Diluent for fluorescent detection of drugs, and preparation method and application thereof

Abstract

This invention discloses a diluent for drug fluorescence detection, its preparation method, and its application. The diluent contains a buffer, a stabilizer, an inorganic salt, and a dispersant. The buffer includes any one or a combination of at least two of tris(hydroxymethyl)aminomethane, phosphates, or carbonates. The stabilizer includes any one or a combination of at least two of bovine serum albumin, casein, or PEG. The dispersant includes any one or a combination of at least two of polyvinyl alcohol, polyethylene glycol, or lauryl alcohol. This invention creatively discovers that a diluent composed of these four components—buffer, stabilizer, inorganic salt, and dispersant—can significantly improve the specificity and uniformity of drug detection reagents, increase product stability, reduce inter-product variability, and improve detection accuracy. It is suitable for rapid screening in public places. Furthermore, the components are relatively safe and will not cause environmental pollution.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, and relates to a diluent for drug fluorescence detection, its preparation method, and its application, particularly a diluent for drug fluorescence detection in hair, its preparation method, and its application. Background Technology

[0002] Traditional rapid drug testing techniques, primarily using colloidal gold methods on urine, have an detection time of only about 24–48 hours. Compared to biological samples such as urine, blood, and saliva, hair samples offer advantages such as stability, ease of collection, preservation, longer detection time, wider applicability, and lower risk of contamination. While traditional colloidal gold immunoassay techniques can effectively detect drugs and their metabolites in urine at very high levels, hair contains very low concentrations. Therefore, techniques with higher sensitivity and better resistance to interference are needed for hair testing.

[0003] Currently, drug test strips typically use fluorescent microspheres labeled with antibodies or antigens. Fluorescent microspheres are functional microspheres that have fluorescent substances labeled on their surface through chemical reactions or physical adsorption, or have fluorescent substances embedded or polymerized inside the microspheres, thus giving them a fluorescent display effect. Fluorescent microspheres can be of any shape, but are primarily spherical. Currently, types of fluorescent microspheres include quantum dot fluorescent microspheres, rare-earth fluorescent microspheres, and self-emissive fluorescent microspheres, among others. Fluorescent microspheres have advantages such as uniform particle size, good dispersibility, and high luminescence rate, but their stability is poor and they are affected by the external environment and media. Prolonged exposure or strong light irradiation may cause photobleaching or photolysis, leading to signal attenuation. When multiple different fluorescent dyes are used to label fluorescent microspheres, different fluorescent signals may interfere with or overlap, affecting the interpretation and quantitative analysis of results. Currently, the preparation process of fluorescent drug detection reagents often involves diluting the prepared fluorescently labeled antibodies or antigens with buffer solutions or inorganic salt solutions and then coating them. This process can easily lead to low detection sensitivity, poor specificity, and batch-to-batch instability, resulting in false negative and false positive results. For example, CN110850095A discloses a drug trace detection test strip and its preparation method and kit, which uniformly disperses time-resolved fluorescent microsphere-labeled specific monoclonal antibodies in phosphate buffer.

[0004] In conclusion, developing novel diluents for reconstitution and dilution after fluorescent labeling during test strip preparation to improve the stability and uniformity of fluorescence signals is of great significance for the field of drug fluorescence detection. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention relates to a diluent for drug fluorescence detection, its preparation method, and its application, particularly a diluent for drug fluorescence detection in hair, aiming to improve detection stability and accuracy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a diluent for drug fluorescence detection, the diluent containing a buffer, a stabilizer, an inorganic salt, and a dispersant; the buffer includes any one or a combination of at least two of tris(hydroxymethyl)aminomethane, phosphate, and carbonate; the stabilizer includes any one or a combination of at least two of bovine serum albumin, casein, or PEG; and the dispersant includes any one or a combination of at least two of polyvinyl alcohol, polyethylene glycol, or lauryl alcohol.

