CN117147828A

CN117147828A - Diluent for drug fluorescence detection and preparation method and application thereof

Info

Publication number: CN117147828A
Application number: CN202311151190.2A
Authority: CN
Inventors: 方少华; 喻洪江; 孔庆波; 黄伟忠; 李展飞; 麻蓉
Original assignee: Zhejiang Orient Gene Biotech Co Ltd
Current assignee: Zhejiang Orient Gene Biotech Co Ltd
Priority date: 2023-09-07
Filing date: 2023-09-07
Publication date: 2023-12-01

Abstract

The invention discloses a diluent for drug fluorescence detection, a preparation method and application thereof. The diluent comprises a buffering agent, a stabilizing agent, an inorganic salt and a dispersing agent; the buffer comprises any one or a combination of at least two of tris, phosphate or carbonate; the stabilizer comprises any one or a combination of at least two of bovine serum albumin, casein or PEG; the dispersing agent comprises any one or a combination of at least two of polyvinyl alcohol, polyethylene glycol and lauryl alcohol. The invention creatively discovers that the diluent composed of the buffer, the stabilizer, the inorganic salt and the dispersing agent can well promote the specificity and the uniformity of drug detection reagent products, increase the stability of the products, reduce the difference between the products, improve the detection accuracy, be applicable to the rapid screening in public places, and in addition, the components are safer and do not cause environmental pollution.

Description

Diluent for drug fluorescence detection and preparation method and application thereof

Technical Field

The invention belongs to the technical field of drug detection, relates to a diluent for drug fluorescence detection and a preparation method and application thereof, and in particular relates to a diluent for hair poisoning fluorescence detection and a preparation method and application thereof

Background

The traditional rapid detection technology for drug-taking personnel mainly adopts a urine colloidal gold method for only about 24-48 hours. Compared with biological detection materials such as urine, blood, saliva and the like, the hair detection material has the advantages of stable property, convenient material taking, easy storage, long detection time limit, wide application range, less pollution opportunity and the like. Because the content of urine poisoning products and metabolites thereof is very high, the traditional colloidal gold immunoassay technology can be achieved. However, hair intoxicants and their metabolites are very low in content, and therefore, techniques with higher detection sensitivity and better tamper resistance are required for hair detection.

At present, a drug detection test strip usually adopts fluorescent microspheres to mark antibodies or antigens, wherein the fluorescent microspheres are functional microspheres, fluorescent substances are marked on the surfaces of the microspheres through chemical reaction or physical adsorption, or certain fluorescent substances are embedded or polymerized in the microspheres, so that the microspheres have a fluorescent display effect; the fluorescent microsphere can be in any shape, and mainly takes the shape of sphere. Currently, the types of fluorescent microspheres include quantum dot fluorescent microspheres, rare earth fluorescent microspheres, self-luminescent fluorescent microspheres, and the like. The fluorescent microsphere has the advantages of uniform granularity, good dispersibility, high luminous efficiency and the like, but has poor stability and can be influenced by external environment and medium. Photobleaching or photolysis may occur over time or under intense light, resulting in signal decay of the fluorescent microspheres. When a plurality of different fluorescent dyes are used to label the fluorescent microspheres, the different fluorescent signals may interfere or overlap with each other, affecting interpretation and quantitative analysis of the results. In the existing preparation process of fluorescent drug detection reagent, buffer solution or inorganic salt solution is often used for diluting and preparing fluorescent substance-marked antibody or antigen, and coating is carried out, so that low detection sensitivity, poor specificity and batch-to-batch instability are easy to cause false negative results and false positive results, for example, CN110850095A discloses a drug trace detection test strip, a preparation method and a kit thereof, and a time-resolved fluorescent microsphere-marked specific monoclonal antibody is uniformly dispersed in phosphate buffer solution.

In summary, the development of the novel diluent is used for re-dissolving and diluting after the fluorescent label is finished in the preparation process of the detection test strip, so that the stability and uniformity of fluorescent signals are improved, and the novel diluent has important significance in the field of drug fluorescence detection.

Disclosure of Invention

Aiming at the defects and actual demands of the prior art, a diluent for drug fluorescence detection and a preparation method and application thereof, in particular to a diluent for hair poisoning fluorescence detection and a preparation method and application thereof, so as to improve detection stability and accuracy.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a diluent for fluorescence detection of drugs, the diluent comprising a buffer, a stabilizer, an inorganic salt and a dispersant; the buffer comprises any one or a combination of at least two of tris, phosphate and carbonate; the stabilizer comprises any one or a combination of at least two of bovine serum albumin, casein or PEG; the dispersing agent comprises any one or a combination of at least two of polyvinyl alcohol, polyethylene glycol and lauryl alcohol.

