CN110596378A - Multichannel universal chromatography method for detecting small molecules, test strip and kit - Google Patents
Multichannel universal chromatography method for detecting small molecules, test strip and kit Download PDFInfo
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Abstract
The invention belongs to the field of small molecule detection, and relates to a multi-channel universal chromatography method for detecting small molecules, a test strip and a kit. The method comprises the following steps: (1) co-incubating the substance to be detected, the various labeled antibodies and the various complexes to obtain a mixture; wherein each labeled antibody is a specific antibody of a target small molecule and is labeled with a signal substance; each complex is formed by a target small molecule and a recognition element; (2) passing the mixture through a detection zone and a quality control zone in sequence; wherein, the detection area is a plurality of detection areas, and each detection area is fixedly provided with a specific antibody of a recognition element; a secondary antibody of the labeled antibody is fixed on the quality control region; (3) and judging the content of the target small molecules in the object to be detected according to the signal intensity of the detection area and the quality control area. The invention realizes the organic fusion of sensitivity, specificity, universality and high flux of immune reaction.
Description
Technical Field
The invention belongs to the field of small molecule detection, and particularly relates to a multi-channel universal chromatography method for detecting small molecules, a small molecule detection test strip and a small molecule detection kit.
Background
In the field of small molecule detection, the principle of immunochromatographic test paper based on the principle of chromatographic separation is to fix specific antigen of an object to be detected to a test area of the test paper in advance. Under the action of capillary force, the small molecules to be detected and the labeled antibody in the sample flow to the position of the measuring area, and the immobilized antigen and the substance to be detected in the solution compete to bind with the labeled antibody in the solution. The signal intensity of the measuring area is inversely proportional to the content of the small molecules to be measured. Furthermore, the multi-channel immunochromatography detection is mostly realized by arranging a plurality of target substance specificity detection areas on the chromatography test paper.
The chromatographic detection method is more and more widely applied in the fields of food safety, clinical medical treatment and the like because of rapid determination, low cost, intuitive signal response and easy field use. However, the test paper has certain technical defects: 1. the antigen immobilization of the substance to be detected is not beneficial to the full exposure of the binding site and the full progress of the competitive binding reaction, thereby limiting the detection sensitivity. 2. Competitive binding reaction in the detection area of the chromatographic test paper has many influence conditions, is difficult to accurately control and is not beneficial to exerting the detection sensitivity. 3. In multi-channel detection, one detection area corresponds to a specific target substance, and the detection has no wide universality.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a multi-channel universal chromatography method for detecting small molecules, a test strip and a kit aiming at the defects of the existing immunoassay products. The detection method is sensitive in detection, can realize the fusion of multi-channel and general type, has wide application range, is beneficial to providing a more stable detection platform for big data detection, and has wide market prospect.
In order to achieve the above object, a first aspect of the present invention provides a multi-channel general-purpose chromatography method for detecting small molecules, comprising the steps of:
(1) co-incubating the substance to be detected, the various labeled antibodies and the various complexes to obtain a mixture; wherein each labeled antibody is a specific antibody of a target small molecule and is labeled with a signal substance, and the signal substance of each labeled antibody is the same; each complex is formed by a target small molecule and a recognition element, and the recognition element of each complex is different;
(2) enabling the mixture obtained in the step (1) to sequentially pass through a detection area and a quality control area; wherein, the detection area is a plurality of detection areas, and each detection area is fixedly provided with a specific antibody of a recognition element; a secondary antibody of the labeled antibody is fixed on the quality control region;
(3) and judging the content of the target small molecules in the object to be detected according to the signal intensity of the detection area and the quality control area.
In the method of the present invention, the "plurality" is at least two. The number of the labeled antibody, the complex and the detection area is the same and corresponds to the number of the target small molecules which can be detected.
The existing detection methods of small molecules are various, wherein the immunochromatography detection test strip is generally provided with a recognition binding element of the small molecule to be detected in a sample adding area, and then the small molecule is detected through a subsequent detection area and a subsequent quality control area. In practical application, the problems of detection sensitivity, universality and incapability of fusing multiple channels exist. Research shows that the antigen coated on the binding site of the detection area can not be fully exposed, so that the competitive binding reaction time in the detection area is short and uncontrollable, and the detection sensitivity is difficult to be fully improved. In multi-channel detection, each detection area corresponds to a specific target substance, and universal detection is not realized.
