Fluorescence quenching immunochromatography test paper for rapidly detecting methylamphetamine in saliva as well as preparation method and application of fluorescence quenching immunochromatography test paper

Abstract

The invention belongs to the technical field of rapid detection of drugs, and relates to fluorescence quenching immunochromatography test paper, a preparation method and application of the fluorescence quenching immunochromatography test paper in rapid detection of methamphetamine in saliva. A detection line formed by coupling a methylamphetamine antigen with a quantum dot and a quality control line formed by coupling bovine serum albumin with the quantum dot are sequentially arranged on the NC membrane, the gold label pad is coated with an AuNPs-mAb quencher, and the AuNPs-mAb quencher is prepared by coupling colloidal gold with a methylamphetamine antibody. Aiming at the defect that a colloidal gold test strip cannot be accurately quantified, the fluorescence quenching immunochromatography test paper provided by the invention can simultaneously realize qualitative screening and quantitative detection of methylamphetamine residues in a saliva sample; by means of a fluorescent quantitative card reader, a standard curve is established, and accurate quantification of methylamphetamine in saliva is achieved; the method is simple to operate, rapid in detection, convenient to use, high in sensitivity and low in cost.

Description

Technical Field

[0001] The invention belongs to the technical field of rapid drug detection, and specifically relates to a fluorescence quenching immunochromatographic test paper for rapid detection of methamphetamine in saliva, a preparation method and application thereof. The test paper can be used to rapidly detect the content of methamphetamine in a saliva sample. Background Art

[0002] Methamphetamine is modified based on the chemical structure of ephedrine. It looks like pure white crystals, so it is also called ice. Ice is highly addictive and is currently the most harmful synthetic drug in my country. After taking it, it has a strong stimulating effect on the human central nervous system and is highly toxic. It consumes a lot of physical strength, reduces immune function, and seriously damages the heart and brain tissues and even causes death. Addicts can also develop mental disorders, show delusions, aggression, and illusions, which can lead to violent behavior and seriously endanger social order and public safety. Therefore, establishing a quick and accurate method to screen and detect methamphetamine residues in the body can quickly and efficiently identify drug users, which is of great significance for maintaining social order.

[0003] Methamphetamine can be detected using a variety of methods, such as gas chromatography (GC), high performance liquid chromatography (HPLC), gas / liquid chromatography-mass spectrometry (GC / LC-MS-MS), etc. These methods have extremely high sensitivity and accuracy, but the pre-treatment process for the detection sample is complicated and relies on expensive equipment, a long time and professional staff. They are usually used as laboratory tests and are not suitable for rapid screening of a large number of samples. There is no way to quickly screen a large number of samples on site. Enzyme-linked immunosorbent assay (ELISA) and chemiluminescence (CL) have attracted much attention due to their high sample throughput and have been successfully applied to the detection of methamphetamine. However, they are still limited by time-consuming incubation processes and multi-step operations, and have inconvenient disadvantages in on-site detection.

[0004] In contrast, lateral flow immunochromatographic sensor (LFICS) is a disposable, low-cost, rapid, specific and sensitive detection method. Among them, colloidal gold immunochromatography is an immunochromatographic technique established by using colloidal gold as a marker and the specific reaction of antigen and antibody. This technique does not require expensive equipment, is simple to operate, and has a short detection time (3-5min), which is suitable for on-site batch screening of samples. However, although this method has its advantages, traditional test strips using gold nanoparticles usually have relatively low sensitivity due to limited signal amplification; for example, in Chinese patent CN109085347A, colloidal gold immunochromatographic test strips based on gold nanoparticles detect methamphetamine in saliva, and its detection limit is 50ng / mL. Due to the limitation of sensitivity and the negative readout of signal intensity, this method is not satisfactory.

[0005] Colloidal gold immunochromatographic test strips are easy to operate and can be used for rapid on-site testing, but their sensitivity is relatively low and they cannot be accurately quantified. Moreover, the objects of detection are mainly the blood and urine of the subjects. Such samples are prone to inconvenience and embarrassment in sampling and require special places. Samples are easily replaced or adulterated, and test results are easily interfered by common legal drugs and may result in false positive results.

