New antibiotic pollutant detection method combining solid-phase extraction and enzyme-linked immunosorbent assay

Abstract

The invention provides a method for detecting new antibiotic pollutants in a water body sample by combining solid-phase extraction and enzyme-linked immunosorbent assay, which comprises the following steps: (a) selecting an organic solvent, so that R2 of a standard curve obtained by enzyme-linked immunosorbent assay of a standard substance containing the new antibiotic pollutants and an aqueous solution of the organic solvent is greater than 0.98; (b) determining the optimal concentration of the organic solvent by taking the quantitative detection interval, the sensitivity, the detection limit and the precision of enzyme-linked immunosorbent assay as evaluation indexes and combining a CRITIC weight method; and (c) preparing the new antibiotic pollutants enriched by the organic solvent selected in the step (a) through solid-phase extraction of the water body sample and water into a redissolved aqueous solution, and detecting the redissolved aqueous solution through enzyme-linked immunosorbent assay to determine the concentration of the new antibiotic pollutants in the water body sample, wherein the concentration of the organic solvent in the redissolved aqueous solution is the optimal concentration. The invention also provides a method for optimizing enzyme-linked immunosorbent assay.

Description

Technical Field

[0001] This application relates to the field of detection of new antibiotic pollutants, specifically to a method for detecting new antibiotic pollutants in water samples by combining solid-phase extraction and enzyme-linked immunosorbent assay (ELISA). This application also relates to optimizing the ELISA detection method. Background Technology

[0002] New pollutants in the environment often exhibit characteristics such as biotoxicity, environmental persistence, and bioaccumulation. Their hazards are potential and insidious; even at low concentrations, they can pose risks to the ecological environment and human health. New pollutants are diverse and rapidly increasing. Antibiotics, as an important class of new pollutants, are widely distributed in aquatic environments, exerting a sustained impact on the environment. They can also be transferred and bioaccumulated through the food chain, and even generate resistance genes in organisms, causing biological mutations.

[0003] Current detection and analysis methods typically require the collection of large quantities of environmental samples, followed by pretreatment processes such as enrichment, concentration, and interference removal, before qualitative and quantitative analysis using chromatography / mass spectrometry. However, this analytical method is not only time-consuming but also involves expensive equipment, resulting in high analytical costs. It is insufficient for understanding antibiotic concentration levels in large-scale aquatic environments, and even more so for meeting the needs of effective remediation.

[0004] It is also known that the combination of solid phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) can be used to determine novel contaminants in wastewater samples, for example, see CHUN Pu et al, Trace analysis of contraceptive drug levonorgestrel in wastewater samples by a newly developed indirect competitive enzyme-linked immunosorbent assay (ELISA) coupled with solid phase extraction, Analytica Chimica acta 628 (2008) 73-79, which shows that this combined method can provide results similar to those of HPLC detection.

[0005] Currently, there is still a need to improve the detection methods for new antibiotic pollutants, thereby improving the ease of detection, sensitivity, accuracy, and specificity. Summary of the Invention

[0006] The purpose of this application is to improve or optimize the detection method for novel antibiotic contaminants that combines solid-phase extraction and enzyme-linked immunosorbent assay (ELISA), thereby improving the sensitivity, accuracy, and specificity of the ELISA detection process.

[0007] According to a first aspect of this application, a method for detecting novel antibiotic pollutants in water samples by combining solid-phase extraction and enzyme-linked immunosorbent assay (ELISA) is provided, comprising:

[0008] (a) Select an organic solvent such that the R value of the standard curve obtained by enzyme-linked immunosorbent assay (ELISA) containing the standard of the novel antibiotic contaminant and an aqueous solution of the organic solvent is equal to that obtained by ELISA. 2 Greater than 0.98;

[0009] (b) Using the quantitative detection range, sensitivity, limit of detection, and precision of enzyme-linked immunosorbent assay (ELISA) as evaluation indicators, and combining this with the CRITIC weighting method, determine the optimal concentration of the organic solvent selected in step (a); and

[0010] (c) Prepare a reconstituted aqueous solution by mixing the organic solvent selected in step (a), the antibiotic-like new pollutant enriched by solid-phase extraction of the water sample, and detect the reconstituted aqueous solution by enzyme-linked immunosorbent assay (ELISA) to determine the concentration of the antibiotic-like new pollutant in the water sample, wherein the concentration of the organic solvent in the reconstituted aqueous solution is the optimal concentration.

[0011] In some implementations, step (b) includes:

[0012] (i) Prepare standard aqueous solutions with a dual concentration gradient containing a standard of a novel antibiotic contaminant and an organic solvent selected in step (a), wherein a set of standard aqueous solutions containing the standard of the gradient concentration is prepared for each concentration of the organic solvent.

[0013] (ii) Perform enzyme-linked immunosorbent assay (ELISA) on each group of standard aqueous solutions to obtain standard curves corresponding to each concentration of the organic solvent, and calculate the quantitative detection range, sensitivity, limit of detection, and precision values ​​corresponding to each standard curve; and

[0014] (iii) Using the normalized dimensionless values ​​of the quantitative detection range, sensitivity, detection limit and precision as evaluation indicators, the CRITIC weighting method is used to weight each evaluation indicator to obtain the weight corresponding to each evaluation indicator. The concentration of the organic solvent in the standard aqueous solution corresponding to the minimum sum of the values ​​of each evaluation indicator multiplied by the corresponding weight is the optimal concentration.

[0015] In some implementations, step (b) further includes:

[0016] Based on the water quality of the water sample, the ratio range of the organic solvent to water is selected; and

[0017] Prepare the standard aqueous solution with the dual concentration gradient within the specified ratio range.

[0018] In some embodiments, the ratio of the organic solvent to water is in the range of 1-50% by volume of the organic solvent.

[0019] In some implementations, for clean water bodies such as drinking water, surface water, groundwater, seawater, atmospheric water, and glacial water, the ratio of the organic solvent to water is in the range of 1-10% by volume of the organic solvent.

[0020] In some implementations, for polluted water bodies such as domestic sewage, industrial wastewater, agricultural wastewater and medical wastewater, the ratio of the organic solvent to water is in the range of 10-50% by volume of the organic solvent.

[0021] In some embodiments, the method has a detection limit of greater than 0 to 10 ng / L for the novel antibiotic pollutant in the water sample, further to 0.1-5 ng / L, and even further to 0.2-5 ng / L.

[0022] In some implementations, the solid-phase extraction process is achieved via microfluidics.

[0023] In some implementations, the standard curve is fitted to the following equation (1):

[0024]

[0025] In equation (1), x is the concentration of the novel antibiotic pollutant; y is the percentage absorbance value corresponding to x; A1 is the upper asymptote estimate; A2 is the lower asymptote estimate; x0 is the half-maximum inhibitory concentration (IC50). 50 ; and p is the slope of the standard curve at the position corresponding to x0.

[0026] In some implementations, the sensitivity is determined by the upper half-inhibition concentration (IC50) of the standard curve. 50 The slope at the location of ) is represented.

[0027] In some implementations, the normalized dimensionless value of the sensitivity is calculated using the following equation (2):

[0028]

[0029] In equation (2), k represents the upper half-maximal inhibitory concentration (IC) of the standard curve. 50 The reciprocal of the slope at the location of the value; mean(k) is the average value of k corresponding to each standard curve; min(k) is the minimum value of k corresponding to each standard curve; max(k) is the maximum value of k corresponding to each standard curve; k′ represents the normalized dimensionless value of the sensitivity.

[0030] In some implementations, the normalized dimensionless value of the quantitative detection interval is calculated using the following equations (3)-(6).