Compared with the existing detection method, the method has the advantages that the substance to be detected, the labeled antibody and the compound are incubated together, and the competitive binding reaction is carried out in an incubation mode, so that the immune recognition reaction is carried out more fully, and the detection sensitivity is higher. Moreover, the incubation condition is optimized according to the corresponding substance reaction, and the controllability of the reaction can be improved. According to the invention, the specific antibody immobilized in the detection area specifically recognizes the recognition element but not the target small molecule, so that the complex obtained by any small molecule which can be modified by the recognition element can be recognized, and the detection has the characteristic of universal type. On the other hand, the invention designs a plurality of detection areas, and can realize multi-channel universal detection. The invention combines the sensitivity of chromatography detection, multi-channel and universality, and has wider detection application range.
In the invention, if the object to be detected contains the target small molecule, the obtained mixture is a mixture of a labeled antibody-target small molecule and a labeled antibody-antigen (the antigen is a compound obtained by modifying the target small molecule by a recognition element such as BSA or OVA or other proteins and also is expressed as the recognition element-target small molecule); if the analyte does not contain the target small molecule, the mixture is only the labeled antibody-antigen. The content of the small molecular substances in the substance to be detected is judged according to the signal intensity of the detection area and the quality control area, so that the method is more accurate and sensitive. The multiple detection areas can realize the simultaneous detection of multiple target objects, namely the multi-channel universal detection. When the content is lower than the detectable value, the object to be detected is determined not to contain the target small molecule. Therefore, the method of the present invention may be either a qualitative or quantitative detection method.
According to the method of the present invention, the signal substance used for labeling the antibody may employ various signal substances conventional in the art, including but not limited to at least one of fluorescent dyes, phosphorescent materials, magnetic nanomaterials, carbon nanomaterials, fluorescent quantum dots, biotin, radioisotopes, electron-dense substances, colloidal gold, and enzymes; preferably, the fluorescent dye includes at least one of Alexa series dyes, aminoacridine, BODIPY fluorescent dyes, fluorescein and its derivatives, and catechol type fluorescent dyes.
According to the method of the present invention, the recognition element may be a universal recognition element having a property of binding to the target small molecule, and the application range may be expanded by using a universal recognition element substance as a binding substance for recognizing the target small molecule. Preferably, the recognition element is at least one of Ovalbumin (OVA), hemocyanin (KLH), Bovine Serum Albumin (BSA), human serum albumin (THY), and chicken ovalbumin (HAS).
The method of the invention is suitable for detecting a plurality of small molecules, preferably, the target small molecules are at least two of sedatives, dyes, pesticides, veterinary drugs, additives and pollutants; more preferably, the target small molecules are at least two of diazepam, malachite green, organophosphorus pesticides, chloramphenicol drugs, tetracycline drugs, melamine and aflatoxin; further preferably, the target small molecules are diazepam and malachite green.
The present invention is not particularly limited to a specific incubation method, and preferably, in the step (1), the incubation temperature is 25 to 40 ℃, preferably 35 to 37 ℃; the incubation time is 5-20 min; the incubation is performed in a buffer, the composition of which comprises: taking 0.04-0.06mol/L phosphate buffer solution as a reference, adding the following substances in percentage by weight: 1.5-2.5% of sucrose, 4-6% of fructose, 0.8-1.2% of PEG and 202.5-3.5% of Tween.
According to the method of the present invention, the concentration and the amount of each substance in the incubation system can be adjusted as needed, and the present invention is not particularly limited thereto. For example, the concentration of each labeled antibody is independently 0.5-5ng/mL and the concentration of each complex is independently 80-120 ng/mL.
The invention provides a small molecule detection test strip, which comprises a sample pad, a water absorption pad and a bottom plate, wherein the bottom plate is fixed with a nitrocellulose membrane; wherein,
the sample pad is used for bearing co-incubation products of an object to be detected, a plurality of labeled antibodies and a plurality of complexes;
each labeled antibody is a specific antibody of a target small molecule and is labeled with a signal substance, and the signal substance of each labeled antibody is the same; each complex is formed by a target small molecule and a recognition element, and the recognition element of each complex is different;
the nitrocellulose membrane is provided with a detection area and a quality control area; the detection area is multiple, and each detection area is fixedly provided with a specific antibody of a recognition element; and a secondary antibody of the labeled antibody is fixed on the quality control region.
