600 of the indicator bacteria suspension is 3.0-4.0. Controlling the density of the bacterial bodies in the bacterial suspension can on the one hand ensure that the bacterial concentration is moderate and can better reflect the metabolism of toxic substances, and on the other hand can make most of the bacteria in the logarithmic growth phase, have strong metabolic ability, and can effectively decompose the toxic substances in the water body, which is conducive to the detection of toxic products by SERS method, and thus is conducive to the judgment of the toxicity of the water body.

[0037] In some embodiments, when the indicator bacteria is selected from Escherichia coli, the process of preparing the Escherichia coli suspension includes the following steps:

[0038] The purchased Escherichia coli freeze-dried powder is inoculated in 150 mL of high-temperature sterilized nutrient solution and incubated at 37°C for 24 hours, then centrifuged at 5000 r / min at room temperature for 5 min, and the supernatant is removed to obtain Escherichia coli cells.

[0039] The prepared 0.5% sodium chloride solution is used for washing twice to remove the influence of the culture medium or bacterial metabolites on the spectrum.

[0040] The washed bacteria are resuspended in the sodium chloride solution, and the concentration of the bacterial suspension is determined at 600 nm in a UV spectrophotometer to determine the OD 600 of the bacterial suspension is 3.6.

[0041] In specific embodiments, the provided Escherichia coli is selected from Escherichia coli CICC 23689, China Industrial Microbial Culture Collection Center.

[0042] The culture method of the Escherichia coli suspension is as follows: a loop is taken from the 3-10th generation of the preserved strain on the test tube slope, streaked on a plate, and cultured in an incubator for 24 hours. Several test tubes are taken, 5-7 mL (about 1 / 3 of the height of the test tube) of LB solid culture medium is taken in the test tube, the test tube is sealed with cotton paper, and the test tube is sterilized in a high-pressure sterilization pot at 121°C for 21 min. The sterilized test tube slope is placed on a sterile operation table at an angle, the solid culture medium in the test tube is solidified into a slope, and the strain colony required for the experiment is picked from the plate with a loop or a bamboo stick and inoculated in the test tube slope. Then the slope is placed in a constant temperature incubator at 37°C ± 1°C for culture.

[0043] The method for preparing the Escherichia coli suspension is as follows: an appropriate amount of sterilized LB liquid medium is taken in a conical flask, a small amount of activated slope strain is adhered with a loop, the loop is immersed in the LB medium and shaken several times, the above operation is repeated 2-3 times, and then the conical flask is tightly plugged with sterile cotton. The inoculated bacteria are placed in a constant temperature incubator for culture. Then the cultured bacterial liquid is centrifuged (4000 rpm, 5 min) to remove the supernatant, and the bacterial suspension is dispersed in 0.9% NaCl solution for later use.

[0044] In step S02, the sample to be tested is provided, the indicator bacteria suspension is mixed with the sample to be tested for the first time, and the precipitate is separated and collected to obtain the first mixture.

[0045] In some embodiments, the indicating bacteria suspension is mixed with the water sample to be tested in a first mixed culture, and the volume ratio of the indicating bacteria suspension to the water sample to be tested is (7-9): 1. In some specific embodiments, the volume ratio of the indicating bacteria suspension to the water sample to be tested is 9: 1.

[0046] In some embodiments, in the step of mixing the indicating bacteria suspension with the water sample to be tested in a first mixed culture, the temperature of the first mixed culture is the optimal growth temperature of the bacteria in the indicating bacteria suspension, and the culture time is 50-60 minutes. In some specific embodiments, when the indicating bacteria are selected from Escherichia coli, the first mixed culture of the Escherichia coli suspension and the water sample to be tested is performed at 37°C for 50-60 minutes.

[0047] In some embodiments, the separation and collection of the precipitate includes but is not limited to separation and treatment of the precipitate and supernatant by centrifugation.

[0048] In some embodiments, after the separation and collection of the precipitate, the method further comprises: washing the precipitate with a sodium chloride solution, which can help reduce the interference of heavy metals on the spectrum.