In the invention, a specific diluent formula is designed, which contains a buffer, a stabilizer, inorganic salt and a dispersing agent, and is used for diluting an antibody or antigen marked by a fluorescent marker in the coating process of a test paper binding pad (or a fluorescent pad), wherein the buffer and the inorganic salt provide a proper ionic environment for the fluorescent marker by adjusting the ionic concentration of a system; the stabilizer is used for increasing the stability of the fluorescent marker and reducing the result difference of the reagent; the dispersing agent is used for preventing the fluorescent marker particles from depositing or separating out; the stability and uniformity of the hair drug fluorescent reagent are improved, and the specificity of the hair drug fluorescent reagent is not influenced, so that the stability and accuracy of detection are improved.

Preferably, the inorganic salt comprises any one or a combination of at least two of sodium chloride, potassium chloride or sodium borate.

Preferably, the mass percent of buffer in the diluent is 1.5% to 3.5%, including but not limited to 1.6%, 1.7%, 1.8%, 2%, 2.5%, 2.8%, 3%, 3.2% or 3.4%.

Preferably, the mass percent of the stabilizer in the diluent is 0.5% -2%, including but not limited to 0.6%, 0.7%, 0.8%, 1%, 1.5%, 1.6%, 1.8% or 1.9%.

Preferably, the mass percent of the inorganic salt in the diluent is 0.1% -0.3%, including but not limited to 0.15%, 0.18%, 0.2%, 0.25%, 0.28% or 0.29%.

Preferably, the mass percent of dispersant in the diluent is 0.4% to 0.8%, including but not limited to 0.5%, 0.6% or 0.7%.

Preferably, the polyvinyl alcohol comprises PVA-40 or PVA-10.

Preferably, the diluent further comprises a preservative and/or water.

Preferably, the preservative comprises any one or a combination of at least two of Proclin300, sodium azide.

Preferably, the mass percent of the preservative in the diluent is 0.5% -10%, including but not limited to 0.6%, 0.7%, 0.8%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8% or 9%, etc.

Preferably, the diluent for drug fluorescence detection comprises, by mass, 100% of 1.5% -3.5% of buffer, 0.5% -2% of stabilizer, 0.1% -0.3% of inorganic salt, 0.4% -0.8% of dispersing agent, 0.5% -10% of preservative, and the balance of water.

In a second aspect, the present invention provides a method for preparing the diluent for fluorescence detection of drugs according to the first aspect, the method comprising:

and mixing the buffering agent, the stabilizing agent, the inorganic salt and the dispersing agent to obtain the diluent.

In a third aspect, the present invention provides the use of a diluent for fluorescence detection of drugs according to the first aspect for the preparation of a product for detecting drugs.

In a fourth aspect, the present invention provides a method of preparing a drug fluorescence detection test strip, the method comprising:

diluting by mixing the diluent for drug fluorescence detection according to the first aspect with an antibody marked by a fluorescent marker, and coating the mixture on a binding pad; drawing the antigen solutions of drugs on the detection films respectively to form detection lines corresponding to different drugs; and sequentially adhering the sample pad, the combination pad (or the fluorescent pad), the detection membrane and the absorption pad on the surface of the substrate from one end to the other end of the substrate to obtain the drug fluorescent detection test strip.

In the invention, a specific diluent formula is designed, and the diluent formula can be effectively applied to preparing drug fluorescence detection test paper, and is used for diluting an antibody or antigen marked by a fluorescent marker in the process of coating a detection test paper binding pad (or fluorescent pad), so that the detection stability and accuracy of the test paper are obviously improved.

Preferably, the fluorescent marker comprises a fluorescent microsphere or a magnetic bead microsphere.

Preferably, the dilution factor of the dilution is 5-20 times.

Preferably, the drug comprises any one or a combination of at least two of morphine, phenylpropanamine, ketamine, tetrahydrocannabinic acid, cannabinoids or cocaine.

