阅读说明：本技术 一种用于毒品检测的量子点荧光免疫层析检测方法 (Quantum dot fluorescence immunochromatography detection method for drug detection ) 是由 刘晓 毛勇辉 施有猛 于 2021-07-30 设计创作，主要内容包括：本发明涉及毒品检测技术领域,具体是一种用于毒品检测的量子点荧光免疫层析检测方法,具体包括四大步骤。本发明采用量子点荧光免疫层析方法,通过荧光阅读仪阅读检测线和质控线的荧光值,根据不同浓度毒品抗原的检测线和质控线荧光值的不同,得出检测线和质控线的比值,该比值对应毒品抗原的浓度做标准曲线,进而实现对传统毒品和新型毒品的快速检测,具有检测精准、灵敏度高的特点,应用范围高,适合广泛推广,为打击犯罪分子提供有力证据。(The invention relates to the technical field of drug detection, in particular to a quantum dot fluorescence immunochromatography detection method for drug detection, which specifically comprises four steps. The invention adopts a quantum dot fluorescence immunochromatography method, reads the fluorescence values of a detection line and a quality control line through a fluorescence reader, obtains the ratio of the detection line and the quality control line according to the difference of the fluorescence values of the detection line and the quality control line of drug antigens with different concentrations, and makes a standard curve corresponding to the concentration of the drug antigens, thereby realizing the rapid detection of the traditional drugs and the novel drugs.)

1. A quantum dot fluorescence immunochromatography detection method for drug detection is characterized by relating to a sample pad (2), a combination pad (3), an NC membrane (7), a water absorption pad (5) and a PVC bottom plate (1);

the detection method comprises the following steps:

step 1: preparing drug antigen into liquid, spraying the liquid on the binding pad (3), and drying at 35-38 ℃ to form a quantum dot labeled antibody;

step 2: respectively coating the SD-BSA conjugate and the rabbit anti-human polyclonal antibody on an NC membrane (7), and drying at 40-45 ℃ to respectively form a detection line (6) and a quality control line (4);

and step 3: assembling a sample pad (2), a combination pad (3), an NC membrane (7) and a water absorption pad (5) on a PVC bottom plate (1) in sequence to form the quantum dot immunochromatographic test paper;

and 4, step 4: the method comprises the steps of dripping a sample solution on a sample pad (2), moving along the chromatography direction under the action of capillary siphonage, reading fluorescence values of a detection line (6) and a quality control line (4) through a fluorescence reader when the sample solution reaches the detection line (6) and the quality control line (4), obtaining the ratio of the detection line (6) to the quality control line (4) according to the difference of the fluorescence values of the detection line (6) to the quality control line (4) of the drug antigens with different concentrations, and making a standard curve corresponding to the concentration of the drug antigens according to the ratio so as to realize the detection of the drugs.

2. The fluorescence immunochromatography detection method of quantum dots for drug detection according to claim 1, characterized in that the bonding pad (3) is a polyester film fluorescent pad.

3. The method according to claim 1, wherein the method for preparing the drug antigen comprises the following steps:

s1, dissolving 50mg of BSA in 0.13mol/L NaHCO3 to prepare BSA activated solution, and measuring the pH value to be 7.6;

s1, dissolving 1mg of drug antitoxic molecule, 1.073mg of dicyclohexylcarbodiimide and 0.598mg of N-hydroxysuccinimide in anhydrous tetrahydrofuran, oscillating at 30-40 ℃ for 24h, and centrifuging at 4000r/min for 15 min;

s1, after centrifuging, washing and precipitating for 2-3 times by adopting anhydrous tetrahydrofuran, taking supernatant I, dissolving residues in 0.2ml of dimethylformamide after the tetrahydrofuran in the supernatant I is completely volatilized, and taking supernatant II after precipitating;

and S1, merging the supernatant I and the supernatant II, slowly dripping the merged supernatant I and the merged supernatant II into BSA activating solution, placing the mixture on a shaker, shaking the mixture for 4 hours at the temperature of 15-25 ℃, and further purifying to obtain the drug antigen.

