Small molecule compound detection method and application thereof
By combining magnetic nanozyme enrichment with MetaSPR chip and nanozyme catalytic colorimetric method, a dual-mode detection method is developed, which solves the problems of high cost and complex operation of existing small molecule compound detection equipment. This method enables rapid, simple and accurate quantitative detection of small molecule compounds and is suitable for rapid screening of food safety.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-27
- Publication Date
- 2026-03-31
AI Technical Summary
Existing methods for detecting small molecule compounds suffer from problems such as expensive equipment, complex operation, high cost, low sensitivity, and high false positive rate. Furthermore, existing dual-mode detection methods are not applicable to small molecule detection and require multiple devices.
A dual-mode detection method based on magnetic nanozymes was adopted, combining MetaSPR chip and nanozyme catalytic colorimetry. Small molecule compounds were enriched by magnetic nanozymes, and the concentration of small molecules was calculated by using MetaSPR chip to detect the difference in optical density and nanozyme catalytic colorimetry, combined with standard curve equation.
It enables rapid, simple, and accurate quantitative detection of small molecule compounds, lowers the detection limit, and improves the sensitivity and accuracy of detection, making it suitable for rapid screening of food safety.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of detection technology, and more specifically, to a method for detecting small molecule compounds and its application. Background Technology
[0002] Small molecule drug residues (such as pesticide residues, veterinary drug residues, hormones, and illegal additives) in food involve safety issues, so it is necessary to detect and monitor their content. The standard methods for detection are generally liquid chromatography, gas chromatography, and liquid chromatography-mass spectrometry. Although these methods have good stability, high accuracy, and strong repeatability, they require expensive instruments and equipment, professional operators, and complex pretreatment processes. They are also not environmentally friendly and cannot meet the requirements of timely detection and on-site results.
[0003] Among rapid detection methods, the immunochromatographic strip rapid detection method is widely used. Although it is simple, it has a high detection limit, generally only suitable for qualitative detection, and has a relatively high false positive or false negative rate. Other rapid detection methods, such as ELISA, require multiple steps such as incubation, washing, and color development, which are somewhat cumbersome. Furthermore, the HRP enzyme used is a natural enzyme, which is expensive and easily inactivated.
[0004] Dual-mode sensor detection methods, employing two detection approaches, offer advantages such as self-verification, reduced false readings, and improved detection reliability, and have recently garnered widespread attention. However, current dual-mode immunosensors are primarily used in the biomedical field, employing a sandwich detection method that cannot detect small molecules. Secondly, probe preparation is overly complex, requiring the embedding of carbon quantum dots containing fluorescent signals and BSA-AuNPs capable of generating SPR signals. This cumbersome process reduces operability and increases detection errors. Finally, the two detection methods in existing dual-mode detection differ significantly, requiring the use of different instruments, which also hinders application.
[0005] Therefore, further research is needed on detection methods for small molecules. Summary of the Invention
[0006] One of the objectives of this invention is to provide a simpler and more accurate method for detecting small molecule substances.
[0007] This invention provides a method for detecting small molecule compounds, comprising: (1) Sample pretreatment: The sample to be tested is extracted with the small molecule compound to be tested as the enrichment target to obtain the test solution; (2) The small molecule compound antibody to be tested is coupled onto the magnetic nanozyme to obtain the antibody-coated magnetic nanozyme; the magnetic nanozyme is a magnetic material with peroxidase-like catalytic effect; (3) The test solution is mixed with the magnetic nanozyme coated with antibody and then magnetically separated to obtain the test particles; the test particles are reconstituted to obtain the test particle solution. (4) MetaSPR detection: In the detection well of the MetaSPR chip, the optical density of the blank detection liquid at a specific wavelength is detected as the starting optical density. In the detection aperture of the MetaSPR chip, the optical density of the test particle solution after incubation at a specific wavelength is detected as the endpoint optical density. The difference between the starting and ending optical densities is substituted into the standard curve equation to calculate the concentration of the small molecule compound to be tested in the sample. The MetaSPR chip detection wells are coated with haptens of small molecule compounds to be tested. The standard curve equation is obtained by fitting the optical density difference obtained from the detection gradient concentration of the standard solution of the small molecule compound to be tested. (5) Nanozyme-catalyzed colorimetric detection: After the endpoint optical density of the test particle solution is measured, the solution is mixed with TMB colorimetric solution and incubated until the incubation is terminated, and the optical density value is measured. The optical density value is substituted into the standard curve equation two to calculate the concentration value of the small molecule compound to be tested in the sample. The second standard curve equation is obtained by fitting the optical density value of the solution after the endpoint optical density is detected using the standard solution of the small molecule compound with the detection gradient concentration. (6) The average of concentration value one and concentration value two is taken as the final concentration result.