[0006] The invention aims at the disadvantages of existing colloidal gold test paper for methamphetamine, such as low sensitivity, susceptibility to matrix interference, inability to accurately quantify, etc., and provides a preparation method and a quantitative detection method of a fluorescence quenching immunochromatographic test strip for on-site detection of methamphetamine, which is high in sensitivity, simple and fast, and has little matrix interference, and also provides the application of the test strip in a saliva matrix. Summary of the invention

[0007] The purpose of the present invention is to solve the above-mentioned problems existing in the prior art, and proposes a fluorescence quenching immunochromatographic test paper for rapid detection of methamphetamine in saliva, a preparation method and application thereof, quantum dots (QDs) with an emission wavelength of 525 nm are used as fluorescent donors to label antigens and fixed on an NC membrane, colloidal gold (AuNPs) with an absorption wavelength of 525 nm are used as fluorescent receptors to couple with antibodies, and quantum dot fluorescence quenching immunochromatographic test paper is prepared based on the fluorescence resonance energy transfer (FRET) principle. The detection result is positively correlated with the concentration of the analyte, the appearance of the fluorescent signal is the detection limit, the naked eye is easier to capture and the resolution is high, and compared with traditional immunochromatographic test paper, the sensitivity is greatly improved.

[0008] The technical solution of the present invention is:

[0009] The invention provides a fluorescence quenching immunochromatographic test paper for rapidly detecting methamphetamine in saliva, comprising a sample pad, glass fiber filter paper, a gold label pad, an NC membrane and a water absorbent pad, wherein a detection line formed by quantum dots coupling to methamphetamine antigens and a quality control line formed by quantum dots coupling to bovine serum albumin are sequentially arranged on the NC membrane, an AuNPs-mAb quencher is coated on the gold label pad, and the AuNPs-mAb quencher is prepared by coupling colloidal gold with a methamphetamine antibody.

[0010] The invention adds glass fiber filter paper to the composition of conventional test strips to play an online filtering role, realizes direct sample loading and detection of saliva samples, thereby simplifying the operation steps and improving the detection sensitivity.

[0011] Further, the quantum dots include cadmium selenide (CdSe), cadmium telluride (CdTe), cadmium sulfide (CdS), zinc selenide (ZnSe), indium phosphide (InP), indium arsenide (InAs), and nanocrystals or semiconductor nanocrystals composed of a layer of ZnS or CdS wrapped on a CdSe core. The quantum dots serve as fluorescent donors, and the colloidal gold serves as fluorescent acceptors.

[0012] Quantum dots are believed to have excellent photophysical properties such as wide absorption spectrum, large extinction coefficient, narrow and tunable photoluminescence, high emission quantum yield, long fluorescence, and good biocompatibility, which enable quantum dots to play an important role in bioimaging and biosensing. The present invention uses quantum dots for labeling to amplify the labeling signal and increase sensitivity.

[0013] The fluorescent labeling material used in the present invention is a quantum dot with a particle size of 5-20 nm obtained by surface modification of oil-soluble quantum dots using a polymer, which can be uniformly dispersed in an aqueous solution, and the surface carboxyl group has reactive activity.

[0014] Principle of the present invention: Fluorescence quenching immunochromatographic test strip is based on fluorescence resonance energy transfer (FRET) between a fluorescent donor and a fluorescent acceptor. When a fluorescent substance approaches certain nanomaterials, the fluorescent signal can be quenched. In order to achieve the maximum FRET efficiency, the spectra of the fluorescent material and the nanoquencher should show obvious overlap. The maximum emission wavelength of the CdSe / ZnS quantum dots (QDs) selected as the fluorescent donor of the present invention is 525nm, and the colloidal gold (AuNPs) as the fluorescent acceptor has a maximum absorption peak at 525nm. The methamphetamine antigen is labeled with QDs and fixed on the detection line (T line) of the NC membrane to indicate the presence or absence of the object to be tested; BSA is labeled with QDs and fixed on the quality control line (C line) of the NC membrane to indicate the validity of the test strip; AuNPs are coupled with methamphetamine antibodies and sprayed on the gold label pad.

[0015] When the sample does not contain methamphetamine (MET), the gold-labeled antibody (AuNPs-mAb) binds to the methamphetamine antigen (QDs-MET-BSA) coupled to the quantum dots on the T line, shortening the distance between the fluorescent donor and the acceptor, causing FRET to quench the fluorescence of QDs. The green fluorescence band on the T line disappears, while the fluorescence band on the C line is obvious and unchanged.

[0016] When the sample contains MET, MET competes with the antigen on the T line to bind to AuNPs-mAb, which reduces the number of AuNPs-mAb bound to QDs-MET-BSA on the T line, weakens the FRET effect, and causes the T line fluorescence to appear. As the MET concentration increases, the T line fluorescence intensity increases until it is comparable to the brightness of the C line.