[0031]

[0032] △x=x2-x1 (5)

[0033]

[0034] In equations (3)-(6), x1 is the left endpoint of the quantitative detection interval; x2 is the right endpoint of the quantitative detection interval; Y 80 It is 80%; Y 20 It is 20%; △x represents the width of the quantitative detection interval; mean(△x) is the average value of △x corresponding to each standard curve; min(△x) is the minimum value of △x corresponding to each standard curve; max(△x) is the maximum value of △x corresponding to each standard curve; △x′ represents the normalized dimensionless value of the quantitative detection interval; and x0 and p are the same as defined in claim 4; and / or

[0035] The normalized dimensionless value of the detection limit is calculated by the following equations (7)-(10):

[0036]

[0037] In equations (7)-(10), A LOD A is the percentage absorbance value corresponding to the detection limit; average It is the average percentage absorbance value obtained by repeated measurements of a standard aqueous solution of the aforementioned antibiotic-type new pollutant at a concentration of 0; 3s represents 3 times the standard deviation; Y LOD x represents the binding rate corresponding to the detection limit; LOD Indicates the detection limit; x LOD ′ represents the normalized dimensionless value of the detection limit; mean(x) LOD ) represents the x corresponding to each standard curve LOD The average value; min(x) LOD ) represents the x corresponding to each standard curve LOD The minimum value in; max(x) LOD ) represents the x corresponding to each standard curve LOD The maximum value in; and A1, A2, x0, and p are the same as those defined in claim 4; and / or

[0038] The normalized dimensionless value of the precision is calculated by the following equations (11) and (12):

[0039]

[0040] In equations (11) and (12), n represents the number of times the standard aqueous solution of each gradient concentration of the novel antibiotic contaminant is repeatedly measured; x i This represents the concentration obtained from the i-th measurement; The value represents the average concentration obtained from n measurements; S represents the standard deviation; RSD represents the precision; RSD′ represents the normalized dimensionless value of the precision; mean(RSD) represents the average RSD of each standard curve; min(RSD) represents the minimum RSD of each standard curve; and max(RSD) represents the maximum RSD of each standard curve.

[0041] In some implementations, the weighting process using the CRITIC weighting method includes:

[0042] (1) Form an m-row, 4-column matrix X of the normalized dimensionless values ​​of the quantitative detection range, the sensitivity, the detection limit, and the precision, where m is the number of concentration gradients of the optimal organic solvent;

[0043] (2) Calculate the objective weights corresponding to each evaluation index according to the following equations (13)-(19):

[0044]

[0045] In equations (13)-(19), x ij This represents the value of the evaluation index in the i-th row and j-th column of the matrix X; It is the average value of the evaluation indicators in the i-th row; S is the average value of the evaluation indicators in column j; j μ is the standard deviation of the evaluation index in column j, representing the index variability; i μ is the standard deviation of the values ​​in the i-th row; j It is the standard deviation of the values ​​in the j-th column; cov(x i ,x j ) represents the covariance of the matrix X; r ij R represents the correlation coefficient between the i-th row and j-th column of matrix X; j Indicates the conflict of indicators in column j; C j This represents the information content of the evaluation index in column j; and W j Represents the objective weight of the evaluation index in column j; and

[0046] (3) Multiply the four evaluation indicators of each row of the matrix X by the corresponding objective weights, and then sum them up. The gradient concentration of the optimal organic solvent corresponding to the minimum sum is taken as the optimal concentration.

[0047] In some embodiments, the novel antibiotic contaminant is selected from 3-methylquinoline-2-carboxylic acid, avermectin, albendazole, amikacin, amoxicillin, ampicillin, azithromycin, penicillin, β-lactam antibiotics, cephalexin, cefquinoxime, chloramphenicol, chlorpromazine, chlortetracycline, ciprofloxacin, colistin, doramectin, doxycycline, enrofloxacin, erythromycin, florfenicol, fluoroquinolones, furazolidone, gentamicin, isoniazid, kanamycin, and lincomycin. Medicinal compounds, melamine, metronidazole, natamycin, nitrofurantoin, nitrofurantoin, nitrofurazone, oxytetracycline, piracetam, prostaglandin, quinolones, ribavirin, sarafloxacin, zizomycin, streptomycin, sulfadiazine, sulfamethoxazole, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfanilides, sulfaquinoxaline, sulfonamides, tetracycline, thiamphenicol, tilmicosin, trimethoprim, tylosin, and vancomycin; and / or

[0048] The organic solvent is selected from methanol, acetonitrile, and dimethyl sulfoxide, and optionally, the organic solvent is methanol.

[0049] According to another aspect of this application, an optimized method for enzyme-linked immunosorbent assay (ELISA) is provided, comprising:

[0050] Screening for the optimal concentration of the organic solvent in an aqueous solution containing the target substance and the organic solvent to be detected by enzyme-linked immunosorbent assay (ELISA) includes:

[0051] The optimal concentration was determined using the quantitative detection range, sensitivity, limit of detection, and precision of enzyme-linked immunosorbent assay (ELISA) as evaluation indicators, combined with the CRITIC weighting method.

[0052] In some implementations, the method includes:

[0053] (i) Prepare standard aqueous solutions containing a standard of the target substance and the organic solvent in a dual concentration gradient, wherein a set of standard aqueous solutions containing the standard of the gradient concentration is prepared for each concentration of the organic solvent;

[0054] (ii) Perform enzyme-linked immunosorbent assay (ELISA) on each group of standard aqueous solutions to obtain standard curves corresponding to each concentration of the organic solvent, and calculate the quantitative detection range, sensitivity, limit of detection, and precision values ​​corresponding to each standard curve; and

[0055] (iii) Using the normalized dimensionless values ​​of the quantitative detection range, sensitivity, detection limit and precision as evaluation indicators, the CRITIC weighting method is used to weight each evaluation indicator to obtain the weight corresponding to each evaluation indicator. The concentration of the organic solvent in the standard aqueous solution corresponding to the minimum sum of the values ​​of each evaluation indicator multiplied by the corresponding weight is the optimal concentration. Attached Figure Description

[0056] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. The drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0057] Figure 1 The enzyme-linked immunosorbent assay (ELISA) standard curves obtained by preparing gradient standard aqueous solutions in the 0-8% methanol concentration range are shown.

[0058] Figure 2 The standard curve of enzyme-linked immunosorbent assay (ELISA) at a concentration of 1% methanol is shown.

[0059] Figure 3 The enzyme-linked immunosorbent assay (ELISA) standard curves obtained by preparing gradient standard aqueous solutions in the range of 10-50% methanol concentration are shown.

[0060] Figure 4 The standard curve of enzyme-linked immunosorbent assay (ELISA) at a 10% methanol concentration is shown. Detailed Implementation

[0061] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments in this application, all other implementation methods obtained by those skilled in the art without inventive effort are within the scope of protection of this application.

[0062] A combined solid-phase extraction (SPE) and enzyme-linked immunosorbent assay (ELISA) method holds promise as an effective approach for detecting trace amounts of emerging antibiotic pollutants in water samples. SPE enriches emerging antibiotic pollutants in water samples by combining liquid-solid extraction (LiPE) with column liquid chromatography (LCC). Specific target compounds are adsorbed onto a stationary phase, achieving separation, enrichment, and purification of the target compounds. SPE offers advantages such as high separation efficiency, large sample processing capacity, and no need for large amounts of organic solvents. ELISA is used for quantitative and qualitative analysis of antibiotics. By binding antigens or antibodies to the surface of a solid-phase carrier, the specific binding of antigens and antibodies, along with the colorimetric reaction catalyzed by enzymes labeled on the antibody or antigen, determines the antibiotic content in the water, achieving immunoassay for target analyte detection.