[0008] This invention provides a rapid quantitative detection method for small molecules based on a colorimetric and MetaSPR dual-mode detection method using transition metal oxide nanozymes. First, the target small molecule compound is extracted from the sample using reagents. Then, an antibody-coated magnetic nanozyme is used to specifically adsorb and enrich the target small molecule compound, followed by magnetic separation. After reconstitution, the nanozyme is added to the detection wells of a MetaSPR chip. After shaking and incubation, the optical density is measured using a microplate reader. By comparing the optical density with that of the initial blank detection solution and using a standard curve equation, the concentration of the target small molecule compound can be obtained as follows: Next, the free magnetic nanozyme (with the target small molecule compound bound to its antibody) is transferred from the detection wells and added to a TMB chromogenic solution. After incubation and color development, a stop solution is added, and the sample is detected using a microplate reader to obtain the optical density value. This value, combined with the standard curve equation, yields the second concentration value of the target small molecule compound. The two concentration values can be compared and verified to obtain a more accurate detection result.
[0009] The dual-mode detection method of the present invention is simple to operate, has high accuracy, low detection limit, stable performance, and high detection throughput, and is suitable for rapid screening of food safety.
[0010] In this invention, small molecule substances refer to compounds with a single, clearly defined active ingredient and a molecular weight of less than 1,000 Daltons. The method of this invention is preferably used to detect small molecule drugs, so as to realize the detection of drug residues (such as pesticide residues, veterinary drug residues, illegal additives, etc.) in the test substance (such as food), providing strong technical support for food safety production and grassroots supervision.
[0011] In this invention, standard curve equation one and standard curve equation two are obtained by importing the test results of the standard into the ELISA Calc software and fitting them through the Logistic curve fitting (four-parameter) operation.
[0012] In the detection method of the present invention, the magnetic nanozyme is a magnetic metal oxide material, preferably a ferrooxy group (such as Fe3O4) material, a doped iron oxide (such as Co-Fe3O4) material or other magnetic metal oxide (such as NiFe2O4) material, and more preferably Fe3O4 nanoparticles.
[0013] The magnetic nanozyme of the present invention can be synthesized according to conventional techniques in the art, including hydrothermal synthesis, microwave-assisted synthesis, thermal decomposition, etc.
[0014] In the detection method of the present invention, the antibody to be tested is achieved by carboxylating or amylating the magnetic nanozyme.
[0015] Specifically, magnetic nanozymes can be functionalized by carboxylation or amination, and then coupled with small molecule antibody compounds using methods known in the art.
[0016] In step (2) of the detection method of the present invention, when the antibody of the small molecule compound to be tested is coupled to the magnetic nanozyme, the concentration of the magnetic nanozyme is 0.5-5 mg / mL, preferably 1 mg / mL; the concentration of the antibody is 10-50 μg / mL, preferably 20 μg / mL.
[0017] In step (3) of the detection method of the present invention, the volume ratio of the test solution to the magnetic nanozyme coated with antibody is 0.5:(0.01-0.015); the concentration of the test particle solution obtained after reconstitution is 0.10~0.15 mg / mL, preferably 0.12 mg / mL.