4. The method according to claim 3, wherein the anti-virus molecule of the drug is morphine antibody, methamphetamine antibody, K powder antibody or cocaine antibody.

5. The method of claim 1, wherein the SD-BSA conjugate and the rabbit anti-human polyclonal antibody are diluted with a buffer solution to a concentration of 0.3-0.5mg/mL before use.

6. The method according to claim 5, wherein MES is used as the buffer and the pH is 4.5-5.

Technical Field

The invention relates to the technical field of drug detection, in particular to a quantum dot fluorescence immunochromatography detection method for drug detection.

Background

Drugs generally refer to drugs that cause addiction, and the term drug is used herein as a broad concept, mainly referring to opium, heroin, methamphetamine, etc. abused by drug addicts.

In order to combat criminals, detection is usually required for case-involved persons taking drugs, and currently, gold immunochromatography and enzyme-linked immunosorbent assay are mostly used, but the two methods are complicated to operate and have high requirements on environment and operators, and meanwhile, the sensitivity and the accuracy of the two methods are balanced, so that lawless persons taking light-weight drugs often get rid of the network. Therefore, the person skilled in the art provides a quantum dot fluorescence immunochromatography detection method for drug detection to solve the problems set forth in the background art.

Disclosure of Invention

The invention aims to provide a quantum dot fluorescence immunochromatography detection method for drug detection, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a quantum dot fluorescence immunochromatographic assay detection method for drug detection relates to a sample pad, a combination pad, an NC membrane, a water absorption pad and a PVC bottom plate;

the detection method comprises the following steps:

step 1: preparing drug antigen into liquid, spraying the liquid on a bonding pad, and drying at 35-38 ℃ to form a quantum dot labeled antibody;

step 2: respectively coating the SD-BSA conjugate and the rabbit anti-human polyclonal antibody on an NC membrane, and drying at 40-45 ℃ to respectively form a detection line and a quality control line;

and step 3: assembling a sample pad, a combination pad, an NC membrane and a water absorption pad on a PVC (polyvinyl chloride) bottom plate in sequence to form the quantum dot immunochromatography test paper;

and 4, step 4: dropping the sample solution on the sample pad, moving along the chromatography direction under the action of capillary siphon, reading the fluorescence values of the detection line and the quality control line by a fluorescence reader when the sample solution reaches the detection line and the quality control line, obtaining the ratio of the detection line and the quality control line according to the difference of the fluorescence values of the detection line and the quality control line of the drug antigens with different concentrations, and making a standard curve corresponding to the concentration of the drug antigens to further realize the detection of the drugs.

As a further aspect of the invention: the bonding pad adopts a polyester film fluorescent pad.

As a further aspect of the invention: the preparation method of the drug antigen comprises the following steps:

s1, dissolving 50mg of BSA in 0.13mol/L NaHCO3 to prepare BSA activated solution, and measuring the pH value to be 7.6;

s1, dissolving 1mg of drug antitoxic molecule, 1.073mg of dicyclohexylcarbodiimide and 0.598mg of N-hydroxysuccinimide in anhydrous tetrahydrofuran, oscillating at 30-40 ℃ for 24h, and centrifuging at 4000r/min for 15 min;

s1, after centrifuging, washing and precipitating for 2-3 times by adopting anhydrous tetrahydrofuran, taking supernatant I, dissolving residues in 0.2ml of dimethylformamide after the tetrahydrofuran in the supernatant I is completely volatilized, and taking supernatant II after precipitating;

and S1, merging the supernatant I and the supernatant II, slowly dripping the merged supernatant I and the merged supernatant II into BSA activating solution, placing the mixture on a shaker, shaking the mixture for 4 hours at the temperature of 15-25 ℃, and further purifying to obtain the drug antigen.

As a further aspect of the invention: the drug antitoxic molecule adopts morphine antibody, amphetamine antibody, K powder antibody or cocaine antibody.

As a further aspect of the invention: the SD-BSA conjugate and the rabbit anti-human polyclonal antibody need to be diluted to the concentration of 0.3-0.4mg/mL by adopting a buffer solution before use.