The multichannel universal immunochromatographic test paper provided by the invention realizes 'competition' binding reaction by introducing a pre-incubation step, a detection area and a quality control area are positioned on a nitrocellulose membrane pad and are vertically distributed with a test paper long shaft, and the detection area is coated with an identification element antibody capable of identifying an immune complex formed in the pre-incubation step, namely if protein modifying small molecules in antigen is BSA, the detection area antibody is an anti-BSA antibody; if the protein modifying the small molecule in the antigen is OVA, the detection region antibody is an anti-OVA antibody. The quality control region is coated with a secondary antibody corresponding to the labeled antibody, such as a goat anti-mouse secondary antibody.
When an object to be detected is detected, the object to be detected, a labeled antibody and an antigen are incubated, the obtained mixture is added into chromatography test paper, the mixture enters a reaction area through the water absorption force of a water absorption pad, if the object to be detected contains target small molecules, the obtained mixture is labeled antibody-target small molecules and labeled antibody-antigen, the labeled antibody-antigen is combined with an antibody of an anti-recognition element of a detection area, the labeled antibody-target small molecules are combined with a secondary antibody of a corresponding labeled antibody of a quality control area, and the content of the target small molecules in the object to be detected is judged through the signal intensity of the detection area and the signal intensity of the quality control area; if the analyte does not contain the target small molecule, only the labeled antibody-antigen is formed. The test strip for detecting the small molecules provided by the invention provides convenience for detecting the small molecules.
In the invention, the bottom plate can be made of non-water-absorbing material, PVC or other hard materials; the sample pad can be suction filter paper or a glass fiber membrane; the absorbent pad may be absorbent paper.
In addition, in the present invention, the detection region and the quality control region are only used for functional distinction, and the "region" may be in the form of a "line" or a "region" having a certain geometric shape. According to a specific embodiment of the present invention, the detection region and the quality control region are in the form of a detection line and a quality control line.
According to the present invention, the labeled antibody, the recognition element, the target small molecule and the complex involved in the small molecule detection test strip are the same as those described above, and are not described herein again. The amount of each component on the test strip is not particularly limited, and can be determined by one skilled in the art according to the principle of the present invention and according to actual needs.
A third aspect of the present invention provides a small molecule detection kit comprising:
the small molecule detection test strip;
an incubation solution, the composition of which is as follows: taking 0.04-0.06mol/L phosphate buffer solution as a reference, adding the following substances in percentage by weight: 1.5-2.5% of sucrose, 4-6% of fructose, 0.8-1.2% of PEG and 202.5-3.5% of Tween; and the number of the first and second groups,
an optional standard graph card of target small molecules.
In the present invention, the "target small molecule" may also be referred to as "small molecule to be detected", which means a small molecule to be detected, as is well known to those skilled in the art.
The kit provided by the invention can prepare a standard curve graph according to the test condition so as to judge the content of the small molecules according to the signal intensity, and can carry the standard curve graph so as to obtain the content of the small molecules by referring to the standard curve graph.
According to a preferred embodiment of the present invention, the recognition element is BSA, and in the actual detection, the following reaction occurs as the chromatographic process proceeds:
the target micromolecules with a certain content in the solution and the BSA derivatives of the target micromolecules compete to combine with the labeled antibody, the more the content of the target micromolecules in the solution is, the less immune complexes formed by the BSA derivatives of the target micromolecules and the BSA derivatives of the target micromolecules competing with the labeled antibody are, and the weaker signals of the detection area are; namely the content of the target small molecules is inversely proportional to the signal intensity of the detection area; combining the goat anti-mouse secondary antibody in the quality control region with an immune complex of the target small molecule and the labeled antibody; the content of the labeled antibody determines the intensity of signal response, the intensity of the detection area signal is inversely proportional to the content of the target small molecule, and the intensity of the quality control area signal is proportional to the content of the target small molecule, so that the content of the target small molecule can be judged by comparing the relative intensity of the detection area signal.