[0063] Following solid-phase extraction (SPE) and prior to enzyme-linked immunosorbent assay (ELISA), the target compounds enriched by SPE need to be reconstituted in a suitable solvent for ELISA loading. Typically, novel antibiotic contaminants are more soluble in organic solvents than in water; therefore, the presence of organic solvents allows for increased solubility of novel antibiotic contaminants in the reconstituted aqueous solution.

[0064] The inventors of this application discovered in their research that reconstituted solutions, as a linking point between solid-phase extraction and enzyme-linked immunosorbent assay (ELISA), can improve the sensitivity and accuracy of ELISA detection by optimizing the concentration of organic solvents in the reconstituted solution (e.g., a reconstituted aqueous solution).

[0065] According to the implementation scheme of this application, a method for detecting novel antibiotic pollutants in water samples by combining solid-phase extraction and enzyme-linked immunosorbent assay (ELISA) is provided, including:

[0066] (a) Select an organic solvent such that the R0 of the standard curve obtained by enzyme-linked immunosorbent assay (ELISA) for a standard containing a new antibiotic contaminant and an aqueous solution of the organic solvent is such that the R0 of the standard curve is significantly higher than that of the standard containing the new antibiotic contaminant. 2 Greater than 0.98;

[0067] (b) Using the quantitative detection range, sensitivity, limit of detection, and precision of enzyme-linked immunosorbent assay (ELISA) as evaluation indicators, and combining this with the CRITIC weighting method, determine the optimal concentration of the organic solvent selected in step (a); and

[0068] (c) Prepare a reconstituted aqueous solution by mixing the organic solvent selected in step (a), the antibiotic-like new pollutants enriched by solid-phase extraction of water samples, and detect the concentration of antibiotic-like new pollutants in the water sample by enzyme-linked immunosorbent assay (ELISA), wherein the concentration of the organic solvent in the reconstituted aqueous solution is the optimal concentration.

[0069] This application creatively combines the quantitative detection range, sensitivity, limit of detection, and precision of enzyme-linked immunosorbent assay (ELISA), using these four parameters for multidimensional consideration during conditional experiments to comprehensively and accurately evaluate the impact of optimized conditions on the method. This approach surpasses evaluation methods that rely on the parallelism of curves or traditional evaluation metrics (such as IC50). 50 This method can optimize certain conditions and relies on data for judgment, making it more scientific and accurate. It provides theoretical guidance for the use of SPE-ELISA for detection and provides important assurance for the accuracy and reliability of qualitative and quantitative results of antibiotics in water samples.

[0070] In some implementations, when determining the optimal concentration in step (b), the water properties of the water sample and the differences in dissolved substances therein may be further considered, and the concentration of organic solvent in the reconstituted aqueous solution may be adjusted.

[0071] When selecting the organic solvent and its concentration in the reconstituted aqueous solution, the type and concentration of the organic solvent can be appropriately adjusted according to the properties of the water sample and the different dissolved substances in the water. This application innovatively combines the quantitative detection range, sensitivity, limit of detection, and precision of enzyme-linked immunosorbent assay (ELISA) and optimizes the concentration of organic solvents using the CRITIC weighting method, providing improved limits of detection and sensitivity for different water bodies and different substances.

[0072] In some implementations, step (b) includes:

[0073] (i) Prepare standard aqueous solutions with a dual concentration gradient containing a standard of a novel antibiotic contaminant and a selected organic solvent, wherein a set of standard aqueous solutions containing a gradient concentration of the standard is prepared for each concentration of the organic solvent.

[0074] (ii) Perform enzyme-linked immunosorbent assay (ELISA) on each group of standard aqueous solutions to obtain standard curves corresponding to different concentrations of organic solvents. Calculate the quantitative detection range, sensitivity, limit of detection, and precision for each standard curve.

[0075] (iii) The normalized dimensionless values ​​of quantitative detection range, sensitivity, detection limit and precision are used as evaluation indicators. The CRITIC weighting method is used to weight them to obtain the weights corresponding to each evaluation indicator. The concentration of organic solvent in the standard aqueous solution corresponding to the minimum sum of the values ​​of each evaluation indicator multiplied by the corresponding weights is the optimal concentration.

[0076] In some implementation schemes, when selecting an organic solvent in step (a), the solubility of the organic solvent in water for the novel antibiotic contaminant to be tested can also be taken into consideration. Additionally, the water quality of the water body to be tested and the differences in the dissolved substances therein can also be taken into consideration.

[0077] In some implementations, step (b) further includes: selecting a range of organic solvent to water ratios based on the water quality of the water sample; and preparing the aforementioned dual-concentration gradient standard aqueous solution within the selected range. For example, when testing clean water (such as drinking water), the optimal solvent concentration can be selected from a lower organic solvent concentration range (<10%), while when testing water with a higher organic content (such as wastewater), a lower volume fraction of methanol may not be able to dissolve the concentrated wastewater sample, and it is appropriate to select the optimal concentration from a higher organic solvent concentration range (10-50%).

[0078] In some implementations, the ratio of organic solvent to water is in the range of 1-50% by volume of organic solvent.

[0079] In some implementations, for clean water bodies such as drinking water, surface water, groundwater, seawater, atmospheric water, and glacial water, the ratio of organic solvent to water is in the range of 1-10% by volume of organic solvent.

[0080] In some implementation schemes, for polluted water bodies such as domestic sewage, industrial wastewater, agricultural wastewater and medical wastewater, the ratio of organic solvent to water is in the range of 10-50% by volume of organic solvent.

[0081] In some implementations, the method of this application has a detection limit of greater than 0 to 10 ng / L for novel antibiotic pollutants in water samples, further to 0.1-5 ng / L, and even further to 0.2-5 ng / L.

[0082] In some implementations, the solid-phase extraction process in the method of this application can be achieved by microfluidics.

[0083] In some implementations, the operating conditions for enzyme-linked immunosorbent assay (ELISA) in steps (b) and (c) are the same, except that the concentration of organic solvent in the aqueous solution used for sample loading and detection differs.

[0084] In some implementations, the standard curve of ELISA is fitted to the following equation (1):

[0085]

[0086] In equation (1), x is the concentration of the new antibiotic pollutant; y is the percentage absorbance value corresponding to y; A1 is the upper asymptote estimate; A2 is the lower asymptote estimate; x0 is the half-maximum inhibitory concentration (IC50). 50 ; and p is the slope of the standard curve at the position corresponding to x0.

[0087] In some implementations, the standard curve can be fitted using computer software such as Origin.

[0088] In some implementations, the sensitivity of ELISA is determined by the upper half-inhibitory concentration (IC50) of the standard curve. 50 The slope at the location of ) is represented.

[0089] IC 50 The value represents the compound concentration required to achieve a 50% inhibition effect. Typically, IC50... 50 The lower the value, the stronger the inhibitory effect of the compound, meaning the higher the sensitivity of the compound to the biological process. This application innovatively uses IC. 50 Using the slope at the location of the value as a sensitivity indicator can provide more comprehensive and dynamic information. Compared to IC... 50The slope of the curve better reflects the significant change in the reaction caused by a small increase in concentration, and better represents the sensitivity of ELISA to the detection of the target compound.

[0090] In some implementations, the normalized dimensionless value of the sensitivity is calculated using the following equation (2):

[0091]

[0092] In equation (2), k represents the upper half-maximal inhibitory concentration (IC50) of the standard curve. 50 The reciprocal of the slope at the location of the value; mean(k) is the average value of k for each standard curve; min(k) is the minimum value of k for each standard curve; max(k) is the maximum value of k for each standard curve; k′ represents the normalized dimensionless value of the sensitivity.