[0018] In the detection method of the present invention, the amount of hapten of the small molecule compound to be tested coated in the detection well of the MetaSPR chip is 0.02~0.1 μg / well, preferably 0.04 μg / well.
[0019] In step (4) of the detection method of the present invention, the incubation temperature is 37±1℃ and the time is 10-20 min, preferably 15 min; In step (5), the incubation temperature is 37±1℃ and the incubation time is 8-15 min, preferably 10 min.
[0020] In a specific application, the detection method of the present invention uses chloramphenicol as the small molecule compound to be tested and aquatic products as the sample to be tested.
[0021] In step (1), the sample to be tested is first extracted with acetonitrile containing sodium chloride, the supernatant is collected and then the impurities are extracted with n-hexane. Finally, the acetonitrile layer extracted with n-hexane is collected, filtered, dried and reconstituted to obtain the solution to be tested. In step (2), the antibody to be tested is conjugated by carboxylating Fe3O4 nanoparticles; In steps (1) and (3), the reconstitution was performed using a 20 mmol / L Tris-HCl solution with pH 8.3 containing 1% NaCl; In step (4), the specific wavelength is 575nm and / or 595nm; In step (5), the detection wavelength for detecting the optical density value is 450 nm.
[0022] When detecting chloramphenicol, in step (4), at least one of the wavelengths 575nm and 595nm can be used. That is, the content of chloramphenicol can be calculated by the difference in optical density under a single wavelength, or the content of chloramphenicol can be calculated by combining the difference in optical density of two wavelengths (ΔOD595-ΔOD575).
[0023] The present invention also provides the application of the above detection method in the detection of small molecule compounds in food; preferably, the small molecule compound is a small molecule drug residue.
[0024] The beneficial effects of this invention are at least as follows: ① This invention employs a dual-mode detection method, which allows for comparison and verification between the two methods, thereby improving the accuracy of the detection results.
[0025] ② The magnetic nanozyme used in this invention is easy to synthesize, and the process of preparing the MetaSPR detection well (coating hapten) is also very simple, making it suitable for mass production.
[0026] ③ The detection process of the two methods in the dual-mode detection method is relatively simple. Only an ELISA reader is needed to complete the two detection processes. Moreover, the detection throughput is high, making it suitable for large-scale on-site screening.
[0027] ④ The two detection methods in the dual-mode detection method have high sensitivity, low detection limit and good specificity, and are easy to use. Attached Figure Description
[0028] Figure 1 This is a particle size distribution diagram of magnetic nanoparticles.
[0029] Figure 2 This is a zeta potential distribution diagram of carboxylated magnetic nanoparticles.
[0030] Figure 3 This is a schematic diagram of the dual-mode sensing principle.
[0031] Figure 4 The standard curve equation for the MetaSPR method in Example 2 is shown.
[0032] Figure 5 The equation for the standard curve of the nanozyme catalytic colorimetric method in Example 2 is shown.
[0033] Figure 6 This is the specific detection result of the MetaSPR detection method in Example 3.
[0034] Figure 7 The results are the specific detection results of the nanozyme catalytic colorimetric method in Example 3.
[0035] Figure 8 The standard curve equation for the MetaSPR method in Example 3 is shown.
[0036] Figure 9 The equation for the standard curve of the nanozyme catalytic colorimetric method in Example 3 is shown.
[0037] Figure 10 The standard curve equation for the MetaSPR method in Example 5 is shown.
[0038] Figure 11 The equation for the standard curve of the nanozyme catalytic colorimetric method in Example 5 is shown. Detailed Implementation
[0039] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.
[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.
[0041] This invention describes the specific process of detecting small molecules in food using the method described in this invention, taking the detection of chloramphenicol drug residues in aquatic products as an example. The MetaSPR detection equipment was purchased from Liangzhun (Wuhan) Life Science Technology Co., Ltd., model: WeSPR100.
[0042] In this paper, data is processed according to the "round half to even" rounding rule.