Compared with the prior art, the invention has the beneficial effects that:
(1) in the invention, the 'competition' reaction is carried out in the pre-incubation step, the time controllability is strong, the antigen epitope is more fully exposed, and the reaction can be more fully carried out due to more sufficient reaction time and more sufficient epitope exposure, so that the 'competition' reaction has higher sensitivity.
(2) In the invention, the detection area is fixed with the universal identification element, the detection area is not changed by the change of the object to be detected, and the arrangement of multiple detection areas realizes the combination of multiple channels and universality. Therefore, the detection method, the test strip and the kit provided by the invention have wider application range.
(3) The invention realizes the organic fusion of the sensitivity, specificity, universality and high flux of immunoreaction and realizes the high-sensitivity high-flux high-universality quantitative detection of the object to be detected.
(4) The method and the product of the invention can be used in the fields of food safety, clinical diagnosis, inspection and quarantine, etc., and have wide market prospect.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
Fig. 1 is a schematic diagram of a high-sensitivity multichannel universal small molecule detection test strip according to embodiment 1 of the present invention;
FIG. 2 is a standard curve chart in example 2 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.
The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the following examples, the composition of the incubation liquid is as follows: based on 0.05mol/L phosphate buffer solution, the following substances are added in percentage by weight: 2% of sucrose, 5% of fructose, 1% of PEG and 203% of Tween.
Example 1
In this embodiment, the detection of diazepam and malachite green is taken as an example to illustrate the preparation of the test strip.
BSA and OVA are selected as universal identification elements, fluorescent quantum dot microspheres (QB) are selected as signal substances, and the preparation method of the test strip comprises the following steps:
1) respectively coating two detection areas of the NC membrane with an anti-BSA antibody and an anti-OVA antibody, wherein the coating process comprises the following steps: respectively diluting BSA antibody and OVA antibody to 0.5mg/ml by using a mixed solution of an incubation solution and methanol (the content of methanol is 15%), and coating the BSA antibody and the OVA antibody in a detection area on a nitrocellulose membrane by using an Isoflow gold spraying and membrane scratching instrument, wherein the coating amount is 1.0 mu l/cm; and (3) drying the coated reaction membrane for 2 hours at 37 ℃ for later use.
2) Coating a goat anti-mouse secondary antibody in the NC membrane quality control area, diluting the goat anti-mouse secondary antibody to the concentration of 0.5mg/mL by using a mixed solution of an incubation liquid and methanol (the content of the methanol is 15%), and coating the goat anti-mouse secondary antibody in the quality control area on the nitrocellulose membrane by using an Isoflow gold spraying and membrane scratching instrument, wherein the coating amount is 1.0 mu l/cm; and (3) drying the coated reaction membrane for 2 hours at 37 ℃ for later use.
3) The labeled antibody is prepared by a commercial detection method, a QB labeled antibody is prepared by using a carboxylated modified quantum dot microsphere (QB), and a diazepam-BSA compound and a malachite green-OVA compound are purchased.
4) Sequentially assembling an NC film, a glass fiber pad and a water absorption paper pad on a bottom plate to prepare a test strip; the product is stored in an environment with the temperature of 2-8 ℃ and the validity period is 12 months.
Example 2
In this embodiment, the preparation of a standard curve is illustrated by taking the detection of diazepam and malachite green in aquatic products as an example, and the test strip prepared in embodiment 1 is used for the test:
(1) diazepam (DAP) and malachite green (MCG) standards were prepared as different concentrations of solutions with incubations, respectively: 1.28ng/mL, 0.64ng/mL, 0.32ng/mL, 0.16ng/mL, 0.08ng/mL, 0.04ng/mL, 0.02ng/mL, 0.01 ng/mL;
(2) DAP-BSA solution with the final concentration of 0.1 mug/100 muL and a labeled antibody QB-mAb (purchased from Beijing Najing Biotech Co., Ltd., product model: QBB12117, H07187M) with the final concentration of 1ng/mL are respectively mixed with DAP standard substances with different concentrations, and incubation is carried out for 10min at 37 ℃ to form a mixture of immune complexes QB-DAP antibody-DAP-BSA (QB-anti-DAP-mAb-DAP-BSA) and QB-DAP antibody-DAP (QB-anti-DAP-mAb-DAP);
mixing MCG-OVA solution with a final concentration of 0.1 mug/100 mug and labeled antibody QB-mAb (purchased from Beijing Najing Biotechnology Co., Ltd., product model number: QBB12117, H07187M) with a final concentration of 1ng/mL respectively with MCG standard substances with different concentrations, and incubating at 37 ℃ for 10min to form a mixture of immune complexes QB-MCG antibody-MCG-OVA (QB-anti-MCG-mAb-MCG-OVA) and QB-MCG antibody-MCG (QB-anti-MCG-mAb-MCG);
the two incubation reactions are carried out in the same mixed solution to generate the corresponding complex.