[0093] In this application, to facilitate rapid data processing, the quantitative detection interval is mapped to the range of 0 to 1, transforming the dimensional expression into a dimensionless expression for easier comparison and weighting. In some implementations, the normalized dimensionless value of the quantitative detection interval is calculated using the following equations (3)-(6):

[0094] Upper limit of quantitative detection interval

[0095]

[0096] Lower limit of quantitative detection interval

[0097]

[0098] Quantitative detection interval △x

[0099] △x=x2-x1 (5)

[0100] Normalized dimensionless value Δx′ of the quantitative detection interval

[0101]

[0102] In equations (3)-(6), x1 is the left endpoint of the quantitative detection interval; x2 is the right endpoint of the quantitative detection interval; Y 80 It is 80%; Y 20 It is 20%; △x represents the width of the quantitative detection interval; mean(△x) is the average value of △x corresponding to each standard curve; min(△x) is the minimum value of △x corresponding to each standard curve; max(△x) is the maximum value of △x corresponding to each standard curve; △x′ represents the normalized dimensionless value of the quantitative detection interval; x0 and p are the same as defined above.

[0103] In some implementations, the normalized dimensionless value of the detection limit is calculated using the following equations (7)-(10):

[0104]

[0105] In equations (7)-(10), A LOD A represents the percentage absorbance value corresponding to the detection limit. average It is the average percentage absorbance value obtained from repeated measurements of a standard aqueous solution with a concentration of 0 for new antibiotic pollutants; 3s represents 3 times the standard deviation; Y LOD The binding rate corresponding to the detection limit; x LOD Indicates the detection limit; x LOD ′ represents the normalized dimensionless value of the detection limit; mean(x) LOD ) represents the x corresponding to each standard curve LOD The average value; min(x) LOD ) represents the x corresponding to each standard curve LOD The minimum value in; max(x) LOD ) represents the x corresponding to each standard curve LOD The maximum value in; A1, A2, x0, and p are the same as those defined above.

[0106] In some implementations, the normalized dimensionless value of precision is calculated using the following equations (11) and (12):

[0107]

[0108] In equations (11) and (12), n represents the number of times the standard aqueous solution of each gradient concentration of the new antibiotic contaminant is repeatedly measured; x i This represents the concentration obtained from the i-th measurement; The value represents the average concentration obtained from n measurements; S represents the standard deviation; RSD represents the relative standard deviation, which represents precision; RSD′ represents the normalized dimensionless value of precision; mean(RSD) represents the average RSD of each standard curve; min(RSD) represents the minimum RSD of each standard curve; and max(RSD) represents the maximum RSD of each standard curve.

[0109] The CRITIC weighting method is an objective weighting method. Its core idea is to use two indicators: contrast strength and conflict index. Contrast strength is represented by standard deviation; a larger standard deviation indicates greater volatility, and thus a higher weight. Conflict index is represented by correlation coefficient; a larger correlation coefficient indicates less conflict, and thus a lower weight. In weight calculation, the contrast strength and conflict index are multiplied and then normalized to obtain the final weight.

[0110] In some implementations, the weighting process using the CRITIC weighting method in the method of this application includes:

[0111] (1) Form an m-row, 4-column matrix X containing four evaluation indicators (i.e., normalized dimensionless values ​​of quantitative detection range, sensitivity, detection limit, and precision), where m is the number of concentration gradients of the organic solvent, and the elements in the matrix X... ij This represents the value of the evaluation index in the j-th column corresponding to the gradient concentration in the i-th row of the organic solvent, which is the value of the evaluation index in the i-th row and j-th column of matrix X.

[0112] Taking five groups of samples to be evaluated (corresponding to gradient concentrations of organic solvents, such as 0%, 1%, 2%, 4%, and 8%) as an example, the original index data matrix formed by the four evaluation indicators is as follows:

[0113]

[0114] (2) Calculate the objective weights corresponding to each evaluation index according to the following equations (13)-(19):

[0115]

[0116]

[0117] In equations (13)-(19), x ij This represents the value of the evaluation index in the i-th row and j-th column of matrix X; It is the average value of the evaluation indicators in the i-th row; S is the average value of the evaluation indicators in column j; j μ is the standard deviation of the evaluation index in column j, representing the index variability; i μ is the standard deviation of the values ​​in the i-th row; j It is the standard deviation of the values ​​in the j-th column; cov(x i ,x j ) represents the covariance of matrix X; r ij R represents the correlation coefficient between the i-th row and j-th column of matrix X; j Indicates the conflict of indicators in column j; C jThis represents the information content of the evaluation index in column j; and W j Represents the objective weight of the evaluation index in column j; and

[0118] (3) Multiply the four evaluation indicators of each row of matrix X by the corresponding objective weights, and then sum them up. The gradient concentration of the organic solvent corresponding to the minimum sum is taken as the optimal concentration.

[0119] In this text, "new pollutants" refers to toxic and hazardous chemicals that are released into the environment and possess characteristics such as biotoxicity, environmental persistence, and bioaccumulation, posing significant risks to the ecological environment or human health, but which are not yet included in management or whose existing management measures are insufficient. New pollutants mainly include persistent organic pollutants, endocrine disruptors, and antibiotics controlled by international conventions.

[0120] In some implementation schemes, the new antibiotic contaminants are selected from 3-methylquinoline-2-carboxylic acid, avermectin, albendazole, amikacin, amoxicillin, ampicillin, azithromycin, penicillin, β-lactam antibiotics, cephalexin, cefquinoxime, chloramphenicol, chlorpromazine, chlortetracycline, ciprofloxacin, colistin, doramectin, doxycycline, enrofloxacin, erythromycin, florfenicol, fluoroquinolones, furazolidone, gentamicin, isoniazid, kanamycin, and lincomycin. The list includes antibiotics such as melamine, metronidazole, natamycin, nitrofurantoin, nitrofurantoin, nitrofurazone, oxytetracycline, pyrimethanil, prostaglandins, quinolones, ribavirin, sarafloxacin, zizomycin, streptomycin, sulfadiazine, sulfamethoxazole, sulfamethoxypyrimidine, sulfamethoxazole, sulfamethoxazole-trimethoprim, sulfanilamides, sulfaquinoxaline, sulfonamides, tetracycline, thiamphenicol, tilmicosin, trimethoprim, tylosin, and vancomycin. In some implementation plans, the new antibiotic contaminant is sulfamethoxazole.

[0121] In some embodiments, the organic solvent mentioned in the method of this application is selected from methanol, acetonitrile, dimethyl sulfoxide, etc. In some embodiments, methanol is selected as the organic solvent. When selecting a solvent, factors such as its compatibility with reactants and products, solubility, polarity, toxicity and environmental friendliness, and more importantly, its impact on ELISA antibodies, should be considered.

[0122] In some implementation methods, the novel antibiotic contaminant in the detection method of this application is sulfamethoxazole, the organic solvent is methanol, and the optimal volume concentration of methanol is 1% (for clean water bodies with low organic matter content) or 10% (for water bodies with high organic matter content).

[0123] In some implementations, the water sample is filtered through a filter membrane, for example, a filter membrane with a size of 0.45 μm, before solid-phase extraction.

[0124] In some implementations, the water samples are pretreated to remove metal ions before solid-phase extraction, for example by adjusting the pH to 2-3 by adding 0.1% formic acid (500 μL) and adding 0.5% disodium ethylenediaminetetraacetate (EDTA) to complex the metal ions.