[0043] Example 1 In this embodiment, carboxylated Fe3O4 nanoparticles (i.e., magnetic nanoparticles, magnetic nanozymes) were prepared and coated with chloramphenicol antibody; MetaSPR detection wells were prepared.
[0044] The preparation method of carboxylated Fe3O4 nanoparticles is as follows: 6.7 g of ethylene glycol was placed in a small beaker, and 0.06 g of sodium citrate was added and dissolved under magnetic stirring; 0.135 g of ground ferric chloride powder was added and stirred until dissolved; 0.4 g of sodium acetate was added and stirred until dissolved; the solution was rapidly heated (by microwave heating) to 240 °C and reacted for 1 h under stirring at a stirring rate of 900 rpm; after the reaction was completed, the black product was collected with a magnet, washed three times alternately with ultrapure water and ethanol, and finally reconstituted with ultrapure water; 1 mL of the reconstituted solution was taken, dried, and weighed, and the product solution was adjusted to 1 mg / mL for use in coating antibodies.
[0045] The particle size and zeta potential of the prepared carboxylated Fe3O4 nanoparticles were measured as follows: Figure 1 and Figure 2 As shown, the nanoparticles have a particle size of approximately 170 nm and a Zeta potential of -44.41 mV, confirming the successful preparation of carboxylated magnetic nanoparticles.
[0046] The preparation method of chloramphenicol antibody-coated magnetic nanoparticles is as follows: Take 1 mL of magnetic nanoparticles prepared in the previous step with a concentration of 1 mg / mL, wash twice with 50 mmol / L MES 5.5 buffer solution, and reconstitute to 1 mL; add EDC to a final concentration of 10 mmol / L, shake at room temperature for 10 min, and then add NHS to a final concentration of 5 mmol / L for 20 min for activation; magnetically separate and wash twice with 10 mmol / L PBS 7.4 and reconstitute to 1 mL; add 20 μg of chloramphenicol antibody (purchased from Wuxi Ditengmin Biotechnology Co., Ltd.), and incubate at room temperature for 2 h; add ethanolamine solution to a final concentration of 30 mmol / L, and incubate for 1 h; wash twice with 10 mmol / L PBS 7.4 and reconstitute to 1 mL, ready for use.
[0047] The preparation steps for the MetaSPR detection wells are as follows: Chloramphenicol hapten (purchased from Wuxi Ditengmin Biotechnology Co., Ltd.) was diluted to 20 μg / mL with 10 mmol / L PBS 7.4; 2 μL of small molecule antigen was added to each well of the MetaSPR detection plate after washing and drying with ultrapure water, and the antigen was dropped into the center of the well; the plate was covered and incubated overnight at 4°C; 150 μL of blocking buffer (10 mmol / L PBS 7.4, containing 1% BSA, 3% sucrose, and 0.1% Tween) was added to each well, the plate was covered, and the plate was stored at 37°C for 1 hour; the blocking buffer was discarded, the plate was removed, and the plate was dried at 37°C, then vacuum-sealed and stored at 4°C for later use.
[0048] Example 2 In this embodiment, the chloramphenicol antibody-coated magnetic nanoparticles prepared in Example 1, the MetaSPR detection wells, and other relevant reagents were used to plot the standard curve and detect actual samples. A schematic diagram is shown below. Figure 3 The specific steps are as follows: Sample pretreatment: Take 5 g of homogenized fish meat, add 1 g of sodium chloride and 5 mL of acetonitrile, vortex extract for 2 min, centrifuge, and transfer the supernatant to a 15 mL centrifuge tube; add 5 mL of n-hexane, vortex for 1 min, centrifuge and discard the supernatant, repeat the n-hexane treatment once; filter the acetonitrile layer solution through a membrane (pore size 0.45 μm), take 0.5 mL of nitrogen gas to dry it, redissolve it with 0.5 mL of Tris-HCl detection solution (20 mmol / L, pH 8.3, containing 1% NaCl), add 12 μL of chloramphenicol antibody-coated magnetic nanoparticles prepared in Example 1, and shake thoroughly; after magnetic separation, redissolve it with 100 μL of Tris-HCl detection solution, ready for testing.