Preferably, the method for preparing the drug fluorescence detection test strip comprises the following steps:

preparing a sample pad treatment liquid: the formula is as follows: sodium tetraborate 0.5-5%, S17 0.1-10%, casein 0.1-1%, sodium cholate 0.1-1%, proclin300 0.5-10%;

(II) sample pad treatment: the sample pad treatment liquid is fully dissolved and uniformly stirred and then sprayed on glass fiber with proper size, and the glass fiber is placed in an oven with the temperature of 40-50 ℃ to be dried for 12-24 hours, taken out, sealed, dried and stored;

(III) preparation of a fluorescent pad: diluting the fluorescent microsphere marked monoclonal antibody and marked rabbit IgG according to the proportion of 20% -60% and 5% -20% respectively by using the diluent, adding 0.1-0.5 g/mL of sucrose and 0.01-0.1 g/mL of trehalose, preparing and fully and uniformly dissolving, spraying the mixture on a blank pad with the spraying amount of 1.0 mu L/cm, drying the blank pad in an oven at 30-40 ℃ for 12-24 hours, and sealing and storing the blank pad in a dark place;

and (IV) preparing a coating film: diluting a drug-antigen hemoglobin conjugate and goat anti-rabbit IgG to 0.1-1.0 mg/mL by adopting a coating buffer solution, adding coomassie brilliant blue into goat anti-rabbit IgG, preparing the concentration of coomassie brilliant blue to 0.1-1 mg/mL, coating the goat anti-rabbit IgG diluent and the drug-antigen diluent on a quality control line and a test line on a nitrocellulose membrane (NC membrane) respectively according to the coating amount of 1.0 mu L/cm after the preparation, and then drying in an oven at 40-50 ℃ for 20-30 h;

and (fifth) assembling: the sample pad, the fluorescent pad, the NC film and the water absorbing pad are sequentially adhered on the PVC bottom plate, the water absorbing pad is adhered on the upper part of the nitrocellulose film to generate partial overlapping of 1-2 mm, the lower end of the nitrocellulose film is arranged below the fluorescent pad, the partial overlapping of 1-2 mm exists between the nitrocellulose film and the fluorescent pad, the other end of the fluorescent pad is overlapped with the sample pad by 1-2 mm, and the other end of the sample pad is aligned with the PVC lining plate.

In a fifth aspect, the present invention provides a diluent for fluorescence detection of drugs according to the first aspect and an application of a method for preparing a fluorescence detection test strip for drugs according to the fourth aspect in drug detection.

Compared with the prior art, the invention has the following beneficial effects:

the invention creatively discovers that the diluent composed of the buffer, the stabilizer, the inorganic salt and the dispersing agent can well promote the specificity and the uniformity of drug detection reagent products, increase the stability of the products, reduce the difference between the products, improve the detection accuracy, be applicable to the rapid screening in public places, and in addition, the components are safer and do not cause environmental pollution.

Drawings

FIG. 1 is a schematic diagram of the detection of a positive sample in the present invention;

FIG. 2 is a schematic diagram of the detection of a negative sample according to the present invention;

FIG. 3 is a schematic diagram of the structure of the test paper of the present invention.

Detailed Description

The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.

The invention adopts the technical principle that: after being treated, a sample to be detected such as a hair sample and the like is dissolved in a certain amount of lysate, fully and uniformly mixed and reacted for 1-2 min, the sample is dripped at a sample pad of a sample adding area, an antigen to be detected in a positive sample is combined with a fluorescent microsphere marked antibody in a fluorescent pad and is subjected to forward chromatography through capillary action, when the antigen reaches an NC film of a detection area, the antigen fixed on the detection line does not have the fluorescent microsphere marked antibody combined with the antigen, namely, the detection line does not have fluorescent reaction, and a strong positive result is shown as shown in figure 1. The antigen of the object to be detected does not exist in the negative sample, and the fluorescent marked antibody in the fluorescent pad is chromatographed on the NC film along with capillary action and combined with the antigen protein conjugate coated on the detection area, so that a strong fluorescent reaction is shown as shown in figure 2. It can be seen that the amount of the fluorescent microsphere labeled antibody bound on the line is inversely proportional to the amount of the analyte in the sample, the fluorescent label bound on the quality control line is irrelevant to the amount of the analyte in the sample, and other fluorescent labels continue to be chromatographed to reach the absorption region. After chromatography is finished, the fluorescence intensity of a reading detection line and a quality control line of a fluorescence reader is adopted, the T/C value is calculated, and the content of an object to be detected in the sample can be calculated through a standard curve built in the instrument.