[0125] In the method of this application, the extraction conditions of solid-phase extraction, including the extraction column, eluent, and elution operation conditions, can be determined according to the water quality of the water sample to be loaded. For example, the following extraction conditions can be used: Oasis HLB solid-phase extraction column; activation: wash once with 6 mL of methanol, then wash once with 6 mL of pure water; loading: 500 mL of water sample is passed through the solid-phase extraction column at a constant flow rate of 1 drop / s; rinsing: the column is rinsed with 6 mL of methanol-water mixture (methanol:water = 1:9) to remove impurities, and then dried under vacuum for 30 min; elution: elution is performed with 6 mL of methanol. The sample is then purged with nitrogen to near dryness under a 30°C water bath; the solution is reconstituted to 500 μL using an aqueous solution of the optimal concentration of organic solvent for ELISA detection.

[0126] In some implementations, the solid-phase extraction column is selected from HLB or C18 solid-phase extraction columns, and the eluent for solid-phase extraction is selected from methanol, acetonitrile, dimethyl sulfoxide, etc.

[0127] In the method of this application, the ELISA kit used in the ELISA experiment can be selected from kits suitable for various new antibiotic pollutants in the aquatic environment.

[0128] This application also provides an optimized method for enzyme-linked immunosorbent assay (ELISA), including:

[0129] The optimal concentration of organic solvent in the aqueous solution containing the target substance to be detected by enzyme-linked immunosorbent assay (ELISA) was selected, including:

[0130] The optimal concentrations were determined using the quantitative detection range, sensitivity, limit of detection, and precision of enzyme-linked immunosorbent assay (ELISA) as evaluation indicators, combined with the CRITIC weighting method.

[0131] It should be understood that the organic solvent in the method of this application has the effect of increasing the solubility of the target substance in water.

[0132] In some implementation schemes, the optimized enzyme-linked immunosorbent assay (ELISA) method of this application further includes:

[0133] (i) Prepare standard aqueous solutions containing a standard of the target substance and an organic solvent with a dual concentration gradient, wherein a set of standard aqueous solutions containing a standard of gradient concentration is prepared for each concentration of organic solvent.

[0134] (ii) Perform enzyme-linked immunosorbent assay (ELISA) on each group of standard aqueous solutions to obtain standard curves corresponding to different concentrations of organic solvents. Calculate the quantitative detection range, sensitivity, limit of detection, and precision for each standard curve.

[0135] (iii) The normalized dimensionless values ​​of quantitative detection range, sensitivity, detection limit and precision are used as evaluation indicators. The CRITIC weighting method is used to weight them to obtain the weights corresponding to each evaluation indicator. The concentration of organic solvent in the standard aqueous solution corresponding to the minimum sum of the values ​​of each evaluation indicator multiplied by the corresponding weights is the optimal concentration.

[0136] Other implementation schemes of the method for optimizing enzyme-linked immunosorbent assay (ELISA) in this application can refer to the relevant implementation schemes of step (b) of the method for detecting new antibiotic pollutants in water samples by combining solid-phase extraction and ELISA, which will not be described again here.

[0137] The target substance in the optimized enzyme-linked immunosorbent assay (ELISA) method provided in this application is not limited to new pollutants. Any target substance that is suitable for ELISA detection and has a higher solubility in organic solvents than in water can be used with the optimized method provided in this application.

[0138] To address the solubility of various target substances in organic solvents and the tolerance of antibodies in ELISA kits to organic solvents, this application innovatively proposes a multi-dimensional combination of indicators to comprehensively and accurately evaluate the impact of optimized conditions on SPE-ELISA. Compared to evaluating the parallelism of curves or using traditional evaluation indicators (such as IC50), this approach offers a more comprehensive and accurate assessment. 50 This method can optimize certain conditions and relies on data for judgment, making it more scientific and accurate. It provides theoretical guidance for the use of SPE-ELISA for detection and provides important assurance for the accuracy and reliability of qualitative and quantitative results of antibiotics in water samples.

[0139] Compared to solid-phase extraction-chromatography / mass spectrometry (SPE-ELISA), the SPE-ELISA kit offers a simpler and more accurate, reliable, rapid, and specific detection method suitable for rapid screening of large batches of samples containing multiple contaminants. This patent enables the simultaneous enrichment and detection of multiple antibiotics in various water qualities, including wastewater, groundwater, and surface water. It provides a cost-effective, efficient, sensitive, and accurate detection method for wastewater treatment plants, drinking water plants, and environmental monitoring stations in diverse environments and locations.

[0140] The method described in this application has at least the following advantages over using only one-dimensional screening methods:

[0141] 1. Comprehensiveness: Multi-dimensional screening considers multiple relevant factors, enabling a more comprehensive evaluation and comparison of options. This helps avoid relying solely on IC (Information Capacity). 50The risk of bias or neglect of other important factors.

[0142] 2. Accuracy: The method of this application can more accurately detect the concentration of new antibiotic pollutants in water.

[0143] 3. Adaptability: The method of this application is more adaptable to complex and variable aquatic environments. In different aquatic environments, the concentration of organic matter and the aquatic matrix are different. Single-dimensional screening cannot cover every species, while the screening conditions obtained by the method of this application can better adapt to these changes.

[0144] Example

[0145] The test materials used in the embodiments of this application are all conventional test materials in the art, and can be purchased through commercial channels.

[0146] Example 1: Screening of sulfamethoxazole methanol reconstituted solution in relatively clean water (pure water)

[0147] (1) Reagents used:

[0148] Enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Zhenke Biotechnology Co., Ltd., ZK-169, including ELISA plate, antibiotic standards, antibiotic antibody working solution, antibiotic enzyme conjugate, substrate solution A, substrate solution B, stop solution, concentrate dilution solution and concentrate wash solution); methanol and formic acid (both chromatographic grade, Merk, Germany); ultrapure water (conductivity 18.2 MΩ·cm); disodium EDTA (Sigma); Oasis HLB solid phase extraction column (200 mg / 6 mL, Waters); filter membrane (0.45 μm, Shanghai Anpu Scientific Instruments Co., Ltd.); sulfamethoxazole standard (99.9%, Tianjin Alta Technology Co., Ltd.).

[0149] (2) Instruments used:

[0150] Solid phase extraction instrument (USE-24S, Beijing Yousheng United Technology Co., Ltd.); nitrogen blowing instrument (EFAA-DC12, Shanghai Anpu Scientific Instruments Co., Ltd.); multi-functional microplate reader (TECAN, Austria).

[0151] (3) Specific operation process:

[0152] 1) Preparation of standard solution: Weigh an appropriate amount of the standard substance into a 10.0 mL amber volumetric flask, dissolve and dilute to volume with methanol to prepare a 100 mg / L standard stock solution. Transfer the stock solution to a amber reagent bottle and store at -20.0℃ for later use. Transfer 10.0 μL of the standard stock solution into a 10.0 mL amber volumetric flask and dilute to volume with methanol to obtain a 100 μg / L standard working solution. Store at 4℃ for later use. Prepare the standard working solution immediately before use.

[0153] 2) Screening for optimal methanol conditions: Based on the principle of indirect competitive enzyme-linked immunosorbent assay (ELISA) between antigen and antibody, an aqueous methanol solution containing sulfamethoxazole standard was used for detection. Note that each liquid reagent must be brought to room temperature and shaken well before use. Prepare a series of standard aqueous solutions containing 0%, 1%, 2%, 4%, and 8% methanol at sulfamethoxazole concentrations of 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L, and 81 μg / L, respectively.

[0154] Take a microplate and add 50 μL of a series of standard aqueous solutions to the corresponding wells, performing 6 replicates per sample. Add 50 μL of enzyme-labeled reagent and 50 μL of antibody working solution to each well, and gently vortex to mix. Cover the plate with a cover film and incubate at room temperature in the dark for 30 min. Carefully remove the cover film, shake off the liquid in the wells, add excess washing working solution, wash thoroughly several times, and pat dry with absorbent paper. Any remaining air bubbles can be popped with an unused pipette tip. Add 50 μL of substrate solution A to each well and 50 μL of substrate solution A to each well, and gently vortex to mix. Cover the plate with a cover film and incubate in the dark. Add 50 μL of stop solution to each well and gently vortex to mix.