As a further aspect of the invention: MES is adopted as the buffer solution, and the pH value is 4.5-5.

Compared with the prior art, the invention has the beneficial effects that: the invention adopts a quantum dot fluorescence immunochromatography method, reads the fluorescence values of a detection line and a quality control line through a fluorescence reader, obtains the ratio of the detection line and the quality control line according to the difference of the fluorescence values of the detection line and the quality control line of drug antigens with different concentrations, and makes a standard curve corresponding to the concentration of the drug antigens, thereby realizing the rapid detection of the traditional drugs and the novel drugs.

Drawings

FIG. 1 is a schematic structural diagram of a quantum dot immunochromatographic test paper used in a quantum dot fluorescence immunochromatographic detection method for drug detection.

In the figure: 1. a PVC base plate; 2. a sample pad; 3. a bonding pad; 4. a quality control line; 5. a water absorbent pad; 6. detecting lines; 7. and (3) NC film.

Detailed Description

Example 1

Referring to fig. 1, in the embodiment of the present invention, a quantum dot fluorescence immunochromatographic assay for drug detection relates to a sample pad 2, a combination pad 3, an NC membrane 7, a water absorption pad 5, and a PVC base plate 1;

the detection method comprises the following steps:

step 1: preparing the drug antigen into liquid, spraying the liquid on the combination pad 3, and drying the liquid at 35 ℃ to form a quantum dot labeled antibody;

step 2: respectively coating the SD-BSA conjugate and the rabbit anti-human polyclonal antibody on an NC membrane 7, and drying at 40 ℃ to respectively form a detection line 6 and a quality control line 4;

and step 3: assembling a sample pad 2, a combination pad 3, an NC membrane 7 and a water absorption pad 5 on a PVC base plate 1 in sequence to form the quantum dot immunochromatographic test paper;

and 4, step 4: the method comprises the steps of dripping a sample solution on a sample pad 2, moving along the chromatography direction under the action of capillary siphon, reading fluorescence values of a detection line 6 and a quality control line 4 through a fluorescence reader when the sample solution reaches the detection line 6 and the quality control line 4, obtaining the ratio of the detection line 6 to the quality control line 4 according to the difference of the fluorescence values of the detection line 6 and the quality control line 4 of the drug antigens with different concentrations, and making a standard curve corresponding to the concentration of the drug antigens according to the ratio so as to realize the detection of the drugs.

Further, the bonding pad 3 employs a polyester film fluorescent pad.

Further, the preparation method of the drug antigen comprises the following steps:

s1, dissolving 50mg of BSA in 0.13mol/L NaHCO3 to prepare BSA activated solution, and measuring the pH value to be 7.6;

s1, dissolving 1mg of drug antitoxic molecule, 1.073mg of dicyclohexylcarbodiimide and 0.598mg of N-hydroxysuccinimide in anhydrous tetrahydrofuran, oscillating at 30 ℃ for 24 hours, and centrifuging at 4000r/min for 15 min;

s1, after centrifuging, washing and precipitating for 2-3 times by adopting anhydrous tetrahydrofuran, taking supernatant I, dissolving residues in 0.2ml of dimethylformamide after the tetrahydrofuran in the supernatant I is completely volatilized, and taking supernatant II after precipitating;

and S1, merging the supernatant I and the supernatant II, slowly dripping the merged supernatant I and the merged supernatant II into BSA activating solution, placing the mixture on a shaker, shaking the mixture for 4 hours at 15 ℃, and further purifying to obtain the drug antigen.

Furthermore, morphine antibody is adopted as the drug antitoxic molecule.

Furthermore, SD-BSA conjugates and rabbit anti-human polyclonal antibodies were diluted with buffer to a concentration of 0.3mg/mL before use.

Furthermore, MES is used as buffer solution, and the pH value is 4.5-5.