Example 1

The embodiment provides a diluent for detecting hair fluorescent drugs, which comprises the following components in percentage by mass (calculated by 100 percent): TRIS (TRIS) 1.5%, bovine serum albumin (protease-free/IgG BSA) 0.5%, sodium chloride (NaCl) 0.1%, polyvinyl alcohol (PVA-40) 0.4%, preservative (Proclin 300) 0.6% and the balance water.

Example 2

The embodiment provides a diluent for detecting hair fluorescent drugs, which comprises the following components in percentage by mass (calculated by 100 percent): TRIS (TRIS) 2.0%, bovine serum albumin (protease-free/IgG BSA) 1.0%, sodium chloride (NaCl) 0.2%, polyvinyl alcohol (PVA-40) 0.5%, preservative (Proclin 300) 0.6% and the balance water.

Example 3

The embodiment provides a diluent for detecting hair fluorescent drugs, which comprises the following components in percentage by mass (calculated by 100 percent): TRIS (TRIS) 2.5%, bovine serum albumin (protease-free/IgG BSA) 1.5%, sodium chloride (NaCl) 0.3%, polyvinyl alcohol (PVA-40) 0.6%, preservative (Proclin 300) 0.6% and the balance water.

Example 4

The embodiment provides a diluent for a hair fluorescent drug detection test strip, which comprises the following components in percentage by mass (calculated by 100 percent): TRIS (TRIS) 3.0%, bovine serum albumin (protease-free/IgG BSA) 2.0%, sodium chloride (NaCl) 0.4%, polyvinyl alcohol (PVA-40) 0.7%, preservative (Proclin 300) 0.6% and the balance water.

Example 5

The embodiment provides a diluent for a hair fluorescent drug detection test strip, which comprises the following components in percentage by mass (calculated by 100 percent): TRIS (TRIS) 3.5%, bovine serum albumin (protease-free/IgG BSA) 2.5%, sodium chloride (NaCl) 0.5%, polyvinyl alcohol (PVA-40) 0.8%, preservative (Proclin 300) 0.6% and the balance water.

Example 6

The embodiment provides a diluent for detecting hair fluorescent drugs, which comprises the following components in percentage by mass (calculated by 100 percent): sodium borate 2.5%, bovine serum albumin (protease-free/IgG BSA) 1.5%, sodium chloride (NaCl) 0.3%, polyvinyl alcohol (PVA-40) 0.6%, preservative (Proclin 300) 0.6% and the balance water.

Example 7

The embodiment provides a diluent for detecting hair fluorescent drugs, which comprises the following components in percentage by mass (calculated by 100 percent): TRIS (TRIS) 2.5%, casein 1.5%, sodium chloride (NaCl) 0.3%, polyvinyl alcohol (PVA-40) 0.6%, preservative (Proclin 300) 0.6% and the balance water.

Example 8

The embodiment provides a diluent for detecting hair fluorescent drugs, which comprises the following components in percentage by mass (calculated by 100 percent): TRIS (TRIS) 2.5%, bovine serum albumin (protease-free/IgG BSA) 1.5%, sodium chloride (NaCl) 0.3%, PEG 0.6%, preservative (Proclin 300) 0.6% and the balance water.

Example 9

The embodiment provides a diluent for detecting hair fluorescent drugs, which comprises the following components in percentage by mass (calculated by 100 percent): TRIS (TRIS) 2.5%, bovine serum albumin (protease-free/IgG BSA) 0.75%, casein 0.75%, sodium chloride (NaCl) 0.3%, polyvinyl alcohol (PVA-40) 0.6%, preservative (Proclin 300) 0.6%, balance water.

Comparative example 1

This comparative example provides a diluent for a hair fluorescence drug test strip, which differs from example 1 only in that: the stabilizer is not contained, and the weight parts of the stabilizer are distributed into the mass of the buffer solution, the dispersing agent and the inorganic salt in proportion.

Comparative example 2

This comparative example provides a diluent for a hair fluorescence drug test strip, which differs from example 1 only in that: the dispersing agent is not contained, and the mass parts of the dispersing agent are distributed into the mass of the buffer solution, the stabilizer and the inorganic salt in proportion.

Preparation example 1

Preparation of a fluorescence detection kit for detecting drugs in hair.