[0008] In this invention, a specific diluent formulation is designed, containing buffers, stabilizers, inorganic salts, and dispersants. During the coating process of the test strip conjugate pad (or fluorescent pad), it is used to dilute the antibody or antigen labeled with the fluorescent marker. The buffers and inorganic salts provide a suitable ionic environment for the fluorescent marker by adjusting the ion concentration of the system. The stabilizers increase the stability of the fluorescent marker and reduce the variability of reagent results. The dispersants prevent the deposition or precipitation of fluorescent marker particles. This improves the stability and uniformity of the hair drug fluorescent reagent without affecting its specificity, thereby improving the stability and accuracy of the detection.

[0009] Preferably, the inorganic salt includes any one or a combination of at least two of sodium chloride, potassium chloride, or sodium borate.

[0010] Preferably, the mass percentage 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%.

[0011] Preferably, the mass percentage of stabilizer in the diluent is 0.5% to 2%, including but not limited to 0.6%, 0.7%, 0.8%, 1%, 1.5%, 1.6%, 1.8%, or 1.9%.

[0012] Preferably, the mass percentage of inorganic salts in the diluent is 0.1% to 0.3%, including but not limited to 0.15%, 0.18%, 0.2%, 0.25%, 0.28%, or 0.29%.

[0013] Preferably, the mass percentage of the dispersant in the diluted solution is 0.4% to 0.8%, including but not limited to 0.5%, 0.6%, or 0.7%.

[0014] Preferably, the polyvinyl alcohol comprises PVA-40 or PVA-10.

[0015] Preferably, the diluent further contains a preservative and / or water.

[0016] Preferably, the preservative includes any one or a combination of at least two of Proclin 300 and sodium azide.

[0017] Preferably, the mass percentage of the preservative in the diluted solution is 0.5% to 10%, including but not limited to 0.6%, 0.7%, 0.8%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%.

[0018] Preferably, the diluent for drug fluorescence detection contains, by mass percentage, 1.5% to 3.5% buffer, 0.5% to 2% stabilizer, 0.1% to 0.3% inorganic salt, 0.4% to 0.8% dispersant, and 0.5% to 10% preservative, with the balance being water.

[0019] In a second aspect, the present invention provides a method for preparing the diluent for drug fluorescence detection as described in the first aspect, the method comprising:

[0020] The buffer, stabilizer, inorganic salt and dispersant are mixed to obtain the diluted solution.

[0021] Thirdly, the present invention provides the application of the diluent for drug fluorescence detection described in the first aspect in the preparation of products for detecting drugs.

[0022] Fourthly, the present invention provides a method for preparing a fluorescent drug detection test strip, the method comprising:

[0023] The drug fluorescence detection diluent described in the first aspect is mixed with the fluorescently labeled antibody and diluted, and then coated onto the conjugate pad; the drug antigen solution is streaked onto the detection membrane to form detection lines corresponding to different drugs; the sample pad, conjugate pad (or fluorescent pad), detection membrane and absorbent pad are sequentially adhered to the surface of the substrate from one end to the other to obtain the drug fluorescence test strip.

[0024] In this invention, a specific diluent formulation is designed that can be effectively applied in the preparation of fluorescent drug test strips. During the coating process of the test strip conjugate pad (or fluorescent pad), it is used to dilute the antibody or antigen labeled with the fluorescent marker, significantly improving the detection stability and accuracy of the test strip.

[0025] Preferably, the fluorescent marker comprises fluorescent microspheres or magnetic microspheres.

[0026] Preferably, the dilution factor is 5 to 20 times.

[0027] Preferably, the drug includes any one or a combination of at least two of morphine, amphetamines, ketamine, tetrahydrocannabinol, cannabinoids, or cocaine.

[0028] Preferably, the method for preparing the drug fluorescent test strip includes:

[0029] (I) Preparation of sample pad treatment solution: The formula is as follows: 0.5% to 5% sodium tetraborate, 0.1% to 10% S17, 0.1% to 1% casein, 0.1% to 1% sodium cholate, and 0.5% to 10% Proclin 300.

[0030] (II) Sample pad treatment: After the sample pad treatment solution is fully dissolved and stirred evenly, it is sprayed onto the glass fiber of appropriate size, placed in an oven at 40-50℃ and dried for 12-24 hours, then removed, sealed and dried for storage.