[0049] MetaSPR detection of standard solutions and actual samples: Take 0.5 mL of chloramphenicol standard solutions with gradient concentrations (0, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4 ng / mL), add 12 μL of chloramphenicol antibody-coated magnetic nanoparticles prepared in Example 1, shake thoroughly, and after magnetic separation, reconstitute with 100 μL of Tris-HCl detection solution; add 50 μL of Tris-HCl blank detection solution to the MetaSPR detection well, scan the start point, and detect wavelengths of 575 nm and 595 nm, respectively; completely pour out the blank detection solution in the detection well, and then transfer 50 μL of gradient standard detection solution and sample detection solution to the well, respectively. After shaking and incubating at 37℃ for 15 min, scan the endpoint; calculate the difference between the endpoint and the start point (optical density change value) at the two detection wavelengths, and use the optical density change value at 595 nm – The change in optical density at 575 nm was used as the signal value for the MetaSPR detection method, and a fitting equation was plotted between the standard solution concentration (x value) and the signal value (y value) (obtained by importing the results into ELISA Calc software and fitting the logistic curve (four-parameter)). ; Where -0.00065 represents the signal value when the chloramphenicol concentration approaches infinity, 0.15037 represents the signal difference between chloramphenicol concentrations approaching 0 and approaching infinity, 1.63265 is the slope factor, and 0.23759 is the half-maximal effect concentration. The correlation coefficient R0 2 The value is 0.995. Substituting the signal value of the actual sample into the equation, the chloramphenicol content in the sample is calculated. The standard curve equation is shown below. Figure 4 .
[0050] The nanozyme-catalyzed colorimetric detection process for standard solutions and actual samples is as follows: 100 μL TMB single-component colorimetric solution was added to a 96-well plate, and then the reaction-completed detection solution was transferred from each MetaSPR detection well; the plate was incubated at 37℃ for 10 min, 50 μL of stop solution was added, and the detection wavelength was selected as 450 nm for instrument detection; the fitting equation between the standard solution concentration (x value) and the optical density value (y value) was plotted (obtained by importing the results into ELISA Calc software and fitting the logistic curve (four-parameter)): ; Where 0.56847 represents the signal value when the chloramphenicol concentration approaches 0, 0.61552 represents the signal difference between the chloramphenicol concentrations approaching infinity and approaching 0, -0.95279 is the slope factor, and 0.30350 is the half-maximal effect concentration. The correlation coefficient R0 2The value is 0.997. Substituting the actual sample signal value into the equation, the chloramphenicol content in the sample is calculated. The standard curve equation is shown below. Figure 5 .
[0051] The average value of the chloramphenicol content obtained by the two methods was taken as the final result.
[0052] Example 3 In this embodiment, the detection limit, specificity, reproducibility, stability, spiked recovery rate, and consistency of detection results of the two methods were examined.
[0053] MetaSPR detection limit and specificity test: The mean signal of 11 blank measurements was 0.1478. Subtracting three times the blank standard deviation, the value was 0.1446. Substituting this into the standard curve equation in Example 2, the detection limit was found to be 0.06 ng / mL. The blank solution, chloramphenicol solution at a concentration of 2 ng / mL, and sulfonamide, quinolone, and nitrofuran solutions at a concentration of 20 ng / mL were tested according to the method in Example 2. The signal responses are as follows: Figure 6 As shown, the detection method still has good specificity even when the concentration of other small molecules is 10 times that of chloramphenicol.
[0054] Detection limit and specificity test of nanozyme-catalyzed colorimetric method: The mean signal of 11 blank measurements was 0.5734. Adding three times the standard deviation of the blank, the value was 0.6711. Substituting this into the standard curve equation in Example 2, the detection limit was found to be 0.056 ng / mL. The blank solution, chloramphenicol solution at a concentration of 2 ng / mL, and sulfonamide, quinolone, and nitrofuran solutions at a concentration of 20 ng / mL were tested according to the method in Example 2. The signal responses are as follows: Figure 7 As shown, this demonstrates that the nanozyme catalytic colorimetric method also has good specificity.