(1) Preparation of fluorescent-labeled proteins

Preparing 0.05M 2-morpholinoethanesulfonic acid (MES) solution, adjusting pH, adding 10% fluorescent microspheres, rotating at 50rpm for 60min, taking down and sequentially adding 50mg/mL N-hydroxysuccinimide (NHS) and 50mg/mL carbodiimide (EDC) solution, continuing rotating for 60min, centrifuging at 15000rpm for 15min after rotation is finished, removing supernatant, adding MES buffer solution for full dissolution, adding 0.5mg antibody for rotating reaction for 2.5h, adding 10% blocking agent after reaction is finished, rotating for 3h, centrifuging at 15000rpm for 15min, and adding 1mLTris buffer solution for re-dissolution after removing supernatant.

(2) Sample pad treatment

Preparing sample pad treatment fluid, wherein the formula is as follows: 2.8% sodium tetraborate, 3.6% S17,0.4% casein, 0.5% sodium cholate, 4% Proclin300. And (3) fully dissolving and uniformly stirring the treatment liquid, spraying the treatment liquid on glass fibers, drying the glass fibers in a drying oven at 37 ℃ for 24 hours, taking out, sealing, drying and storing.

(3) Fluorescent pad preparation

The fluorescent microsphere-labeled monoclonal antibody and rabbit IgG were diluted with the buffers of examples 1-9 and comparative examples 1-2 at 40% and 10%, respectively, and 0.5g/mL sucrose and 0.2g/mL trehalose were added. After being fully and uniformly dissolved, the mixture is sprayed on a blank pad with the spraying quantity of 1.0 mu L/cm, and then the blank pad is dried in a baking oven at 37 ℃ for 24 hours and stored in a sealed manner in a dark place;

(4) Preparation of coating film

Diluting the drug-antigen hemoglobin conjugate and the goat anti-rabbit IgG to 0.5mg/mL and 0.8mg/mL by adopting a coating buffer solution, adding coomassie brilliant blue into the goat anti-rabbit IgG, preparing the concentration to be 1.0mg/mL, coating the goat anti-rabbit IgG diluent and the drug-antigen diluent on a quality control line and a test line on an NC film respectively according to the coating amount of 1.0 mu L/cm after the preparation, and then placing the mixture in a 45 ℃ oven for drying for 24 hours and then sealing and preserving at normal temperature;

(5) Hair lysate preparation

Preparing a lysate, wherein the formula is as follows: 0.3% PVP-10000,0.6% sodium sulfite, 0.4% casein, 1.2% keratinase, 4% Proclin300. And (3) fully dissolving and uniformly stirring the treatment liquid, canning, sealing, drying and storing.

The fittings such as the sample pad, the fluorescent pad, the NC film, the water absorbing paper and the like are sequentially adhered on the PVC bottom plate as required, as shown in figure 3, the water absorbing pad is adhered on the upper part of the nitrocellulose film to generate partial overlapping of 2mm, the lower end of the nitrocellulose film is arranged below the fluorescent pad, the partial overlapping of 2mm exists between the nitrocellulose film and the nitrocellulose film, the other end of the fluorescent pad is overlapped with the sample pad by 2mm, and the other end of the sample pad is aligned with the PVC lining plate. Cutting into 4.0mm strips after assembly, and placing into hair reagent card for pressing.

Preparation example 2

Preparation of morphine hair detection reagent

The morphine antibody and rabbit IgG are diluted by the buffer solutions of the examples 1-9 and the comparative examples 1-2 according to the proportion of 40% and 10%, then trehalose and sucrose are added, after full shaking and dissolution, the mixture is sprayed on a blank pad in a spraying amount of 1.0 mu L/cm, the blank pad is dried in an oven at 37 ℃ for 24 hours, the mixture is sealed and stored at normal temperature after the drying is finished, and the sample reaches a detection area and then reaches a quality control line according to the flowing direction of the sample, as shown in figure 1, the reagent line composition is as follows: the detection line is a shaking pill detection line, morphine antigen-bovine serum albumin conjugate is coated, the concentration is 0.3mg/mL, the dividing amount is 1.0 mu L/cm, the quality control line is coated with 0.8mg/mL goat anti-rabbit IgG, and the dividing amount is 1.0 mu L/cm. And (5) after coating, putting the plate film into a drying oven at 45 ℃ for drying for 24 hours, and sealing and storing at normal temperature after drying.