[0155] The absorbance of each well was measured using dual-wavelength detection at 450nm / 630nm, and data were read within 5 minutes. Using Origin software, standard curves were plotted with sulfamethoxazole concentration on the x-axis and the measured percentage absorbance on the y-axis for different methanol concentrations, obtaining standard curves for each methanol concentration, as shown below. Figure 1 As shown. Based on the plotted standard curve and the fitting equation (1), the upper and lower limits of the quantitative detection interval and the IC are calculated. 50 Sensitivity (IC) 50 The corresponding curve slopes, detection limits, and precision are shown in Table 1 below.

[0156] Table 1 shows the ELISA parameter values ​​obtained from experiments in relatively clean water bodies with methanol concentrations ranging from 0% to 8%.

[0157]

[0158] The values ​​of four evaluation indicators are calculated by combining equations (1)-(19), including the normalized value of the quantitative detection interval, sensitivity (IC). 50 The normalized values ​​of the curve slope, detection limit, and precision are calculated using the CRITIC method. The weights of each index are then calculated, and the sum of the index values ​​corresponding to each methanol concentration multiplied by the weights is obtained, as shown in Table 2 below.

[0159] Table 2 shows the ELISA parameter values ​​calculated using the CRITIC method for relatively clean water bodies in the 0-8% methanol concentration range.

[0160]

[0161] Finally, based on the results, the methanol concentration corresponding to the minimum weighted sum was selected, i.e., 1% methanol concentration was the optimal concentration.

[0162] 3) Water sample filtration: Collect 500 mL of water sample (tap water) and filter it through a 0.45 μm filter membrane;

[0163] 4) Water sample pretreatment: After filtration, add 0.1% formic acid (500μL) to the sample to adjust its pH value to 2-3, and add 0.5% disodium ethylenediaminetetraacetate (EDTA) to complex metal ions.

[0164] 5) The sample was enriched by solid-phase extraction using a solid-phase extraction column:

[0165] Connect the solid-phase extraction (SPE) apparatus using an Oasis HLB SPE column. Activation: Wash once with 6 mL of methanol, then once with 6 mL of water. Sample loading: Pass 500 mL of water sample through the SPE column at a constant flow rate of 1 drop / s. Washing: Wash the column with 6 mL of methanol-water mixture (methanol:water = 1:9) to remove impurities, then dry under vacuum for 30 min. Elution: Elute with 6 mL of methanol. Blow dry with nitrogen gas in a 30°C water bath until nearly dry. Reconstitute with 1% methanol aqueous solution to a final volume of 500 μL, based on the concentration of the water sample.

[0166] 6) After solid-phase extraction and enrichment, the reconstituted solution was tested using an ELISA kit and a microplate reader; based on the principle of indirect competitive enzyme-linked immunosorbent assay (ELISA) between antigen and antibody, the reconstituted solution was used for detection.

[0167] Note that each liquid reagent must be brought to room temperature and shaken well before use. Prepare a series of standard aqueous solutions containing 1% methanol at concentrations of 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L, and 81 μg / L of sulfamethoxazole standard material, and mix well. Take an ELISA plate and add 50 μL each of the series of standard aqueous solutions and sample reconstitution solutions to the corresponding wells, with 6 replicates per sample. Add 50 μL of enzyme-labeled material and 50 μL of antibody working solution to each well, and gently vortex to mix. Cover the plate with a cover film and incubate at room temperature in the dark for 30 min. Carefully remove the cover film, shake off the liquid in the wells, add excess washing working solution, wash thoroughly several times, and pat dry with absorbent paper. Any air bubbles that are not removed after patting can be popped with an unused pipette tip. Add 50 μL of substrate solution A to each well and 50 μL of substrate solution A to each well, and gently vortex to mix. Cover the plate with a cover film and incubate in the dark. Add 50 μL of stop solution to each well and gently vortex to mix. Set the microplate reader to 450 nm or dual wavelength 450 nm / 630 nm, measure the absorbance of each well, and read the data within 5 minutes.

[0168] A standard curve was plotted with the concentration of sulfamethoxazole standard material on the x-axis and the measured percentage absorbance value on the y-axis. The content of sulfamethoxazole in the sample reconstituted solution was calculated by referring to the standard curve, and then the content of sulfamethoxazole in the actual water sample was calculated by dividing by the enrichment factor.

[0169] The standard curve obtained using a 1% methanol solution is as follows: Figure 2 As shown, R 2 R can reach 0.99 2 The requirement of a linear range greater than 0.98 and good linearity indicate that the method disclosed in this application yields relatively accurate results. This further demonstrates that the method screens optimal methanol conditions, ensuring detection stability without disrupting the interaction between the ELISA antigen and antibody, and significantly improving the sensitivity of the immunoassay, thus meeting the needs for the analysis of ultra-trace antibiotic-like novel pollutants in water. Furthermore, by using organic solvents to reconstitute the target substance enriched through solid-phase extraction, followed by ELISA detection, the detection limit for sulfamethoxazole in water samples can reach the ng / L level.

[0170] Example 2: Screening of sulfamethoxazole methanol reconstituted solution for water bodies with high organic content (such as sewage)

[0171] (1) Reagents used:

[0172] Enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Zhenke Biotechnology Co., Ltd., ZK-169, including ELISA plate, antibiotic standards, antibiotic antibody working solution, antibiotic enzyme conjugate, substrate solution A, substrate solution B, stop solution, concentrate dilution solution and concentrate wash solution); methanol and formic acid (both chromatographic grade, Merk, Germany); ultrapure water (conductivity 18.2 MΩ·cm); disodium EDTA (Sigma); Oasis HLB solid phase extraction column (200 mg / 6 mL, Waters); filter membrane (0.45 μm, Shanghai Anpu Scientific Instruments Co., Ltd.); sulfamethoxazole standard (99.9%, Tianjin Alta Technology Co., Ltd.).

[0173] (2) Instruments used:

[0174] Solid phase extraction instrument (USE-24S, Beijing Yousheng United Technology Co., Ltd.); nitrogen blowing instrument (EFAA-DC12, Shanghai Anpu Scientific Instruments Co., Ltd.); multi-functional microplate reader (TECAN, Austria).

[0175] (3) Specific operation process:

[0176] 1) Preparation of standard solution: Weigh an appropriate amount of the standard substance into a 10.0 mL amber volumetric flask, dissolve and dilute to volume with methanol to prepare a 100 mg / L standard stock solution. Transfer the stock solution to a amber reagent bottle and store at -20.0℃ for later use. Transfer 10.0 μL of the standard stock solution into a 10.0 mL amber volumetric flask and dilute to volume with methanol to obtain a 100 μg / L standard working solution. Store at 4℃ for later use. Prepare the standard working solution immediately before use.

[0177] 2) Screening for optimal methanol conditions: Based on the principle of indirect competitive enzyme-linked immunosorbent assay (ELISA) between antigen and antibody, an aqueous methanol solution containing sulfamethoxazole standard was used for detection. Note that each liquid reagent must be brought to room temperature and shaken well before use. Prepare a series of standard aqueous solutions with concentrations of 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L, and 81 μg / L, containing 10%, 25%, and 50% methanol by volume, respectively, and mix well.