[0055] The spiking recovery experiment of chloramphenicol in blank fish samples was conducted according to the method in Example 2. Two concentrations were spiked, and each concentration was measured six times using both methods. The results are shown in Table 1. The relative standard deviations of the detection results were all ≤5.4%, indicating good precision and good method stability. In addition, the recoveries of both methods were between 90% and 105%, and the detection results showed good consistency.
[0056] Table 1. Spike recovery experiment of blank samples This embodiment further simulates the detection performance of the detection chip and reagents after one year of storage at 4°C through a high-temperature storage accelerated aging test (stored at 37°C for 6 days). The standard curves of the two detection methods of this invention are shown below. Figure 8 (MetaSPR method) and Figure 9As shown in the (catalytic colorimetric method), the signal values of the two methods did not decrease significantly, and the correlation coefficient R... 2 A value ≥0.994 indicates that both methods have reliable stability.
[0057] Example 4 The detection effects of the present invention were compared with those of commercially available reagent kits, and the specific results are shown in Table 2 below.
[0058] Table 2 As shown in Table 2, this invention exhibits a lower limit of detection and a wider detection range, with significantly better correlation coefficients and spiked recoveries compared to other methods. Compared to immunochromatography, this invention achieves accurate quantification and high throughput. ELISA uses horseradish peroxidase, which is easily inactivated, while the nanozyme used in this invention is more stable. Furthermore, ELISA requires incubation, washing, and color development, making the detection process cumbersome; this invention only requires extraction and sample loading to complete the detection. Moreover, the two methods can be mutually verified, improving the accuracy of the detection results.
[0059] Example 5 In this embodiment, the method described in Example 1 was used to prepare magnetic nanoparticles coated with enrofloxacin antibody (purchased from Wuxi Ditengmin Biotechnology Co., Ltd.), MetaSPR detection wells (enrofloxacin hapten purchased from Wuxi Ditengmin Biotechnology Co., Ltd.), and other related reagents. Then, the duck meat sample pretreatment and detection method were constructed according to the description in Example 2, and the spiked recovery experiment was performed according to the method in Example 3.
[0060] Take 0.5 mL of enrofloxacin standard solutions with gradient concentrations (0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 ng / mL) and sample processing solutions, and obtain detection signals according to the detection process in Example 2. Plot a fitting equation between the standard solution concentration (x value) and the signal value (y value): ; Correlation coefficient R 2 The value is 0.993. Substituting the signal value of the actual sample into the equation, the enrofloxacin content in the sample is calculated. The standard curve equation is shown below. Figure 10 .
[0061] The fitting equation for the standard solution concentration (x value) and optical density value (y value) in the catalytic colorimetric method: ; Correlation coefficient R 2 The value is 0.995. Substituting the signal value of the actual sample into the equation, the enrofloxacin content in the sample is calculated. The standard curve equation is shown below. Figure 11 .
[0062] The average value of the enrofloxacin content obtained by the two methods was taken as the final result.
[0063] The results of the spiked recovery experiments on actual samples are shown in Table 3: Table 3 From Table 3 and Figure 10 , Figure 11 It is evident that this method still exhibits good detection performance for enrofloxacin in duck meat, with relative standard deviations of ≤4.3% between the results and those obtained using the national standard method. Furthermore, compared to the standard method, this method does not require specialized laboratory equipment and personnel, and its manpower, material, and time costs are relatively low, making it well-suited for large-scale screening and providing a reliable basis for further validation of the standard method.