[0049] In some specific embodiments, in the step of separating and collecting the precipitate to obtain the first mixture, the method comprises: after the first mixed culture of the indicating bacteria suspension and the water sample to be tested, high-speed centrifugation is performed at 8000-12000 r / min for 5-10 minutes to remove the supernatant and collect the precipitate; and then the precipitate is washed with a sodium chloride solution to obtain the first mixture.

[0050] In step S03, a probe solution is provided, the probe solution is mixed with the first mixture in a second mixed culture, and the supernatant is separated and collected to obtain a test mixture. The provided probe solution is an indicator for the respiration of microorganisms, which can facilitate SERS analysis of whether the microorganisms are affected by toxic substances in the water body.

[0051] In some embodiments, the probe solution is selected from a p-benzoquinone solution. After the microorganisms react with the benzoquinone solution, metabolic reduction products hydroquinone are produced, so it is beneficial to use SERS analysis to determine the concentration of the reduction product hydroquinone.

[0052] In some embodiments, the volume ratio of the probe solution to the first mixture is 1:(10-12). Controlling the volume ratio of the probe solution and the first mixture can facilitate the reduction of the microorganisms in the first mixture to the probe solution.

[0053] In some embodiments, the second mixed culture of the probe solution and the first mixture is performed under conditions in which the second mixed culture is at an optimal temperature for growth of the indicator bacteria in the bacterial suspension, and the culture time is 40-60 minutes. In some specific embodiments, when the indicator bacteria are selected from E. coli, the second mixed culture of the probe solution and the first mixture is performed under conditions in which the second mixed culture is at 37°C for 40-60 minutes.

[0054] In some embodiments, the separation and collection of the supernatant includes, but is not limited to, separation and treatment of the precipitate and the supernatant by centrifugation.

[0055] In some specific embodiments, the step of separating and collecting the supernatant to obtain the test mixture includes: after the second mixed culture of the probe solution and the first mixture, performing high-speed centrifugation at 10,000-12,000 r / min for 5-10 minutes, collecting the supernatant, and obtaining the test mixture.

[0056] In step S04, a surface-enhanced Raman scattering substrate is provided, the test mixture and the surface-enhanced Raman scattering substrate are mixed, the characteristic peak intensity of the probe reduction product in the test mixture is detected using a Raman instrument, and the water toxicity is analyzed by calculation through an inhibition rate formula.

[0057] In some embodiments, the provided surface-enhanced Raman scattering substrate includes, but is not limited to, a silver nanoparticle substrate, a silver nanoparticle and halide mixture substrate, etc.

[0058] In some embodiments, in the step of detecting the characteristic peak intensity of the probe reduction product in the test mixture using a Raman instrument, the Raman detection conditions are: using a 633 nm laser, performing Raman detection under the conditions of a laser power of 0.5 mw, an integration time of 5 s*4 times, a grating of 1200 line, a center line of 1200, and an objective lens of 50x lens. The characteristic peak intensity of the probe reduction product, hydroquinone, can be analyzed.

[0059] In some embodiments, the provided detection method further includes: configuring standard solutions of the probe reduction product, hydroquinone, at different concentrations, detecting using the provided Raman detection conditions, and drawing a standard curve of the probe reduction product, hydroquinone.

[0060] In some specific embodiments, the preparation method of the standard solutions of the probe reduction product, hydroquinone, at different concentrations includes: obtaining a 1 mM silver nitrate solution by reducing silver nitrate with sodium citrate; configuring a 1 mM KBr solution to modify Ag NPs; configuring standard solutions of the probe reduction product, hydroquinone, at different concentrations; wherein the different concentrations include: 1000 μM, 500 μM, 100 μM, 50 μM, 10 μM, 5 μM, 1 μM, and 0 μM; and measuring the SERS spectra of HQ at different concentrations, respectively, at 1260 cm-1.-1 Linear fitting of the frequency band, the linear equation is I = 9.52973C1 + 494.24753(R 2 = 0.97963), the detection limit can be as low as 1 μM.

[0061] In some embodiments, the characteristic peak intensity of the probe determined by the blank control group is determined by replacing the water sample with a blank aqueous solution.