[0178] Take a microplate and add 50 μL of the series of standards to the corresponding wells, performing 6 replicates per sample. Add 50 μL of enzyme-labeled protein and 50 μL of antibody working solution to each well, and gently vortex to mix. Cover the plate with a cover film and incubate at room temperature in the dark for 30 min. Carefully remove the cover film, shake off the liquid in the wells, add excess washing working solution, wash thoroughly several times, and pat dry with absorbent paper. Any remaining air bubbles can be popped with an unused pipette tip. Add 50 μL of substrate solution A to each well and 50 μL of other substrate solutions to each well, and gently vortex to mix. Cover the plate with a cover film and incubate in the dark. Add 50 μL of stop solution to each well and gently vortex to mix.

[0179] The absorbance of each well was measured using dual-wavelength detection at 450nm / 630nm, and data were read within 5 minutes. Using Origin software, standard curves were plotted with sulfamethoxazole concentration on the x-axis and the measured percentage absorbance on the y-axis for different methanol concentrations, obtaining standard curves for each methanol concentration, as shown below. Figure 3 As shown. Based on the plotted standard curve and the fitting equation (1), the upper and lower limits of the quantitative detection interval and the IC are calculated. 50 Sensitivity (IC) 50 The corresponding curve slopes, detection limits, and precision are shown in Table 3 below.

[0180] Table 3 shows the ELISA parameter values ​​obtained from experiments in water bodies with high organic matter content within the methanol concentration range of 10-50% in the 10-50% methanol concentration range.

[0181]

[0182] The values ​​of the four evaluation indicators are calculated by combining equations (1)-(19), including the normalized value of the interval width, sensitivity (IC). 50 The normalized values ​​of the curve slope, detection limit, and precision are calculated according to the CRITIC method. The weights of each index are then calculated, and the index values ​​corresponding to each methanol concentration are multiplied by their weights and summed. The results are shown in Table 4 below.

[0183] Table 4 shows the ELISA parameter values ​​calculated using the CRITIC method for water bodies with high organic matter content in the methanol concentration range of 10-50%.

[0184]

[0185] Finally, based on the results, the methanol concentration corresponding to the minimum weighted sum was selected, i.e., 10% methanol concentration was the optimal concentration.

[0186] 3) Water sample filtration: 500 mL of water sample (obtained from the urban domestic sewage network) was collected and filtered through a 0.45 μm filter membrane;

[0187] 4) Water sample pretreatment: After filtration, add 0.1% formic acid (500μL) to the sample to adjust its pH value to 2-3, and add 0.5% disodium ethylenediaminetetraacetate (EDTA) to complex metal ions.

[0188] 5) The sample was enriched by solid-phase extraction using a solid-phase extraction column:

[0189] Connect the solid-phase extraction (SPE) apparatus using an Oasis HLB SPE column. Activation: Wash once with 6 mL of methanol, then once with 6 mL of water. Sample loading: Pass 500 mL of water sample through the SPE column at a constant flow rate of 1 drop / s. Washing: Wash the column with 6 mL of methanol-water mixture (methanol:water = 1:9) to remove impurities, then dry under vacuum for 30 min. Elution: Elute with 6 mL of methanol. Blow dry with nitrogen gas in a 30°C water bath until nearly dry. Reconstitute with 10% methanol aqueous solution to a final volume of 500 μL, based on the concentration of the water sample.

[0190] 6) After solid-phase extraction enrichment, the reconstituted solution was analyzed using an ELISA kit and a microplate reader. Based on the principle of indirect competitive enzyme-linked immunosorbent assay (ELISA) between antigen and antibody, the reconstituted solution was used for detection. Note that each liquid reagent must be brought to room temperature and shaken well before use. Prepare a series of standard aqueous solutions containing 10% methanol at concentrations of 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L, and 81 μg / L, and mix well. Take an ELISA plate and add 50 μL of the series of standard aqueous solutions and sample reconstituted solutions to the corresponding wells, performing 6 replicates per sample. Add 50 μL of enzyme-labeled reagent and 50 μL of antibody working solution to each well, and gently shake to mix. Cover the plate with a cover film and incubate at room temperature in the dark for 30 min. Carefully remove the cover film, shake off the liquid in the wells, add excess washing working solution, wash thoroughly several times, and pat dry with absorbent paper. Any remaining air bubbles after patting dry can be popped with an unused pipette tip. Add 50 μL of substrate solution A to each well and gently vortex to mix. Cover the wells with the cover plate membrane and incubate in a dark environment. Add 50 μL of stop solution to each well and gently vortex to mix. Set the microplate reader to 450 nm or dual wavelength 450 nm / 630 nm and measure the absorbance of each well. Read the data within 5 minutes.

[0191] A standard curve was plotted with the concentration of sulfamethoxazole standard material on the x-axis and the measured percentage absorbance value on the y-axis. The content of sulfamethoxazole in the sample reconstituted solution was calculated by referring to the standard curve, and then the content of sulfamethoxazole in the actual water sample was calculated by dividing by the enrichment factor.

[0192] The standard curve obtained using a 10% methanol solution is as follows: Figure 4 As shown, R 2 R can reach 0.99 2The linear range is good, with a requirement greater than 0.98. Results obtained using this patented method for wastewater are relatively accurate. This demonstrates that the patented method screens optimal methanol conditions, ensuring detection stability without disrupting the interaction between ELISA antigen and antibody, and significantly improving the sensitivity of immunoassay, meeting the needs of analyzing ultra-trace antibiotic-like novel pollutants in water. Furthermore, by using organic solvents to reconstitute the target substance enriched through solid-phase extraction, followed by ELISA detection, the detection limit for sulfamethoxazole in water samples can reach the ng / L level.

[0193] The above description is merely a specific embodiment of the invention covered by this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. Methods for detecting novel antibiotic pollutants in water samples by combining solid-phase extraction and enzyme-linked immunosorbent assay (ELISA), including: (a) Select an organic solvent such that the R value of the standard curve obtained by enzyme-linked immunosorbent assay (ELISA) containing the standard of the novel antibiotic contaminant and an aqueous solution of the organic solvent is equal to that obtained by ELISA. 2 Greater than 0.98; (b) Using the quantitative detection range, sensitivity, limit of detection, and precision of enzyme-linked immunosorbent assay (ELISA) as evaluation indicators, and combining this with the CRITIC weighting method, determine the optimal concentration of the organic solvent selected in step (a); and (c) Prepare a reconstituted aqueous solution by mixing the organic solvent selected in step (a), the antibiotic-like new pollutant enriched by solid-phase extraction of the water sample, and detect the reconstituted aqueous solution by enzyme-linked immunosorbent assay (ELISA) to determine the concentration of the antibiotic-like new pollutant in the water sample, wherein the concentration of the organic solvent in the reconstituted aqueous solution is the optimal concentration.

2. The method according to claim 1, wherein, Step (b) includes: (i) Prepare standard aqueous solutions containing the new antibiotic contaminant and the organic solvent selected in step (a) with a dual concentration gradient, wherein a set of standard aqueous solutions containing the gradient concentrations of the standard is prepared for each concentration of the organic solvent; (ii) Perform enzyme-linked immunosorbent assay (ELISA) on each group of standard aqueous solutions to obtain standard curves corresponding to each concentration of the organic solvent, and calculate the quantitative detection range, sensitivity, limit of detection, and precision values ​​corresponding to each standard curve; and (iii) Using the normalized dimensionless values ​​of the quantitative detection range, sensitivity, detection limit and precision as evaluation indicators, the CRITIC weighting method is used to weight each evaluation indicator to obtain the weight corresponding to each evaluation indicator. The concentration of the organic solvent in the standard aqueous solution corresponding to the minimum sum of the values ​​of each evaluation indicator multiplied by the corresponding weight is the optimal concentration.