The morphine hair detection agent is produced as follows:

coating morphine antibody and rabbit IgG fluorescent marker on polyester fiber film, spot-coating morphine antigen and quality control line antibody on detection zone and quality control zone of nitrocellulose film by spot-coating machine, fully drying to make nitrocellulose film firmly adsorb raw material, compounding the above-mentioned polyester fiber film, glass fiber and nitrocellulose film on PVC plastic sheet, placing the compounded plastic sheet on cutting machine, cutting into single test paper, placing single test paper into plastic box matched with single test paper, placing aluminium foil bag, pyrolysis liquor, instruction book and ID card into packaging bag, sealing, waiting to be detected, and making the sample detection of the product to be detected be qualified and leaving factory, and its specific reagent structure diagram is shown in figure 3.

Preparation example 3

Preparation of methamphetamine hair detection reagent:

diluting methamphetamine antibody and rabbit IgG by using the buffer solutions of the examples 1-9 and the comparative examples 1-2 according to the proportion of 40% and 10%, respectively, adding trehalose and sucrose, sufficiently shaking for dissolution, spraying on a blank pad in a spraying amount of 1.0 mu L/cm, drying in a drying oven at 37 ℃ for 24 hours, sealing and storing at normal temperature, and when the flow direction of a sample is, the sample reaches a detection area and then reaches a quality control line, as shown in figure 1, the reagent line composition is as follows: the detection line is a methamphetamine detection line, the methamphetamine antigen-bovine serum albumin conjugate is coated, the concentration is 0.5mg/mL, the dividing amount is 1.0 mu L/cm, the quality control line is coated with 0.8mg/mL of goat anti-rabbit IgG, and the dividing amount is 1.0 mu L/cm. And (5) after coating, putting the plate film into a drying oven at 45 ℃ for drying for 24 hours, and sealing and storing at normal temperature after drying.

The production steps of the methamphetamine hair detection reagent are as follows:

coating a polyester fiber film with a methamphetamine antibody and a rabbit IgG fluorescent marker, spot-coating the methamphetamine antigen and the quality control line antibody on a detection zone and a quality control zone of a nitrocellulose film by a spot-coating machine, sufficiently drying to ensure that the nitrocellulose film firmly adsorbs raw materials, compositing the polyester fiber film, glass fiber and the nitrocellulose film on a PVC plastic sheet, placing the composited plastic sheet on a cutting machine, cutting into single test paper, placing the single test paper into a plastic box matched with the single test paper, placing an aluminum foil bag, a lysate, a specification and an ID card into a packaging bag, sealing, waiting to be detected, and performing spot-checking on the waiting to be detected product to ensure that the sensitivity, the specificity and the stability of the waiting to be detected product are qualified, wherein a specific reagent structure diagram is shown in figure 3.

Test case

(1) The fluorescent pads treated by the treatment solutions in the examples 1-9 and the comparative examples 1-2 are respectively prepared into morphine hair detection kit and methamphetamine hair detection kit, 10 human negative hair lysates are respectively detected, the test is repeated for 5 times, the supernatant of the negative hair lysates is sucked by a dropper, 3 drops of the supernatant are dripped into a sample-adding hole of a reagent plate, a reagent card is reacted for 3 minutes outside the machine after the sample is added, the reagent card is immediately inserted into an instrument after the reaction is finished, a test button is clicked, and the system automatically reads the card and gives a test result to record the negative accuracy.

(2) The fluorescent pads treated by the treatment solutions in the examples and the comparative examples are respectively prepared into morphine hair detection kit and methamphetamine hair detection kit, 5 positive hair (positive samples are confirmed by LCMS) lysate is respectively detected, the test is repeated for 10 times, the supernatant of the positive hair lysate is sucked by a dropper, 3 drops of the supernatant are dripped into a sample-adding hole of a reagent plate, a reagent card is shown in fig. 3, the reagent card is reacted for 3 minutes outside the machine after the sample is added, the reagent card is immediately inserted into an instrument after the reaction is finished, a test button is clicked, and the system automatically reads the card and gives a test result to record the positive accuracy.

(3) Morphine and methamphetamine fluorescent pads prepared from dilutions of examples 1-9 and comparative examples 1-2 above were prepared as morphine hair fluorescence detection kit and methamphetamine hair fluorescence detection kit, respectively, and tested 10 times per concentration point for morphine reference 2ng/mL and methamphetamine reference 2ng/mL with negative hair lysate.

The supernatant of the negative hair lysate is sucked by a dropper, 3 drops of the supernatant are dripped into a sample adding hole of a reagent plate, a reagent card is shown in fig. 3, the reagent card is reacted outside the machine for 3 minutes after the sample is added, the reagent card is immediately inserted into an instrument after the reaction is finished, a test button is clicked, the system automatically reads the card and gives a test result, the fluorescent value reacted by the instrument is recorded, and the CV value of the T/C is calculated according to the ratio T/C of the fluorescent value of a detection line to the fluorescent value of a quality control line.