[0062] In some embodiments, the characteristic peak intensity of the probe determined by the blank control group is determined by replacing the water sample with a blank aqueous solution.

[0063] In some embodiments, the characteristic peak intensity of the probe determined by the blank control group is determined by replacing the water sample with a blank aqueous solution.

[0064] The following will be described in conjunction with specific embodiments.

[0065] Example 1

[0066] Exploring factors affecting bacterial toxicity inhibition rate

[0067] Test process:

[0068] Four different factors, namely: A (probe BQ concentration: 1, 2, 3, 4 represent 0.1, 0.3, 0.6, 1 mM), B (bacterial concentration: 1, 2, 3 represent OD 600 = 2.4, 3.6, 4.8), C (sodium chloride concentration: 1, 2, 3, 4 represent 0.1%, 0.3%, 0.5%, 0.7%), D (reaction time: 1, 2, 3, 4 represent 10, 30, 50, 70 min) were subjected to orthogonal experiment, 16 experimental schemes were generated from the orthogonal array (as shown in Table 1), to realize the comprehensive analysis of the inhibition rate. The specific reaction process is as follows: according to the generated orthogonal table (Table 1), 16 groups of experiments were carried out for multi-factor and multi-level optimization experiment; when carrying out the experiment, according to the corresponding optimization data in the table, the incubated bacteria were washed with optimized concentration of sodium chloride solution to remove the culture medium, and were resuspended in the corresponding concentration of sodium chloride solution; the optimized concentration of p-benzoquinone solution was added to the optimized concentration of bacterial solution, and the optimized time was incubated, and the final supernatant was collected for SERS test

[0069] Table 1

[0070]

[0071] Result analysis:

[0072] As shown in Table 1, the relationship between the influence of various factors on the toxicity inhibition rate is: B>A>D>C; the best combination selected is: BQ concentration of 0.6mM, bacterial concentration of OD 600 =3.6, reaction time 50 min and sodium chloride concentration 0.5%.

[0073] First, regarding the effect of factor A (probe BQ concentration) on the toxicity inhibition rate, its k value (the quality of the specific level of the factor) first increases (0.1-0.6mM) and then decreases (0.6-1.0mM) as the BQ concentration increases. Because BQ itself is somewhat toxic, when a certain concentration of BQ is added to bacteria, the accumulated toxic effect reaches a certain tolerance value of the bacteria, which will have a certain impact on the bacterial respiration. According to the results, a BQ concentration of 0.6mM is the most preferred.

[0074] Second, with respect to factor B (bacterial concentration), when the bacterial concentration gradually increases, the k value increases accordingly; when the bacterial concentration is too high, there will be species competition within the bacterial solution itself, the nutrients will be consumed too quickly, and metabolites will accumulate, which will change the activity of the bacteria themselves.

[0075] Third, regarding factor C (sodium chloride concentration), the results indicate that as the concentration of NaCl solution increases within the 0.1-0.7% range, its level k value reaches a maximum of 0.44 and then begins to decrease. Therefore, a 0.5% NaCl solution was selected as the final concentration for the toxicity test. This is because E. coli, a freshwater bacterium, is susceptible to changes in cellular osmotic pressure due to high or low NaCl concentrations, which can lead to decreased bacterial activity or even death.

[0076] Fourth, for factor D (reaction time), the entire experimental reaction reached stability within 50 minutes, and the k value of the influencing factor level of time varied in a very small range (0.38-0.43).

[0077] Finally, the best combination of influencing factor levels is obtained (such as Figure 2 As shown), the BQ concentration is 0.6mM, the E. coli concentration OD 600 =3.6, incubation time was 50 min, and sodium chloride concentration was 0.5%.

[0078] Example 2

[0079] Toxicity testing and analysis

[0080] In order to determine the applicability of the surface-enhanced Raman scattering method for detecting acute toxicity in water and analyze its sensitivity, this example provides a method for detecting acute toxicity in water containing different concentrations of the simulated poison Cu. 2+The water solution of the probe reduction product hydroquinone is configured as a sample (0 mg / L, 0.2 mg / L, 0.4 mg / L, 0.8 mg / L, 1.6 mg / L, 3.2 mg / L, 6.3 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L) for analysis of the method for detecting acute toxicity of water bodies by surface-enhanced Raman scattering technology.