(3) Adding the incubated solution into a glass fiber pad, sequentially passing through a detection area and a quality control area of a reaction membrane (NC membrane) under the suction action of a water absorption pad, and combining a QB-DAP antibody-DAP-BSA immune complex with a BSA antibody in the detection area when passing through the detection area; the QB-MCG antibody-MCG-OVA immune complex is combined with the OVA antibody in the detection area; when passing through the quality control region, QB-DAP antibody-DAP and QB-MCG antibody-MCG are combined with goat anti-mouse secondary antibody.
(4) And (3) adding the incubated solution into immunochromatographic test paper for chromatographic reaction for 10min, reading data by using a reading instrument, carrying out result quantitative interpretation through the relative signal intensity of the color development strips of the detection area and the quality control area of the detector under the excitation of 365nm ultraviolet light, and analyzing and recording the result.
(5) The data obtained were plotted against a standard curve as shown in FIG. 2.
As can be seen from the calculation of the standard curve in FIG. 2, the lowest detected concentration of DAP was 0.01ng mL-1. The minimum detection concentration of MCG is 0.006ng mL-1。
Example 3
This example is used to illustrate the detection of a sample to be detected (adding DAP and MCG to fish meat), and the following steps are performed:
(1) the test paper prepared in example 1 was equilibrated to room temperature;
(2) diluting the fish meat subjected to DAP and MCG labeling with mixed solution of incubation liquid and methanol (methanol content is 10%) in an equal volume, and performing an immunochromatography process on the obtained diluent;
(3) incubating the diluted solution obtained by diluting the fish meat with the standard sample with the labeled antibody and the antigen for 11min at 37 ℃, wherein the concentration and incubation conditions of all components are the same as those in example 2, and forming a mixture of immune complexes QB-anti-DAP-mAb-DAP and QB-anti-DAP-mAb-DAP-BSA and QB-anti-MCG-mAb-MCG-OVA;
(4) after incubation, adding the solution containing the mixture into a glass fiber pad, sequentially passing through two detection areas and a quality control area of a reaction membrane (NC membrane) under the suction action of a water absorption pad, and combining QB-anti-DAP-mAb-DAP-BSA with BSA antibodies in the detection areas when passing through the detection areas; QB-anti-MCG-mAb-MCG-OVA is combined with OVA antibody in the detection area; when the sample passes through a quality control region, QB-anti-DAP-mAb-DAP and QB-anti-MCG-mAb-MCG are combined with a goat anti-mouse secondary antibody;
(5) and (3) under the excitation of 365nm ultraviolet light, carrying out result quantitative interpretation by detecting the relative signal intensity of the chromogenic bands of the measurement area and the quality control area. The results of the measurements are shown in Table 1, according to the standard curve in example 2.
TABLE 1 results of sample testing
aMean ± standard deviation.
As can be seen from the detection result, the detection method provided by the invention has high detection accuracy.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Claims (10)
1. A multi-channel universal chromatography method for detecting small molecules is characterized by comprising the following steps:
(1) co-incubating the substance to be detected, the various labeled antibodies and the various complexes to obtain a mixture; wherein each labeled antibody is a specific antibody of a target small molecule and is labeled with a signal substance, and the signal substance of each labeled antibody is the same; each complex is formed by a target small molecule and a recognition element, and the recognition element of each complex is different;
(2) enabling the mixture obtained in the step (1) to sequentially pass through a detection area and a quality control area; wherein, the detection area is a plurality of detection areas, and each detection area is fixedly provided with a specific antibody of a recognition element; a secondary antibody of the labeled antibody is fixed on the quality control region;
(3) and judging the content of the target small molecules in the object to be detected according to the signal intensity of the detection area and the quality control area.