And 3 drops of morphine reference and methamphetamine reference are dripped into a sample adding hole of the reagent plate by using a dropper, the reagent card is shown in figure 3, the reagent card is reacted outside the machine for 3 minutes after the sample is added, the reagent card is immediately inserted into an instrument after the reaction is finished, a 'test' button is clicked, the system automatically reads the card and gives a test result, the fluorescence value reacted by the instrument is recorded, and the CV value of the T/C is calculated according to the ratio T/C of the fluorescence value of the detection line to the fluorescence value of the quality control line.

The morphine hair detection kit negative sample results are shown in table 1, the methamphetamine hair detection kit negative sample results are shown in table 2, the LCMS confirms the positive sample concentration results are shown in table 3, the morphine hair detection kit positive sample results are shown in table 4, the methamphetamine hair detection kit positive sample results are shown in table 5, the morphine hair detection kit detection reference results are shown in table 6, and the methamphetamine hair detection kit detection reference results are shown in table 7.

TABLE 1

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TABLE 2

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TABLE 3 Table 3

Sample of	Morphine detection result ng/mg	Methamphetamine assay ng/mg
			Positive hair a	0.61	N/A
Positive hair b	1.55	N/A
			Positive hair c	4.73	N/A
Positive hair d	0.44	N/A
			Positive hair e	5.12	N/A
Positive hair f	N/A	5.88
			Positive hair g	N/A	4.71
Positive hair h	N/A	0.34
			Positive hair i	N/A	0.51
Positive hair j	N/A	3.05

TABLE 4 Table 4

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TABLE 5

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TABLE 6

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TABLE 7

The results show that:

(1) The test paper prepared by using the treatment liquid in the embodiment 1-9 has no false positive result in the detection of a negative sample, which shows that the drug fluorescent diluent disclosed by the invention can promote the release of fluorescent particles, so that the release of fluorescent particles is complete, and compared with the test paper prepared by using the treatment liquid in the comparative embodiment 1-2, 1 component (dispersing agent PVA-40 or stabilizer bovine serum albumin (no protease/IgG BSA)) is absent, and partial false positive condition can appear in the detection of the negative sample, which shows that the dispersing agent PVA-40 and the stabilizer bovine serum albumin (no protease/IgG BSA) have a synergistic effect in promoting the release of hair poisoning products and the strip running effect of the sample, so that the drug fluorescent diluent disclosed by the invention can promote the release of fluorescent particles, so that the background is white and clean after the detection, and the false positive result is reduced.

(2) The test paper prepared by the treatment liquid in the embodiment 1-9 has a positive detection rate of more than 95% in the detection of positive samples, which shows that the drug fluorescent diluent can promote the release of fluorescent particles in a fluorescent pad, so that the release of the fluorescent particles is complete, and compared with the test paper prepared by the treatment liquid in the embodiment 1-2, the test paper prepared by the treatment liquid in the embodiment 1-2 lacks 1 component (dispersing agent PVA-40 or stabilizer bovine serum albumin (no protease/IgG BSA)), partial false negative condition can occur in the detection of positive and negative samples, the positive detection rate is less than 80%, which shows that the dispersing agent PVA-40 or stabilizer bovine serum albumin (no protease/IgG BSA) has a synergistic effect in promoting the release and strip-running effects of the hair poisoning sample, and shows that the drug fluorescent diluent designed by the invention can promote the release of the fluorescent particles and improve the positive detection rate.

(3) The CV value of the fluorescent value T/C of the standard substance of 2ng/mL is less than 10% by using the test paper prepared by the treatment liquid of examples 1-9, which shows that the fluorescent diluent of the hair drug can reduce the reagent difference, the fluorescent diluent of the hair drug promotes the uniform release of fluorescent particles, improves the specific binding of drug antigens and antibodies, has strong anti-interference capability, compared with the test paper prepared by the treatment liquid of comparative examples 1-9, 1 component (dispersing agent PVA-40 or stabilizer bovine serum albumin (no protease/IgG BSA)) is absent, the CV value of the fluorescent diluent of the hair drug of the invention is greater than 20% by using the fluorescent diluent of the hair drug of the invention, which shows that the fluorescent diluent of the hair drug designed by the invention promotes the uniform release of fluorescent particles, improves the specific binding of drug antigens and antibodies and reduces other non-specific interference.