[0017] If there is no fluorescence on the C and T lines, the test paper is invalid. Figure 1 .

[0018] The present invention also provides a method for preparing the fluorescence quenching immunochromatographic test paper, which is characterized by comprising the following steps:

[0019] (1) Preparation of AuNPs-mAb quencher: The precipitate obtained by centrifugation of colloidal gold was re-dissolved in PB buffer, the pH was adjusted to 7-8, and then methamphetamine antibody was added and incubated in the dark for 30-60 min; BSA solution was added to block the blank sites of colloidal gold and incubated for 30-60 min. The blocked solution was centrifuged at 4°C, the supernatant was discarded, and the precipitate was re-dissolved in resuspension buffer and stored at 4°C in the dark;

[0020] (2) Preparation of QDs-MET-BSA: The concentration of QDs was adjusted to 1-2 μmol / L with ultrapure water, and a 100 mg / mL LEDC / NHS mixture was added, and the mixture was incubated in the dark for 20-30 min; methamphetamine antigen was added, and the mixture was incubated in the dark for 40-60 min; 100 mg / mL BSA solution was added, and the mixture was incubated in the dark for 40-60 min; the blocked solution was centrifuged, the supernatant was discarded, and the precipitate was dispersed in PB buffer and stored at 4°C in the dark.

[0021] (3) Preparation of QDs-BSA: QDs were added to ultrapure water to a concentration of 1-2 μmol / L, and then a 100 mg / mL EDC / NHS mixture was added and incubated in the dark for 20-30 min; BSA was added and incubated in the dark for 40-60 min, centrifuged, the supernatant was discarded, the precipitate was dispersed in PB buffer, and stored at 4°C in the dark;

[0022] (4) Preparation of fluorescence quenching immunochromatographic test strips: The prepared QDs-MET-BSA and QDs-BSA were coated on the NC membrane at a rate of 1 μL / cm, designated as the detection line and quality control line, respectively, and dried at 37°C for 12 h; the AuNPs-mAb quencher was sprayed on the gold label pad at 6 μL / cm and dried at 37°C for 3 h; the sample pad and glass fiber filter paper were treated with a PB solution containing 1% BSA, 5% sucrose and 0.6% Tween-20, and dried at 60°C; then the sample pad, glass fiber filter paper, gold label pad, NC membrane and absorbent pad were overlapped by 2 mm in sequence and combined on a PVC plate.

[0023] Furthermore, the PB buffer solution was prepared by dissolving 0.01 M Na2HPO4, 0.72 g Na2HPO4 and 0.12 g KH2PO4 in 500 mL water.

[0024] Furthermore, in step (1), the preparation of the colloidal gold labeled monoclonal antibody (AuNPs-mAb) quencher comprises the following steps:

[0025] Concentration: The colloidal gold was centrifuged at 10,000 rpm for 30 min, and the precipitate was redissolved in PB buffer (0.01 M, 0.72 g Na2HPO4 and 0.12 g KH2PO4 dissolved in 500 mL water);

[0026] Adjust pH: Use K2CO3 (0.2 mol / L) to adjust colloidal gold to pH 7-8;

[0027] Conjugated antibody: Add methamphetamine antibody (MET-mAb) and incubate with gentle stirring in the dark for 30-60 minutes;

[0028] Blocking: Add BSA solution (10%, w / v) to block the blank sites of colloidal gold and incubate for 30-60 min;

[0029] Resuspension: Centrifuge the blocked solution at 4°C, 12000rpm, discard the supernatant, and re-dissolve the precipitate with resuspension buffer (0.01MPB, containing 1% BSA, 0.1% Tween20, 5% sucrose, 0.2% PEG20000) and store at 4°C in the dark.

[0030] Furthermore, in step (2), the preparation of the quantum dot marker QDs-MET-BSA comprises the following steps:

[0031] Activation: Take 0.5-1 nmol QDs, adjust the concentration to 1-2 μmol / L with ultrapure water, then add 60-100 μL of LEDC / NHS mixture (100 mg / mL), and incubate in the dark for 20-30 min;

[0032] Conjugated antigen: Add 300-400 μg of methamphetamine antigen and incubate in the dark for 40-60 min;

[0033] Blocking: Add 100-200 μL BSA solution (100 mg / mL) and incubate in the dark for 40-60 min;

[0034] Resuspension: Centrifuge the blocked solution at 9000 rpm for 15-20 minutes, discard the supernatant, disperse the precipitate in PB buffer, and store at 4°C in the dark for later use.