[0031] (III) Preparation of fluorescent pads: The fluorescent microsphere-labeled monoclonal antibody and labeled rabbit IgG were diluted with the diluent at a ratio of 20%–60% and 5%–20%, respectively. 0.1–0.5 g / mL of sucrose and 0.01–0.1 g / mL of trehalose were added. After thorough dissolution, the mixture was sprayed onto blank pads at a spray rate of 1.0 μL / cm. The pads were then dried in an oven at 30–40 °C for 12–24 h and stored in a sealed container away from light.

[0032] (iv) Preparation of coating membrane: The drug-antigen hemoglobin conjugate and goat anti-rabbit IgG were diluted with PBS to a concentration of 0.1-1.0 mg / mL using coating buffer. Coomassie Brilliant Blue was added to the goat anti-rabbit IgG to a concentration of 0.1-1 mg / mL. After preparation, the above goat anti-rabbit IgG dilution and drug-antigen dilution were coated onto the control line and test line of a nitrocellulose membrane (NC membrane) at a coating amount of 1.0 μL / cm, and then dried in an oven at 40-50℃ for 20-30 h.

[0033] (V) Assembly: On the PVC base plate, the sample pad, fluorescent pad, NC membrane and absorbent pad are attached in sequence. The absorbent pad is attached to the upper part of the nitrocellulose membrane, with a partial overlap of 1-2 mm. The lower end of the nitrocellulose membrane is placed below the fluorescent pad, with a partial overlap of 1-2 mm between the two. The other end of the fluorescent pad overlaps with the sample pad by 1-2 mm, and the other end of the sample pad is aligned with the PVC backing plate.

[0034] Fifthly, the present invention provides the application of the diluent for drug fluorescence detection described in the first aspect and the method for preparing drug fluorescence test strips described in the fourth aspect in drug detection.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This invention creatively discovers that a diluent composed of four components—buffer, stabilizer, inorganic salt, and dispersant—can significantly improve the specificity and uniformity of drug testing reagents, increase product stability, reduce inter-product variability, and improve detection accuracy. It is suitable for rapid screening in public places. Furthermore, each component is relatively safe and will not cause environmental pollution. Attached Figure Description

[0037] Figure 1 This is a schematic diagram illustrating the principle of positive sample detection in this invention;

[0038] Figure 2 This is a schematic diagram illustrating the principle of negative sample detection in this invention;

[0039] Figure 3 This is a schematic diagram of the test strip structure of the present invention. Detailed Implementation

[0040] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0041] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0042] The technical principle of this invention is as follows: After processing, the sample to be tested, such as hair sample, is dissolved in a certain amount of lysis buffer. After thorough mixing and reaction for 1-2 minutes, it is dropped onto the sample pad in the sample application area. The analyte antigen in the positive sample binds to the fluorescent microsphere-labeled antibody in the fluorescent pad and is chromatographyd forward through capillary action. When it reaches the NC membrane in the detection area, the antigen immobilized on the detection line does not bind to the fluorescent microsphere-labeled antibody, meaning there is no fluorescence reaction on the detection line, resulting in a strong positive result. Figure 1 As shown. In negative samples, the analyte antigen is absent. The fluorescently labeled antibody in the fluorescent pad is chromatographically deposited onto the NC membrane via capillary action, binding to the antigen-protein conjugate coated in the detection area, exhibiting a strong fluorescent reaction, as shown. Figure 2As shown, the amount of fluorescently labeled antibody bound to the detection line is inversely proportional to the amount of analyte in the sample, while the amount of fluorescent label bound to the control line is independent of the amount of analyte in the sample. Other fluorescent labels continue chromatography to reach the absorption region. After chromatography, the fluorescence intensity of the detection line and control line is read using a fluorescence reader, and the T / C value is calculated. The content of the analyte in the sample can then be calculated using the instrument's built-in standard curve.