(4) Example 6 shows that the effect of TRIS (TRIS) is better than that of sodium tetraborate, as compared with example 3, by the difference in buffer composition, the integrated detection rate result and the CV value of fluorescence value T/C of 2ng/mL standard. Example 7 the stabilizer composition was different from example 3 and the integrated detection rate results and CV value of fluorescence value T/C of 2ng/mL standard showed that bovine serum albumin (protease free/IgG BSA) results were superior to casein. Example 8 shows that PVA-40 results are superior to PEG as compared to example 3 in that the dispersant composition is different. Example 9 shows that the composition of the stabilizer is different from that of example 3, and the result shows that the bovine serum albumin (no protease/IgG BSA) and casein are synergistically matched, and the effect of the stabilizer is superior to that of the bovine serum albumin (no protease/IgG BSA).

In summary, the invention creatively designs the diluent comprising the buffer solution, the stabilizer, the inorganic salt, the dispersing agent and the preservative, and the fluorescent marker is treated by using the diluent, so that the specificity and the uniformity of the hair fluorescent drug detection reagent product can be improved, the stability of the product is improved, the difference among the products is reduced, the detection accuracy is improved, the diluent is suitable for rapid screening in public places, and in addition, the components are safer and cannot cause environmental pollution.

The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims

1. A diluent for drug fluorescence detection, which is characterized by comprising a buffering agent, a stabilizing agent, inorganic salt and a dispersing agent;

the buffer comprises any one or a combination of at least two of tris, phosphate or carbonate;

the stabilizer comprises any one or a combination of at least two of bovine serum albumin, casein or PEG;

the dispersing agent comprises any one or a combination of at least two of polyvinyl alcohol, polyethylene glycol and lauryl alcohol.

2. The diluent for fluorescence detection of drugs of claim 1, wherein the inorganic salt comprises any one or a combination of at least two of sodium chloride, potassium chloride or borate.

3. The diluent for drug fluorescence detection according to claim 1 or 2, wherein the mass percentage of the buffer in the diluent is 1.5% -3.5%;

preferably, the mass percentage of the stabilizer in the diluent is 0.5% -2%;

preferably, the mass percentage of the inorganic salt in the diluent is 0.1-0.3%;

preferably, the mass percentage of the dispersing agent in the diluent is 0.4-0.8%.

4. A diluent for fluorescence detection of drugs according to any of claims 1-3, wherein said diluent further comprises a preservative and/or water;

preferably, the preservative comprises any one or a combination of at least two of Proclin300, sodium azide;

preferably, the mass percentage of the preservative in the diluent is 0.5-10%;

preferably, the diluent comprises, by mass, 100% of 1.5% -3.5% of buffer, 0.5% -2% of stabilizer, 0.1% -0.3% of inorganic salt, 0.4% -0.8% of dispersing agent, 0.5% -10% of preservative, and the balance of water.

5. A method of preparing a diluent for fluorescence detection of drugs according to any one of claims 1-4, comprising:

6. Use of a diluent for fluorescence detection of drugs according to any of claims 1-4 for the manufacture of a product for detecting drugs.

7. A method of preparing a drug fluorescence detection test strip, the method comprising:

diluting by mixing the diluent for drug fluorescence detection according to any one of claims 1-4 with an antibody labeled with a fluorescent marker, and coating the mixture on a binding pad; drawing the antigen solutions of drugs on the detection films respectively to form detection lines corresponding to different drugs;

and sequentially adhering the sample pad, the binding pad, the detection membrane and the absorption pad on the surface of the substrate from one end to the other end of the substrate to obtain the drug fluorescence detection test strip.

8. The method of preparing a drug fluorescence detection test strip of claim 7, wherein said fluorescent label comprises a fluorescent microsphere or a magnetic bead microsphere;

preferably, the dilution factor of the dilution is 5-20 times.

9. The method of preparing a drug fluorescence detection test strip of claim 7 or 8, wherein the drug comprises any one or a combination of at least two of morphine, anilines, ketamine, tetrahydrocannabinic acid, a cannabinoid or cocaine.

10. Use of a diluent for fluorescence detection of drugs according to any one of claims 1 to 4 and a method for preparing a fluorescence detection test strip for drugs according to any one of claims 7 to 9 in drug detection.