2. The multi-channel universal chromatography method for detecting small molecules according to claim 1, wherein the signal substance comprises at least one of fluorescent dye, phosphorescent material, magnetic nanomaterial, carbon nanomaterial, fluorescent quantum dot, biotin, radioisotope, electron dense substance, colloidal gold, and enzyme;
preferably, the fluorescent dye includes at least one of Alexa series dyes, aminoacridine, BODIPY fluorescent dyes, fluorescein and its derivatives, and catechol type fluorescent dyes.
3. The multi-channel universal chromatography method for detecting small molecules according to claim 1, wherein the identification element is a universal identification element; preferably at least one of ovalbumin, hemocyanin, bovine serum albumin, human serum albumin and chicken egg albumin.
4. The multi-channel universal chromatographic method for detecting small molecules, according to claim 1, wherein the target small molecule is at least two of a sedative, a dye, a pesticide, a veterinary drug, an additive and a pollutant; preferably, the target small molecules are at least two of diazepam, malachite green, organophosphorus pesticides, chloramphenicol drugs, tetracycline drugs, melamine and aflatoxin; further preferably, the target small molecules are diazepam and malachite green.
5. The method according to claim 1, wherein in step (1), the incubation temperature is 25-40 ℃, preferably 35-37 ℃; the incubation time is 5-20 min; the incubation is performed in a buffer, the composition of which comprises: taking 0.04-0.06mol/L phosphate buffer solution as a reference, adding the following substances in percentage by weight: 1.5-2.5% of sucrose, 4-6% of fructose, 0.8-1.2% of PEG and 202.5-3.5% of Tween;
the concentration of each labeled antibody in the incubation system is 0.5-5ng/mL, and the concentration of each complex is 80-120 ng/mL.
6. The test strip for detecting the small molecules is characterized by comprising a sample pad, a water absorption pad and a bottom plate, wherein the bottom plate is fixed with a nitrocellulose membrane; wherein,
the sample pad is used for bearing co-incubation products of an object to be detected, a plurality of labeled antibodies and a plurality of complexes;
each labeled antibody is a specific antibody of a target small molecule and is labeled with a signal substance, and the signal substance of each labeled antibody is the same; each complex is formed by a target small molecule and a recognition element, and the recognition element of each complex is different;
the nitrocellulose membrane is provided with a detection area and a quality control area; the detection area is multiple, and each detection area is fixedly provided with a specific antibody of a recognition element; and a secondary antibody of the labeled antibody is fixed on the quality control region.
7. The small molecule test strip of claim 6, wherein the signal substance comprises at least one of a fluorescent dye, a phosphorescent material, a magnetic nanomaterial, a carbon nanomaterial, a fluorescent quantum dot, biotin, a radioisotope, an electron dense substance, colloidal gold, and an enzyme;
preferably, the fluorescent dye includes at least one of Alexa series dyes, aminoacridine, BODIPY fluorescent dyes, fluorescein and its derivatives, and catechol type fluorescent dyes.
8. The small molecule test strip of claim 6, wherein the recognition element is a universal recognition element; preferably at least one of ovalbumin, hemocyanin, bovine serum albumin, human serum albumin and chicken egg albumin.
9. The small molecule test strip of claim 1, wherein the target small molecule is at least two of a sedative, a dye, a pesticide, a veterinary drug, an additive, and a contaminant; preferably, the target small molecules are at least two of diazepam, malachite green, organophosphorus pesticides, chloramphenicol drugs, tetracycline drugs, melamine and aflatoxin; further preferably, the target small molecules are diazepam and malachite green.
10. A small molecule detection kit, comprising:
the small molecule test strip of any one of claims 6-9;
an incubation solution, the composition of which is as follows: taking 0.04-0.06mol/L phosphate buffer solution as a reference, adding the following substances in percentage by weight: 1.5-2.5% of sucrose, 4-6% of fructose, 0.8-1.2% of PEG and 202.5-3.5% of Tween; and the number of the first and second groups,
an optional standard graph card of target small molecules.
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