[0043] Example 1

[0044] This embodiment provides a diluent for detecting fluorescent drugs in hair, the components of which are as follows (by mass ratio, based on 100%): 1.5% tris(hydroxymethyl)aminomethane (TRIS), 0.5% bovine serum albumin (protease-free / IgG BSA), 0.1% sodium chloride (NaCl), 0.4% polyvinyl alcohol (PVA-40), 0.6% preservative (Proclin 300), and the balance being water.

[0045] Example 2

[0046] This embodiment provides a diluent for detecting fluorescent drugs in hair, the components of which are as follows (by mass ratio, based on 100%): Tris(hydroxymethyl)aminomethane (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 being water.

[0047] Example 3

[0048] This embodiment provides a diluent for detecting fluorescent drugs in hair, the components of which are as follows (by mass ratio, based on 100%): Tris(hydroxymethyl)aminomethane (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 being water.

[0049] Example 4

[0050] This embodiment provides a diluent for a hair fluorescent drug test strip, the components of which are as follows by mass ratio (based on 100%): Tris(hydroxymethyl)aminomethane (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 being water.

[0051] Example 5

[0052] This embodiment provides a diluent for a hair fluorescent drug test strip, the components of which are as follows (by mass ratio, based on 100%): Tris(hydroxymethyl)aminomethane (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 being water.

[0053] Example 6

[0054] This embodiment provides a diluent for detecting fluorescent drugs in hair, the components of which are as follows by mass ratio (based on 100%): 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 being water.

[0055] Example 7

[0056] This embodiment provides a diluent for detecting fluorescent drugs in hair, the components of which are as follows by mass ratio (based on 100%): 2.5% tris(hydroxymethyl)aminomethane (TRIS), 1.5% casein, 0.3% sodium chloride (NaCl), 0.6% polyvinyl alcohol (PVA-40), 0.6% preservative (Proclin 300), and the balance being water.

[0057] Example 8

[0058] This embodiment provides a diluent for detecting fluorescent drugs in hair, the components of which are as follows (by mass ratio, based on 100%): Tris(hydroxymethyl)aminomethane (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 being water.

[0059] Example 9

[0060] This embodiment provides a diluent for detecting fluorescent drugs in hair, the components of which are as follows by mass ratio (based on 100%): Tris(hydroxymethyl)aminomethane (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%, and the balance being water.

[0061] Comparative Example 1

[0062] This comparative example provides a diluent for a hair fluorescent drug test strip, which differs from Example 1 only in that it does not contain a stabilizer and its mass fractions are proportionally allocated to the buffer, dispersant, and inorganic salt.

[0063] Comparative Example 2

[0064] This comparative example provides a diluent for a hair fluorescent drug test strip, which differs from Example 1 only in that it does not contain a dispersant and its mass fractions are proportionally allocated to the mass fractions of the buffer, stabilizer, and inorganic salt.

[0065] Preparation Example 1

[0066] Preparation of a fluorescent detection kit for detecting drugs in hair.

[0067] (1) Preparation of fluorescently labeled proteins

[0068] Prepare a 0.05M 2-morpholinoethanesulfonic acid (MES) solution, adjust the pH, add 10% fluorescent microspheres, and rotate at 50 rpm for 60 min. Remove the microspheres and add 50 mg / mL N-hydroxysuccinimide (NHS) and 50 mg / mL carbodiimide (EDC) solutions sequentially. Continue rotating for 60 min. After rotation, centrifuge at 15000 rpm for 15 min, remove the supernatant, add MES buffer to dissolve the microspheres completely, add 0.5 mg of antibody, and rotate for 2.5 h. After the reaction, add 10% blocking agent, rotate for 3 h, centrifuge at 15000 rpm for 15 min, remove the supernatant, and then reconstitute with 1 mL Tris buffer.

[0069] (2) Sample pad treatment

[0070] Prepare the sample pad treatment solution with the following formula: 2.8% sodium tetraborate, 3.6% S17, 0.4% casein, 0.5% sodium cholate, and 4% Proclin 300. After thoroughly dissolving and stirring the solution, spray it onto the glass fiber and dry it in an oven at 37°C for 24 hours. Then, remove it, seal it, and store it in a dry place.