Test example 1

And (3) testing groups: example 1, reference example 1 (morphine colloidal gold immunochromatographic strip), reference example 2 (enzyme-linked immunosorbent assay);

the testing personnel: collecting 150 hair samples of morphine-sucking persons, and dividing the hair samples into 3 groups (50 persons in each group);

the test method comprises the following steps: respectively detecting saliva/blood/urine/hair samples (the hair samples need to be placed into a hair digestion solution and vibrated for 45S) through 3 groups of products;

the test results were as follows:

item	Number of samples	Number of detected people
			Example 1	50 persons	50
Reference example 1	50 persons	26
			Reference example 2	50 persons	28

The data are combined to obtain that the accuracy of the method 1 is higher than that of reference examples 1 and 2 when the morphine is detected, and powerful evidence is provided for capturing criminals.

Example 2

Referring to fig. 1, in the embodiment of the present invention, a quantum dot fluorescence immunochromatographic assay for drug detection relates to a sample pad 2, a combination pad 3, an NC membrane 7, a water absorption pad 5, and a PVC base plate 1;

the detection method comprises the following steps:

step 1: preparing the drug antigen into liquid, spraying the liquid on the combination pad 3, and drying the liquid at 36 ℃ to form a quantum dot labeled antibody;

step 2: respectively coating the SD-BSA conjugate and the rabbit anti-human polyclonal antibody on an NC membrane 7, and drying at 41 ℃ to respectively form a detection line 6 and a quality control line 4;

and step 3: assembling a sample pad 2, a combination pad 3, an NC membrane 7 and a water absorption pad 5 on a PVC base plate 1 in sequence to form the quantum dot immunochromatographic test paper;

and 4, step 4: the method comprises the steps of dripping a sample solution on a sample pad 2, moving along the chromatography direction under the action of capillary siphon, reading fluorescence values of a detection line 6 and a quality control line 4 through a fluorescence reader when the sample solution reaches the detection line 6 and the quality control line 4, obtaining the ratio of the detection line 6 to the quality control line 4 according to the difference of the fluorescence values of the detection line 6 and the quality control line 4 of the drug antigens with different concentrations, and making a standard curve corresponding to the concentration of the drug antigens according to the ratio so as to realize the detection of the drugs.

Further, the bonding pad 3 employs a polyester film fluorescent pad.

Further, the preparation method of the drug antigen comprises the following steps:

s1, dissolving 50mg of BSA in 0.13mol/L NaHCO3 to prepare BSA activated solution, and measuring the pH value to be 7.6;

s1, dissolving 1mg of drug antitoxic molecule, 1.073mg of dicyclohexylcarbodiimide and 0.598mg of N-hydroxysuccinimide in anhydrous tetrahydrofuran, oscillating at 35 ℃ for 24 hours, and centrifuging at 4000r/min for 15 min;

s1, after centrifuging, washing and precipitating for 2-3 times by adopting anhydrous tetrahydrofuran, taking supernatant I, dissolving residues in 0.2ml of dimethylformamide after the tetrahydrofuran in the supernatant I is completely volatilized, and taking supernatant II after precipitating;

and S1, merging the supernatant I and the supernatant II, slowly dripping the merged supernatant I and the merged supernatant II into BSA activating solution, placing the mixture on a shaker, shaking the mixture for 4 hours at 18 ℃, and further purifying to obtain the drug antigen.

Furthermore, the anti-virus molecules of the drugs adopt ice virus antibodies.

Furthermore, SD-BSA conjugates and rabbit anti-human polyclonal antibodies were diluted with buffer to a concentration of 0.38mg/mL before use.

Furthermore, MES is used as buffer solution, and the pH value is 4.5-5.

Test example 2

And (3) testing groups: example 1, reference example 1 (ice toxicity colloidal gold immunochromatographic test paper), reference example 2 (enzyme-linked immunosorbent assay);

the testing personnel: collecting 150 hair samples of ice drug addicts, and dividing the hair samples into 3 groups (50 persons in each group);

the test method comprises the following steps: respectively detecting saliva/blood/urine/hair samples (the hair samples need to be placed into a hair digestion solution and vibrated for 45S) through 3 groups of products;

the test results were as follows:

item	Number of samples	Number of detected people
			Example 1	50 persons	50
Reference example 1	50 persons	18
			Reference example 2	50 persons	23

The data are combined to obtain that the accuracy of the method 2 is higher than that of the reference examples 1 and 2 when the method is used for detecting the ice toxins, and powerful evidence is provided for capturing criminals.