[0035] Furthermore, in step (3), the preparation of the quantum dot marker QDs-BSA comprises the following steps:

[0036] Activation: Take 0.5-1 nmol QDs, add ultrapure water to a concentration of 1-2 μmol / L, then add 60-100 μL of LEDC / NHS mixture (100 mg / mL), and incubate in the dark for 20-30 min;

[0037] Conjugated BSA: add 10-20 mg BSA and incubate in the dark for 40-60 min;

[0038] Resuspension: Centrifuge the solution at 9000 rpm for 15-20 minutes, discard the supernatant, disperse the precipitate in PB buffer, and store at 4°C in the dark for later use.

[0039] The present invention also provides the use of the fluorescence quenching immunochromatographic test paper described in any one of the above items or the fluorescence quenching immunochromatographic test paper prepared by any one of the above preparation methods in the rapid detection of methamphetamine in saliva.

[0040] Furthermore, the fluorescence quenching immunochromatographic test paper is used for qualitative screening of methamphetamine in saliva, and the detection steps include:

[0041] Drop the saliva sample onto the sample pad of the test strip. After 1 minute, observe the intensity of fluorescence at the T line and C line under ultraviolet light, compare it with the standard color card, and preliminarily judge the residual level of methamphetamine.

[0042] Furthermore, the fluorescence quenching immunochromatographic test paper is used to quantitatively detect methamphetamine in saliva, and the detection method includes:

[0043] (1) Drawing a standard curve: a series of different concentrations of methamphetamine standard solutions were prepared with artificial saliva and dropped onto the sample pad of the test strip. After 1 min, the fluorescence signals at the T line and the C line were collected using a fluorescence immunoassay analyzer to obtain the corresponding fluorescence intensity ratio of the T line to the C line. The Log value of the concentration of the standard solution was used as the horizontal coordinate x, and the fluorescence intensity ratio of the T line to the C line was used as the vertical coordinate y to draw a standard curve y=ax+b;

[0044] (2) Test strip test: Take a saliva sample and drop it onto the sample pad of the test strip. After 1 minute, insert it into the fluorescence detector for detection to obtain the fluorescence intensity ratio of the T line to the C line of the saliva sample;

[0045] (3) Result processing: Substitute the fluorescence intensity ratio of the T line to the C line of the saliva sample into the formula to obtain the methamphetamine concentration in the sample to be tested.

[0046] Beneficial effects of the present invention:

[0047] (1) The test strip provided by the present invention is simple to operate, quick to detect, easy to use, highly sensitive and low in cost.

[0048] The simple operation and easy use are reflected in: the saliva sample is directly dropped into the sample pad of the test strip without dilution or other pre-treatment steps, the fluorescence intensity of the C and T lines is observed by naked eyes under ultraviolet light for semi-quantification, and the signal is collected by a fluorescent immunoassay analyzer for quantification.

[0049] Rapid detection is reflected in the following aspects: the saliva sample is added to the sample pad, and the fluorescence signal becomes stable after 1 minute, and the result can be read.

[0050] The high sensitivity is reflected in the detection limit of 1ng / mL, which is significantly higher than similar products currently reported.

[0051] (2) In view of the shortcoming that colloidal gold test strips cannot be accurately quantified, the fluorescence quenching immunochromatographic test strips provided by the present invention can simultaneously realize the qualitative screening and quantitative detection of methamphetamine residues in saliva samples. The quantitative method of the present invention, with the help of a fluorescence quantitative card reader, establishes a standard curve, realizes the accurate quantification of methamphetamine in saliva, and the recovery rate and precision both meet the quantitative requirements.

[0052] (3) In view of the problem that the test strips currently used for methamphetamine detection on the market are mostly suitable for urine, the present invention provides a rapid test strip for methamphetamine in saliva matrix, which overcomes the various disadvantages of traditional drug urine testing technology, such as inconvenient and embarrassing sampling, the need for special places, the need for female law enforcement officers to supervise female suspects on the spot, the samples are easily swapped and adulterated, and the test results are easily interfered by common legal drugs to produce false positive results, etc. When the test strip of the present invention is used to quantitatively detect methamphetamine residues, it has high accuracy and high stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a schematic diagram of the detection principle of methamphetamine fluorescence quenching immunochromatographic test strip; wherein, AuNPs-mAb: gold-labeled antibody; BSA: bovine serum albumin; QDs-BSA: quantum dots coupled with bovine serum albumin; MET: methamphetamine; MET-BSA: methamphetamine antigen; QDs-MET-BSA: quantum dots coupled with methamphetamine antigen; Excitation light: excitation light; Emission light: emission light; FRET: fluorescence resonance energy transfer.