3. The method according to claim 2, wherein step (b) further comprises: Based on the water quality of the water sample, select the range of the ratio of organic solvent to water; as well as Prepare the dual-concentration gradient standard aqueous solution within the stated ratio range. Optionally, the ratio of the organic solvent to water is in the range of 1-50% by volume of the organic solvent. Optionally, for clean water bodies such as drinking water, surface water, groundwater, seawater, atmospheric water and glacial water, the ratio of the organic solvent to water is in the range of 1-10% by volume of the organic solvent. Optionally, for polluted water bodies such as domestic sewage, industrial wastewater, agricultural wastewater and medical wastewater, the ratio of the organic solvent to water is in the range of 10-50% by volume of the organic solvent. Optionally, the detection limit of the method for the new antibiotic pollutant in the water sample is greater than 0 to 10 ng / L, further 0.1-5 ng / L, and even further 0.2-5 ng / L; Optionally, the solid-phase extraction process is achieved through microfluidics.

4. The method according to claim 2 or 3, wherein the standard curve fitting is based on the following equation (1): In equation (1), x is the concentration of the novel antibiotic pollutant; y is the percentage absorbance value corresponding to x; A1 is the upper asymptote estimate; A2 is the lower asymptote estimate; x0 is the half-maximum inhibitory concentration (IC50). 50 ; and p is the slope of the standard curve at the position corresponding to x0.

5. The method of claim 4, wherein the sensitivity is determined by the upper half-inhibition concentration (IC50) of the standard curve. 50 The slope at the location of ) indicates, Optionally, the normalized dimensionless value of the sensitivity is calculated by the following equation (2): In equation (2), k represents the upper half-maximal inhibitory concentration (IC) of the standard curve. 50 The reciprocal of the slope at the location of the value; mean(k) is the average value of k corresponding to each standard curve; min(k) is the minimum value of k corresponding to each standard curve; max(k) is the maximum value of k corresponding to each standard curve; k′ represents the normalized dimensionless value of the sensitivity.

6. The method according to claim 4 or 5, wherein The normalized dimensionless value of the quantitative detection interval is calculated using the following equations (3)-(6). △x=x2-x1 (5) In equations (3)-(6), x1 is the left endpoint of the quantitative detection interval; x2 is the right endpoint of the quantitative detection interval; Y 80 It is 80%; Y 20 It is 20%; △x represents the width of the quantitative detection interval; mean(△x) is the average value of △x corresponding to each standard curve; min(△x) is the minimum value of △x corresponding to each standard curve; max(△x) is the maximum value of △x corresponding to each standard curve; △x′ represents the normalized dimensionless value of the quantitative detection interval; and x0 and p are the same as defined in claim 4; and / or The normalized dimensionless value of the detection limit is calculated by the following equations (7)-(10): In equations (7)-(10), A LOD A is the percentage absorbance value corresponding to the detection limit; average It is the average percentage absorbance value obtained by repeated measurements of a standard aqueous solution of the aforementioned antibiotic-type new pollutant at a concentration of 0; 3s represents 3 times the standard deviation; Y LOD x represents the binding rate corresponding to the detection limit; LOD Indicates the detection limit; x LOD ′ represents the normalized dimensionless value of the detection limit; mean(xLOD) represents the x corresponding to each standard curve. LOD The average value; min(x) LOD ) represents the x corresponding to each standard curve LOD The minimum value in; max(x) LOD ) represents the x corresponding to each standard curve LOD The maximum value in; and A1, A2, x0, and p are the same as those defined in claim 4; and / or The normalized dimensionless value of the precision is calculated by the following equations (11) and (12): In equations (11) and (12), n represents the number of times the standard aqueous solution of each gradient concentration of the novel antibiotic contaminant is repeatedly measured; x i This represents the concentration obtained from the i-th measurement; The value represents the average concentration obtained from n measurements; S represents the standard deviation; RSD represents the precision; RSD′ represents the normalized dimensionless value of the precision; mean(RSD) represents the average RSD of each standard curve; min(RSD) represents the minimum RSD of each standard curve; and max(RSD) represents the maximum RSD of each standard curve.

7. The method according to any one of claims 4-6, wherein the weighting process using the CRITIC weighting method comprises: (1) Form an m-row, 4-column matrix X of the normalized dimensionless values ​​of the quantitative detection range, the sensitivity, the detection limit, and the precision, where m is the number of concentration gradients of the optimal organic solvent; (2) Calculate the objective weights corresponding to each evaluation index according to the following equations (13)-(19): In equations (13)-(19), x ij This represents the value of the evaluation index in the i-th row and j-th column of the matrix X; It is the average value of the evaluation indicators in the i-th row; S is the average value of the evaluation indicators in column j; j μ is the standard deviation of the evaluation index in column j, representing the index variability; i μ is the standard deviation of the values ​​in the i-th row; j It is the standard deviation of the values ​​in the j-th column; cov(x i ,x j ) represents the covariance of the matrix X; r ij R represents the correlation coefficient between the i-th row and j-th column of matrix X; j Indicates the conflict of indicators in column j; C j This represents the information content of the evaluation indicators in column j; and W j This represents the objective weight of the evaluation index in column j; as well as (3) Multiply the four evaluation indicators of each row of the matrix X by the corresponding objective weights, and then sum them up. The gradient concentration of the optimal organic solvent corresponding to the minimum sum is taken as the optimal concentration.

8. The method according to any one of claims 1-7, wherein The novel antibiotic contaminants are selected from 3-methylquinoline-2-carboxylic acid, avermectin, albendazole, amikacin, amoxicillin, ampicillin, azithromycin, penicillin, β-lactam antibiotics, cephalexin, cefquinoxime, chloramphenicol, chlorpromazine, chlortetracycline, ciprofloxacin, colistin, doramectin, doxycycline, enrofloxacin, erythromycin, florfenicol, fluoroquinolones, furazolidone, gentamicin, isoniazid, kanamycin, lincomycin, and melamine. Amines, metronidazole, natamycin, nitrofurantoin, nitrofurantoin, nitrofurazone, oxytetracycline, piracetam, prostaglandins, quinolones, ribavirin, sarafloxacin, zithromycin, streptomycin, sulfadiazine, sulfamethoxazole, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfanilides, sulfaquinoxaline, sulfonamides, tetracyclines, thiamphenicol, tilmicosin, trimethoprim, tylosin, and vancomycin; and / or The organic solvent is selected from methanol, acetonitrile, and dimethyl sulfoxide, and optionally, the organic solvent is methanol.

9. Optimize the enzyme-linked immunosorbent assay (ELISA) method, including: Screening for the optimal concentration of the organic solvent in an aqueous solution containing the target substance and the organic solvent to be detected by enzyme-linked immunosorbent assay (ELISA) includes: The optimal concentration was determined using the quantitative detection range, sensitivity, limit of detection, and precision of enzyme-linked immunosorbent assay (ELISA) as evaluation indicators, combined with the CRITIC weighting method.

10. The method of claim 9, comprising: (i) Prepare standard aqueous solutions containing a standard of the target substance and the organic solvent in a dual concentration gradient, wherein a set of standard aqueous solutions containing the standard of the gradient concentration is prepared for each concentration of the organic solvent; (ii) Perform enzyme-linked immunosorbent assay (ELISA) on each group of standard aqueous solutions to obtain standard curves corresponding to each concentration of the organic solvent, and calculate the quantitative detection range, sensitivity, limit of detection, and precision values ​​corresponding to each standard curve; and (iii) Using the normalized dimensionless values ​​of the quantitative detection range, sensitivity, detection limit and precision as evaluation indicators, the CRITIC weighting method is used to weight each evaluation indicator to obtain the weight corresponding to each evaluation indicator. The concentration of the organic solvent in the standard aqueous solution corresponding to the minimum sum of the values ​​of each evaluation indicator multiplied by the corresponding weight is the optimal concentration.

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