Example 3

Referring to fig. 1, in the embodiment of the present invention, a quantum dot fluorescence immunochromatographic assay for drug detection relates to a sample pad 2, a combination pad 3, an NC membrane 7, a water absorption pad 5, and a PVC base plate 1;

the detection method comprises the following steps:

step 1: preparing the drug antigen into liquid, spraying the liquid on the combination pad 3, and drying the liquid at 38 ℃ to form a quantum dot labeled antibody;

step 2: respectively coating the SD-BSA conjugate and the rabbit anti-human polyclonal antibody on an NC membrane 7, and drying at 43 ℃ to respectively form a detection line 6 and a quality control line 4;

and step 3: assembling a sample pad 2, a combination pad 3, an NC membrane 7 and a water absorption pad 5 on a PVC base plate 1 in sequence to form the quantum dot immunochromatographic test paper;

and 4, step 4: the method comprises the steps of dripping a sample solution on a sample pad 2, moving along the chromatography direction under the action of capillary siphon, reading fluorescence values of a detection line 6 and a quality control line 4 through a fluorescence reader when the sample solution reaches the detection line 6 and the quality control line 4, obtaining the ratio of the detection line 6 to the quality control line 4 according to the difference of the fluorescence values of the detection line 6 and the quality control line 4 of the drug antigens with different concentrations, and making a standard curve corresponding to the concentration of the drug antigens according to the ratio so as to realize the detection of the drugs.

Further, the bonding pad 3 employs a polyester film fluorescent pad.

Further, the preparation method of the drug antigen comprises the following steps:

s1, dissolving 50mg of BSA in 0.13mol/L NaHCO3 to prepare BSA activated solution, and measuring the pH value to be 7.6;

s1, dissolving 1mg of drug antitoxic molecule, 1.073mg of dicyclohexylcarbodiimide and 0.598mg of N-hydroxysuccinimide in anhydrous tetrahydrofuran, oscillating at 35 ℃ for 24 hours, and centrifuging at 4000r/min for 15 min;

s1, after centrifuging, washing and precipitating for 2-3 times by adopting anhydrous tetrahydrofuran, taking supernatant I, dissolving residues in 0.2ml of dimethylformamide after the tetrahydrofuran in the supernatant I is completely volatilized, and taking supernatant II after precipitating;

and S1, merging the supernatant I and the supernatant II, slowly dripping the merged supernatant I and the merged supernatant II into BSA activating solution, placing the mixture on a shaker, shaking the mixture for 4 hours at the temperature of 22 ℃, and further purifying to obtain the drug antigen.

Furthermore, K powder antibody is adopted as the drug antitoxic molecule.

Furthermore, SD-BSA conjugates and rabbit anti-human polyclonal antibodies were diluted with buffer to a concentration of 0.46mg/mL before use.

Furthermore, MES is used as buffer solution, and the pH value is 4.5-5.

Test example 3

And (3) testing groups: example 1, reference example 1(K powder colloidal gold immunochromatographic test paper), reference example 2 (enzyme-linked immunosorbent assay);

the testing personnel: collecting 150 hair samples of people sucking K powder, and dividing the hair samples into 3 groups (50 people in each group);

the test method comprises the following steps: respectively detecting saliva/blood/urine/hair samples (the hair samples need to be placed into a hair digestion solution and vibrated for 45S) through 3 groups of products;

the test results were as follows:

item	Number of samples	Number of detected people
			Example 1	50 persons	47
Reference example 1	50 persons	32
			Reference example 2	50 persons	15

The data are combined to obtain that the accuracy of the method 3 is higher than that of reference examples 1 and 2 when the K powder drugs are detected, and powerful evidence is provided for capturing criminals.