[0071] (3) Preparation of fluorescent pads

[0072] The fluorescent microsphere-labeled monoclonal antibody and rabbit IgG were diluted at ratios of 40% and 10% using the buffer solutions from Examples 1-9 and Comparative Examples 1-2, respectively. 0.5 g / mL sucrose and 0.2 g / mL trehalose were then added. After thorough dissolution, the mixture was sprayed onto blank pads at a rate of 1.0 μL / cm, and then dried in a 37°C oven for 24 hours before being stored in a sealed container away from light.

[0073] (4) Preparation of coating membrane

[0074] The drug-antigen hemoglobin conjugate and goat anti-rabbit IgG were diluted to 0.5 mg / mL and 0.8 mg / mL with PBS using coating buffer. Coomassie Brilliant Blue was added to the goat anti-rabbit IgG to prepare a concentration of 1.0 mg / mL. After preparation, the above goat anti-rabbit IgG dilution and drug-antigen dilution were coated onto the control line and test line of the NC membrane at a coating amount of 1.0 μL / cm, respectively. The membrane was then dried in a 45℃ oven for 24 h and then stored at room temperature in a sealed container.

[0075] (5) Preparation of hair lysis solution

[0076] Prepare the lysis buffer with the following formula: 0.3% PVP-10000, 0.6% sodium sulfite, 0.4% casein, 1.2% keratinase, and 4% Proclin 300. After thoroughly dissolving and mixing the solution, bottle it, seal it, and store it in a dry place.

[0077] On the PVC base plate, attach the sample pad, fluorescent pad, NC film, absorbent paper, and other accessories in sequence as required. Figure 3 As shown, the absorbent pad is attached to the upper part of the nitrocellulose membrane, creating a 2mm overlap. The lower end of the nitrocellulose membrane is placed below the fluorescent pad, with a 2mm overlap between them. The other end of the fluorescent pad overlaps the sample pad by 2mm, and the other end of the sample pad is aligned with the PVC backing. After assembly, the sample pad is cut into 4.0mm reagent strips, placed in the hair reagent card, and pressed firmly for later use.

[0078] Preparation Example 2

[0079] Preparation of morphine hair test reagent

[0080] Morphine antibody and rabbit IgG were diluted at ratios of 40% and 10% respectively using the buffer solutions from Examples 1-9 and Comparative Examples 1-2. Trehalose and sucrose were then added, and the mixture was thoroughly shaken to dissolve. The solution was then sprayed onto blank pads at a rate of 1.0 μL / cm and dried in an oven at 37°C for 24 hours. After drying, the pads were sealed and stored at room temperature. Following the flow direction of the samples, the samples first reached the detection zone and then the control line. Figure 1 As shown, the reagent lines are composed as follows: the test line is an ecstasy test line, coated with morphine antigen-bovine serum albumin conjugate at a concentration of 0.3 mg / mL, with a stroke volume of 1.0 μL / cm; the control line is coated with 0.8 mg / mL goat anti-rabbit IgG, with a stroke volume of 1.0 μL / cm. After coating, the plates are dried in an oven at 45℃ for 24 hours, and then sealed and stored at room temperature.

[0081] The production steps of morphine hair follicle testing reagent are as follows:

[0082] Morphine antibody and rabbit IgG fluorescent marker were coated onto a polyester fiber membrane. Morphine antigen and control line antibody were spotted onto the detection and control zones of the nitrocellulose membrane using a dotting machine. After thorough drying, the nitrocellulose membrane was allowed to firmly adsorb the raw materials. The polyester fiber membrane, glass fiber, and nitrocellulose membrane were then laminated onto a PVC plastic sheet. The laminated plastic sheet was placed on a cutting machine and cut into single-use test strips. The single-use test strips were placed into a matching plastic box. An aluminum foil bag, lysis buffer, instruction manual, and ID card were placed into the packaging bag, sealed, and ready for testing. Samples were randomly selected for testing to ensure their sensitivity, specificity, and stability were met before shipment. The specific reagent structure diagram is shown below. Figure 3 As shown.