[0064] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A method for detecting small molecule compounds, characterized in that, include: (1) Sample pretreatment: The sample to be tested is extracted with the small molecule compound to be tested as the enrichment target to obtain the test solution; (2) The small molecule compound antibody to be tested is coupled onto the magnetic nanozyme to obtain the antibody-coated magnetic nanozyme; the magnetic nanozyme is a magnetic material with peroxidase-like catalytic effect; (3) The test solution is mixed with the magnetic nanozyme coated with antibody and then magnetically separated to obtain the test particles; the test particles are reconstituted to obtain the test particle solution. (4) MetaSPR detection: In the detection well of the MetaSPR chip, the optical density of the blank detection liquid at a specific wavelength is detected as the starting optical density. In the detection aperture of the MetaSPR chip, the optical density of the test particle solution after incubation at a specific wavelength is detected as the endpoint optical density. The difference between the starting and ending optical densities is substituted into the standard curve equation to calculate the concentration of the small molecule compound to be tested in the sample. The MetaSPR chip detection wells are coated with haptens of small molecule compounds to be tested. The standard curve equation is obtained by fitting the optical density difference obtained from the detection gradient concentration of the standard solution of the small molecule compound to be tested. (5) Nanozyme-catalyzed colorimetric detection: After the endpoint optical density of the test particle solution is measured, the solution is mixed with TMB colorimetric solution and incubated until the incubation is terminated, and the optical density value is measured. The optical density value is substituted into the standard curve equation two to calculate the concentration value of the small molecule compound to be tested in the sample. The second standard curve equation is obtained by fitting the optical density value of the solution after the endpoint optical density is detected using the standard solution of the small molecule compound with the detection gradient concentration. (6) The average of concentration value one and concentration value two is taken as the final concentration result.
2. The detection method according to claim 1, characterized in that, The magnetic nanoenzyme is a magnetic metal oxide material, preferably a ferrooxide material or a doped ferrooxide material, and more preferably Fe3O4 nanoparticles.
3. The detection method according to claim 1 or 2, characterized in that, The conjugation of small molecule compounds to be tested to antibodies can be achieved by carboxylating or amylating magnetic nanozymes.
4. The detection method according to any one of claims 1-3, characterized in that, In step (2), when the antibody to be tested is coupled onto the magnetic nanozyme, the concentration of the magnetic nanozyme is 0.5-5 mg / mL, preferably 1 mg / mL; the concentration of the antibody is 10-50 μg / mL, preferably 20 μg / mL.
5. The detection method according to any one of claims 1-4, characterized in that, In step (3), the volume ratio of the test solution to the magnetic nanozyme coated with antibody is 0.5:(0.01-0.015); the concentration of the test particle solution obtained after reconstitution is 0.10~0.15 mg / mL, preferably 0.12 mg / mL.
6. The detection method according to any one of claims 1-5, characterized in that, The amount of hapten of the small molecule compound to be tested coated in the detection well of the MetaSPR chip is 0.02~0.1 μg / well, preferably 0.04 μg / well.
7. The detection method according to any one of claims 1-6, characterized in that, In step (4), the incubation temperature is 37±1℃ and the incubation time is 10-20 min, preferably 15 min; In step (5), the incubation temperature is 37±1℃ and the incubation time is 8-15 min, preferably 10 min.
8. The detection method according to any one of claims 1-7, characterized in that, The small molecule compound to be tested is chloramphenicol; the sample to be tested is aquatic product.
9. The detection method according to claim 8, characterized in that, In step (1), the sample to be tested is first extracted with acetonitrile containing sodium chloride, the supernatant is collected and then the impurities are extracted with n-hexane. Finally, the acetonitrile layer after n-hexane extraction is collected, filtered, dried and reconstituted to obtain the solution to be tested. In step (2), the antibody to be tested is conjugated by carboxylating Fe3O4 nanoparticles; In steps (1) and (3), the reconstitution was performed using a 20 mmol / L Tris-HCl solution with pH 8.3 containing 1% NaCl; In step (4), the specific wavelength is 575nm and / or 595nm; In step (5), the detection wavelength for detecting the optical density value is 450 nm.
10. The application of the detection method according to any one of claims 1-9 in the detection of small molecule compounds in food; preferably, the small molecule compound is a small molecule drug residue.