Example 4

Referring to fig. 1, in the embodiment of the present invention, a quantum dot fluorescence immunochromatographic assay for drug detection relates to a sample pad 2, a combination pad 3, an NC membrane 7, a water absorption pad 5, and a PVC base plate 1;

the detection method comprises the following steps:

step 1: preparing the drug antigen into liquid, spraying the liquid on the combination pad 3, and drying the liquid at 38 ℃ to form a quantum dot labeled antibody;

step 2: respectively coating the SD-BSA conjugate and the rabbit anti-human polyclonal antibody on an NC membrane 7, and drying at 45 ℃ to respectively form a detection line 6 and a quality control line 4;

and step 3: assembling a sample pad 2, a combination pad 3, an NC membrane 7 and a water absorption pad 5 on a PVC base plate 1 in sequence to form the quantum dot immunochromatographic test paper;

and 4, step 4: the method comprises the steps of dripping a sample solution on a sample pad 2, moving along the chromatography direction under the action of capillary siphon, reading fluorescence values of a detection line 6 and a quality control line 4 through a fluorescence reader when the sample solution reaches the detection line 6 and the quality control line 4, obtaining the ratio of the detection line 6 to the quality control line 4 according to the difference of the fluorescence values of the detection line 6 and the quality control line 4 of the drug antigens with different concentrations, and making a standard curve corresponding to the concentration of the drug antigens according to the ratio so as to realize the detection of the drugs.

Further, the bonding pad 3 employs a polyester film fluorescent pad.

Further, the preparation method of the drug antigen comprises the following steps:

s1, dissolving 50mg of BSA in 0.13mol/L NaHCO3 to prepare BSA activated solution, and measuring the pH value to be 7.6;

s1, dissolving 1mg of drug antitoxic molecule, 1.073mg of dicyclohexylcarbodiimide and 0.598mg of N-hydroxysuccinimide in anhydrous tetrahydrofuran, oscillating at 40 ℃ for 24 hours, and centrifuging at 4000r/min for 15 min;

s1, after centrifuging, washing and precipitating for 2-3 times by adopting anhydrous tetrahydrofuran, taking supernatant I, dissolving residues in 0.2ml of dimethylformamide after the tetrahydrofuran in the supernatant I is completely volatilized, and taking supernatant II after precipitating;

and S1, merging the supernatant I and the supernatant II, slowly dripping the merged supernatant I and the merged supernatant II into BSA activating solution, placing the mixture on a shaker, shaking the mixture for 4 hours at the temperature of 25 ℃, and further purifying to obtain the drug antigen.

Further, cocaine antibody is used as drug anti-toxin molecule.

Furthermore, SD-BSA conjugates and rabbit anti-human polyclonal antibodies were diluted with buffer to a concentration of 0.5mg/mL before use.

Furthermore, MES is used as buffer solution, and the pH value is 4.5-5.

Test example 4

And (3) testing groups: example 1, reference example 1 (cocaine colloidal gold immunochromatographic test paper), reference example 2 (enzyme-linked immunosorbent assay);

the testing personnel: collecting 150 hair samples of cocaine-sucking persons, and dividing the hair samples into 3 groups (50 persons in each group);

the test method comprises the following steps: respectively detecting saliva/blood/urine/hair samples (the hair samples need to be placed into a hair digestion solution and vibrated for 45S) through 3 groups of products;

the test results were as follows:

item	Number of samples	Number of detected people
			Example 1	50 persons	49
Reference example 1	50 persons	19
			Reference example 2	50 persons	31

The above data can be combined to obtain that the accuracy of the detection of cocaine poison in the embodiment 4 is higher than that of the reference examples 1 and 2, and powerful evidence is provided for capturing criminals.

In summary, the following steps: the invention adopts a quantum dot fluorescence immunochromatography method, reads the fluorescence values of a detection line and a quality control line through a fluorescence reader, obtains the ratio of the detection line and the quality control line according to the difference of the fluorescence values of the detection line and the quality control line of drug antigens with different concentrations, and makes a standard curve corresponding to the concentration of the drug antigens, thereby realizing the rapid detection of the traditional drugs and the novel drugs.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention are equivalent to or changed within the technical scope of the present invention.