[0083] Preparation Example 3

[0084] Preparation of methamphetamine hair follicle test reagent:

[0085] Methamphetamine antibody and rabbit IgG were diluted at ratios of 40% and 10% respectively using the buffer solutions from Examples 1-9 and Comparative Examples 1-2. Trehalose and sucrose were then added, and the mixture was thoroughly shaken to dissolve. The solution was then sprayed onto blank pads at a rate of 1.0 μL / cm and dried in an oven at 37°C for 24 hours. After drying, the pads were sealed and stored at room temperature. Following the flow direction of the samples, the samples first reached the detection zone and then the control line. Figure 1 As shown, the reagent lines are composed as follows: the detection line is a methamphetamine detection line coated with methamphetamine antigen-bovine serum albumin conjugate at a concentration of 0.5 mg / mL, with a stroke volume of 1.0 μL / cm; the control line is coated with 0.8 mg / mL goat anti-rabbit IgG, with a stroke volume of 1.0 μL / cm. After coating, the plates are dried in an oven at 45°C for 24 hours, and then sealed and stored at room temperature.

[0086] The production steps of the methamphetamine hair follicle test kit are as follows:

[0087] Methamphetamine antibody and rabbit IgG fluorescent marker were coated onto a polyester fiber membrane. Methamphetamine antigen and control line antibody were spotted onto the detection and control zones of the nitrocellulose membrane using a spotting machine. The membrane was thoroughly dried to ensure strong adsorption of the raw materials. The polyester fiber membrane, glass fiber, and nitrocellulose membrane were then laminated onto a PVC plastic sheet. The laminated plastic sheet was placed on a cutting machine and cut into single-use test strips. Each test strip was placed into a matching plastic box. An aluminum foil bag, lysis buffer, instruction manual, and ID card were placed inside the packaging bag, sealed, and ready for testing. Samples were randomly selected for testing to ensure sensitivity, specificity, and stability; those that passed were released from the factory. A detailed reagent structure diagram is shown below. Figure 3 As shown.

[0088] Test case

[0089] (1) The fluorescent pads treated with the solutions in Examples 1-9 and Comparative Examples 1-2 were used to prepare morphine hair follicle detection kits and methamphetamine hair follicle detection kits, respectively. Ten samples of negative hair lysis buffer were tested, and the tests were repeated five times. The supernatant of the negative hair lysis buffer was aspirated with a dropper and added 3 drops to the sample wells of the reagent plate. The reagent card was placed as shown in the image. Figure 3 As shown, after adding the sample, react outside the instrument for 3 minutes. After the reaction is complete, immediately insert the reagent card into the instrument and click the "Test" button. The system will automatically read the card and give the test result, recording the negative accuracy rate.

[0090] (2) The fluorescent pads treated with the above-mentioned solutions in the examples and comparative examples were used to prepare morphine hair detection kits and methamphetamine hair detection kits, respectively. Five samples of positive hair lysates (concentration of positive samples confirmed by LCMS) were tested, and the tests were repeated 10 times. The supernatant of the positive hair lysate was aspirated with a dropper and added 3 drops to the sample wells of the reagent plate. The reagent card was then... Figure 3 As shown, after adding the sample, react outside the instrument for 3 minutes. After the reaction is complete, immediately insert the reagent card into the instrument and click the "Test" button. The system will automatically read the card and give the test result, and record the positive accuracy rate.

[0091] (3) Using the morphine fluorescent pads and methamphetamine fluorescent pads prepared by diluting the above Examples 1-9 and Comparative Examples 1-2, the gold pads were used to prepare morphine hair fluorescence detection kits and methamphetamine hair fluorescence detection kits, respectively. The negative hair lysis buffer was tested 10 times repeatedly. The negative hair lysis buffer was used to prepare morphine reference standard 2 ng / mL and methamphetamine reference standard 2 ng / mL, and each concentration point was tested 10 times repeatedly.