[0081] Test process:

[0082] The specific reaction steps of the method for detecting acute toxicity of water bodies by surface-enhanced Raman scattering technology are as follows:

[0083] (1) The preparation method of the standard solution of the probe reduction product hydroquinone with different concentrations includes: obtaining a 1 mM silver nitrate solution by the method of reducing silver nitrate with sodium citrate; configuring a 1 mM KBr solution for modifying Ag NPs; configuring a standard solution of the probe reduction product hydroquinone with different concentrations; wherein the different concentrations include: 1000 μM, 500 μM, 100 μM, 50 μM, 10 μM, 5 μM, 1 μM, and 0 μM; and the SERS spectrum of HQ with different concentrations is measured respectively at 1260 cm -1 The linear fitting of the frequency band is performed to obtain a linear equation;

[0084] (2) Configure an indicator bacteria suspension; wherein, Escherichia coli is provided as the indicator bacteria, the Escherichia coli is cultured in 150 mL of high-temperature sterilized nutrient solution at 37°C for 24 h, and then centrifuged at 5000 r / min for 5 min at room temperature to remove the supernatant to obtain Escherichia coli cells; the bacteria are washed with a prepared sodium chloride solution with a concentration of 0.5%; the washed bacteria are resuspended in the sodium chloride solution, and the concentration of the bacteria suspension is measured at 600 nm in a UV spectrophotometer to determine that the OD600 of the Escherichia coli is 3.6;

[0085] (3) Provide a water sample to be tested (i.e., a water solution containing different concentrations of simulated toxicant Cu 2+ , wherein the concentrations include 0 mg / L, 0.2 mg / L, 0.4 mg / L, 0.8 mg / L, 1.6 mg / L, 3.2 mg / L, 6.3 mg / L, 12.5 mg / L, 25 mg / L, and 50 mg / L), mix 0.9 mL of the indicator bacteria suspension with 0.1 mL of the water sample to be tested for first mixed culture at 37°C for 50 min, centrifuge the mixture at high speed, and remove the supernatant. Wash the bacteria with a sodium chloride solution twice to obtain a first mixture;

[0086] (4) providing a probe solution of 1 ml of a BQ aqueous solution, wherein the concentration of BQ in the BQ aqueous solution is 0.6 mM; after mixing the probe solution with the first mixture, the second mixture is cultured at 37°C for 40 minutes, after the culture is completed, the mixture is centrifuged at a speed of 10,000 r / min for 10 minutes to obtain supernatant, and the obtained supernatant is the mixture to be detected;

[0087] (5) providing a surface-enhanced Raman scattering substrate, mixing the mixture to be detected and the surface-enhanced Raman scattering substrate, and detecting the characteristic peak intensity of the probe reduction product in the mixture to be detected by using a Raman instrument, wherein the Raman detection conditions are as follows: a 633 nm laser, a laser power of 0.5 mw, an integral time of 5 s*4 times, a grating of 1200 line, a center line of 1200, and an objective lens of 50* lens; and the inhibition rate is calculated by using an inhibition rate formula: inhibition rate % = [(the characteristic peak intensity of the probe detected in the blank control group - the characteristic peak intensity of the probe reduction product detected in the sample group to be detected) / the characteristic peak intensity of the probe detected in the blank control group] * 100%; and the toxicity of the water body is analyzed.

[0088] Result analysis:

[0089] 1. The linear equation of the probe reduction product hydroquinone is as shown in the following formula (I): Figure 3 The linear equation is I = 9.52973C1 + 494.24753 (R2 = 0.97963), and the detection limit can be as low as 1 μM.

[0090] 2. The spectrum of the water sample to be detected (i.e. an aqueous solution containing different concentrations of simulated toxicant Cu2+, wherein the concentrations include 0 mg / L, 0.2 mg / L, 0.4 mg / L, 0.8 mg / L, 1.6 mg / L, 3.2 mg / L, 6.3 mg / L, 12.5 mg / L, 25 mg / L, and 50 mg / L) inhibiting the reaction of bacterial reduction of BQ to produce HQ is as shown in the following formula (II): Figure 4 It can be seen that the intensity of the characteristic peak is negatively correlated with the concentration of Cu 2+ , which indicates that the greater the toxicity of the water body, the greater the impact on the activity or metabolic process of the bacteria.

[0091] The quantity of the reduction medium is quantified according to the Raman intensity within a stable time of 50 minutes; and the inhibition rate of the toxicant is calculated according to the inhibition rate formula, as shown in the following formula (III): Figure 5 The curve of the inhibition rate and the concentration is obtained, and the value of the half maximal inhibitory concentration (IC50) reflects the toxicity level of Cu 2+ (0.94 mg / L). The sensitivity of the mediated toxicity bioassay is related to the properties of the medium, the microorganism, and the toxicant. In general, a lower IC50 value corresponds to a higher sensitivity of the toxicity detection, and the sensitivity of the toxicity detection of Cu 2+The value of the sensitivity of the method is 1-10 times or more than that of other methods. This is because in the present application, the bacteria are dispersed in a liquid with greater freedom of flow and greater effective specific surface area, which improves the mass transfer efficiency of the bacteria and the medium, thereby showing higher sensitivity.

[0092] In summary, the method for detecting acute toxicity of water provided by the present application is based on a bacterial-medium SERS method to comprehensively evaluate the acute toxicity of water. The microorganism provided is ubiquitous, low-cost, easy to cultivate, and reacts quickly to toxic substances. In addition, the method of mediating microbial respiration through a medium avoids the influence of the turbidity of the actual water on the detection. The method of evaluating the acute toxicity of water by combining SERS with a biosensor shows good sensitivity and can quickly detect and evaluate the acute toxicity of water in a short time. The detection method is easy to operate and is conducive to widespread use.

[0093] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting acute toxicity of a water body using surface enhanced Raman scattering technique, characterized in that, The method comprises the following steps: Prepare indicator bacteria suspension; the indicator bacteria is Escherichia coli CICC 23689, and the OD of the suspension is 600 3.0~4.0; A water sample to be tested is provided, the indicator bacteria suspension and the water sample to be tested are mixed at a volume ratio of (7-9):1 for first mixed culture, and are cultured at 37°C for 50-60 minutes, and then are separated and collected, are washed with 0.5%-1.0% sodium chloride solution, and a first mixture is obtained; A 0.6mM p-benzoquinone solution is provided as a probe solution, the probe solution and the first mixture are mixed at a volume ratio of 1:(10-12) for second mixed culture, and are cultured at 37°C for 40-60 minutes, and then are separated and collected, and a supernatant is obtained as a mixture to be tested; The surface enhanced Raman scattering substrate is provided as a composite substrate modified by silver nanoparticles and potassium bromide, the mixture to be detected and the surface enhanced Raman scattering substrate are mixed, and the characteristic peak intensity of hydroquinone at 1260 cm -1 is detected under the conditions of a 633 nm laser, a laser power of 0.5 mW, an integral time of 5 s x 4 times, a grating of 1200 line, and an objective lens of 50 x, and the inhibition rate is calculated by an inhibition rate formula, wherein the inhibition rate formula is: inhibition rate % = [(the characteristic peak intensity of the probe detected in the blank control group - the characteristic peak intensity of the probe reduction product detected in the sample group to be detected) / the characteristic peak intensity of the probe detected in the blank control group] x 100%. The water toxicity is analyzed, the characteristic peak intensity of the reduction product is reduced, the obtained inhibition rate is increased, and the water toxicity is relatively strong.

2. The method of detecting acute toxicity of a water body using surface enhanced Raman scattering technique according to claim 1, wherein, In the step of configuring the indicator bacteria suspension, the indicator bacteria are cultured at an optimal growth temperature of the indicator bacteria for 22-24 hours, a sodium chloride solution is provided for washing and resuspension treatment, and the indicator bacteria suspension is configured.

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