[0092] Using a dropper, draw up the supernatant of the negative hair lysis buffer and add 3 drops to the sample well of the reagent plate. The reagent card should be inserted as shown in the image. Figure 3 As shown, after adding the sample, react outside the instrument for 3 minutes. After the reaction is complete, immediately insert the reagent card into the instrument and click the "Test" button. The system will automatically read the card and give the test result. Record the fluorescence value of the instrument reaction. Calculate the CV value of T / C by using the ratio of the fluorescence value of the detection line to the fluorescence value of the control line.

[0093] Using a dropper, pipette 2 ng / mL morphine reference standard and 2 ng / mL methamphetamine reference standard, and add 3 drops to each well of the reagent plate. The reagent card should be inserted as shown in the image. Figure 3 As shown, after adding the sample, react outside the instrument for 3 minutes. After the reaction is complete, immediately insert the reagent card into the instrument and click the "Test" button. The system will automatically read the card and give the test result. Record the fluorescence value of the instrument reaction. Calculate the CV value of T / C by using the ratio of the fluorescence value of the detection line to the fluorescence value of the control line.

[0094] Table 1 shows the results of negative samples from the morphine hair follicle test kit; Table 2 shows the results of negative samples from the methamphetamine hair follicle test kit; Table 3 shows the concentration results of positive samples confirmed by LCMS; Table 4 shows the results of positive samples from the morphine hair follicle test kit; Table 5 shows the results of positive samples from the methamphetamine hair follicle test kit; Table 6 shows the results of the morphine hair follicle test kit reference material; and Table 7 shows the results of the methamphetamine hair follicle test kit reference material.

[0095] Table 1

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] Table 2

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] Table 3

[0108] sample Morphine test result ng / mg Methamphetamine test result 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

[0109] Table 4

[0110]

[0111]

[0112]

[0113] Table 5

[0114]

[0115]

[0116]

[0117] Table 6

[0118]

[0119]

[0120] Table 7

[0121]

[0122] The results show that:

[0123] (1) The test strips prepared using the treatment solutions of Examples 1-9 showed no false positive results in the detection of negative samples, indicating that the drug fluorescent diluent of the present invention can promote the release of fluorescent particles and ensure complete release of fluorescent particles. The test strips prepared using the treatment solutions of Comparative Examples 1-2 lacked one component (dispersant PVA-40 or stabilizer bovine serum albumin (without protease / IgG BSA)) compared with Example 3, and some false positives occurred in the detection of negative samples. This indicates that dispersant PVA-40 and stabilizer bovine serum albumin (without protease / IgG BSA) have a synergistic effect in promoting drug release in hair and sample strip running efficiency. This shows that the drug fluorescent diluent designed in the present invention can promote the release of fluorescent particles, making the background white and clean after detection, and reducing false positive results.

[0124] (2) The test strips prepared using the treatment solutions of Examples 1-9 showed a positive detection rate of >95% in the detection of positive samples, indicating that the drug fluorescent diluent of the present invention can promote the release of fluorescent particles in the fluorescent pad and ensure complete release of fluorescent particles. The test strips prepared using the treatment solutions of Comparative Examples 1-2 lacked one component (dispersant PVA-40 or stabilizer bovine serum albumin (without protease / IgG BSA)) compared with Example 3, and some false negatives occurred in the detection of positive and negative samples, with a positive detection rate of <80%. This indicates that the dispersant PVA-40 or stabilizer bovine serum albumin (without protease / IgG BSA) has a synergistic effect in promoting the release of drug samples in hair and the strip running effect, indicating that the drug fluorescent diluent designed in the present invention can promote the release of fluorescent particles and improve the positive detection rate.

[0125] (3) The CV value of the test strips prepared using the treatment solutions of Examples 1-9 for detecting the fluorescence value T / C of 2 ng / mL standard was <10%, indicating that the hair drug fluorescent diluent of the present invention can reduce the variability of reagents. This shows that the drug fluorescent diluent of the present invention promotes the uniform release of fluorescent particles, improves the specific binding of drug antigens and antibodies, and has strong anti-interference ability. The test strips prepared using the treatment solutions of Comparative Examples 1-2 lack one component (dispersant PVA-40 or stabilizer bovine serum albumin (without protease / IgG BSA)) compared with Example 3. The CV value of the fluorescence value T / C of 2 ng / mL standard for detecting the lack of dispersant PVA-40 or stabilizer bovine serum albumin (without protease / IgG BSA) was >20%, indicating that dispersant PVA-40 or stabilizer bovine serum albumin (without protease / IgG BSA) has a synergistic effect in promoting the specific binding of drugs in hair. This shows that the drug fluorescent diluent designed in the present invention promotes the uniform release of fluorescent particles, improves the specific binding of drug antigens and antibodies, and reduces other non-specific interference.

[0126] (4) In Example 6, the buffer composition differed from that in Example 3. The overall detection rate results and the CV value of the fluorescence value (T / C) of the 2 ng / mL standard indicated that tris(hydroxymethyl)aminomethane (TRIS) was more effective than sodium tetraborate. In Example 7, the stabilizer composition differed from that in Example 3. The overall detection rate results and the CV value of the fluorescence value (T / C) of the 2 ng / mL standard indicated that bovine serum albumin (protease-free / IgG BSA) was more effective than casein. In Example 8, the dispersant composition differed from that in Example 3. The experimental results showed that PVA-40 was more effective than PEG. In Example 9, the stabilizer composition differed from that in Example 3. The results showed that bovine serum albumin (protease-free / IgG BSA) and casein worked synergistically, resulting in a better effect than bovine serum albumin (protease-free / IgG BSA).

[0127] In summary, this invention creatively designs a diluent containing four components: buffer solution, stabilizer, inorganic salt, dispersant, and preservative. Using this diluent to treat fluorescent markers significantly improves the specificity and uniformity of hair follicle fluorescent drug detection reagents, increases product stability, reduces inter-product variability, and enhances detection accuracy. It is suitable for rapid screening in public places. Furthermore, the components are relatively safe and will not cause environmental pollution.

[0128] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A diluent for fluorescent detection of drugs, characterized in that, The diluted solution contains, by mass percentage, 1.5%~3.5% buffer, 0.5%~2% stabilizer, 0.1%~0.3% inorganic salt, 0.4%~0.8% dispersant and 0.5%~10% preservative, with the balance being water; The buffer is tris(hydroxymethyl)aminomethane; The stabilizer is a combination of bovine serum albumin and casein; The dispersant is polyvinyl alcohol.

2. The diluent for drug fluorescence detection according to claim 1, characterized in that, The inorganic salt includes 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, characterized in that, The preservatives include any one or a combination of at least two of Proclin 300 and sodium azide.

4. The method for preparing the diluent for drug fluorescence detection according to any one of claims 1-3, characterized in that, The preparation method includes: The buffer, stabilizer, inorganic salt, dispersant, preservative and water are mixed to obtain the diluted solution.

5. The use of the diluent for drug fluorescence detection as described in any one of claims 1-3 in the preparation of products for drug detection.

6. A method for preparing a fluorescent drug detection test strip, characterized in that, The method includes: The drug fluorescence detection diluent according to any one of claims 1-3 is mixed with the antibody labeled with a fluorescent marker and diluted, and then coated onto the conjugate pad; the drug antigen solution is streaked onto the detection membrane to form detection lines corresponding to different drugs; The sample pad, conjugate pad, detection membrane, and absorbent pad are sequentially adhered to the surface of the substrate from one end to the other to obtain the drug fluorescent test strip.

7. The method for preparing a fluorescent drug detection test strip according to claim 6, characterized in that, The fluorescent markers include fluorescent microspheres or magnetic microspheres.

8. The method for preparing a fluorescent drug detection test strip according to claim 6, characterized in that, The dilution factor is 5 to 20 times.

9. The method for preparing a fluorescent drug detection test strip according to claim 6, characterized in that, The drugs include any one or a combination of at least two of the following: morphine, amphetamines, ketamine, tetrahydrocannabinol, cannabinoids, or cocaine.

10. The application of the diluent for drug fluorescence detection according to any one of claims 1-3 and the method for preparing drug fluorescence test strips according to any one of claims 6-9 in drug detection.

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