[0054] Figure 2 is the fluorescence intensity ratio of C and T lines when the fluorescence quenching immunochromatographic test paper detects the sample (FI T / FI C ) changes over time.

[0055] Figure 3 When the fluorescence quenching immunochromatographic test paper is used for detection, the fluorescence intensity changes with the concentration of methamphetamine (standard color card); the concentrations from left to right are 0, 1, 2, 4, 6, 8, 10, 12, 16, 20, 25, 50, and 100 ng / mL.

[0056] Figure 4 is a quantitative linear equation.

[0057] Figure 5 To evaluate the quantitative accuracy of fluorescence quenching immunochromatographic test strips.

[0058] Figure 6 To evaluate the stability of fluorescence quenching immunochromatographic test strips. DETAILED DESCRIPTION

[0059] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] In order to further understand the present invention, the present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0061] Example 1

[0062] The present embodiment provides a fluorescence quenching immunochromatographic test strip, which consists of a sample pad, glass fiber filter paper, a gold label pad, an NC membrane, and a water absorbent pad. Glass fiber filter paper is added to the composition of a conventional immunochromatographic test strip to achieve the purpose of online filtration, thereby eliminating the pretreatment step of the sample before loading, and realizing direct loading and detection of saliva samples, thereby simplifying the operation steps and improving the detection sensitivity.

[0063] The detection line (T line) and quality control line (C line) are sequentially set on the NC membrane. The detection line is formed by coating quantum dots coupled with methamphetamine antigen (QDs-MET-BSA) on the NC membrane. The quantum dots are CdSe / ZnS quantum dots. The quality control line is formed by coating quantum dots coupled with bovine serum albumin (QDs-BSA) on the NC membrane. The gold label pad is coated with AuNPs-mAb quencher, which is prepared by coupling colloidal gold with methamphetamine antibody.

[0064] The sample pad, glass fiber filter paper, gold label pad, NC membrane and absorbent pad were overlapped by 2 mm in sequence and assembled on a PVC plate. Finally, the assembled test strips were cut into 4 mm widths and stored in a desiccator for later use.

[0065] Example 2

[0066] This embodiment provides a method for preparing a fluorescence quenching immunochromatographic test paper, comprising:

[0067] 1. Preparation of colloidal gold (AuNPs) and colloidal gold labeled monoclonal antibody (AuNPs-mAb) quencher

[0068] Add 200mL of distilled water and 1mL of 2% chloroauric acid solution to a 500mL three-necked flask, place it on a temperature-controlled stirring electric heating mantle, stir and heat to reflux, keep the stirring speed and temperature constant, and when the solution boils, quickly and accurately add 6.5mL of 1% trisodium citrate solution. As the reaction progresses, the color of the solution gradually turns gray, then purple, and then gradually turns bright red. When the color of the solution remains bright, continue stirring for 5 minutes, stop heating, take out and cool to room temperature. Transfer to a clean conical flask, protect from light, and store at 4°C for later use.

[0069] To obtain satisfactory sensitivity, 15-20 mL of colloidal gold was centrifuged at 10000 rpm for 30 min, and the precipitate was redissolved with PB buffer (0.01 M, 0.72 g Na2HPO4 and 0.12 g KH2PO4 dissolved in 500 mL water), and the pH of the colloidal gold was adjusted to 7-8 with K2CO3 (0.2 mol / L), and 100-200 μg of methamphetamine antibody (MET-mAb) was added dropwise to the solution, and incubated for 60 min with gentle stirring in the dark. The blank sites of the colloidal gold were blocked with BSA solution (10%, w / v). After incubation for 60 min, the solution was centrifuged at 4°C and 12000 rpm for 25 min. The supernatant was discarded, and the precipitate was fixed to 1.5-2 mL of resuspension buffer (0.01 M PB, containing 1% BSA, 0.1% Tween20, 5% sucrose, 0.2% PEG20000), and stored at 4°C in the dark.

[0070] 2. Preparation of quantum dot marker QDs-MET-BSA

[0071] Take 0.5-1 nmol QDs and add 500 μL ultrapure water to mix, then add 60-100 μL EDC / NHS mixture (100 mg / mL) and incubate in the dark for 30 min. Add 300-400 μg MET-BSA, incubate in the dark for 1 h, then add 100-200 μL BSA solution (100 mg / mL) and incubate for 1 h. Centrifuge at 9000 rpm for 15 min at 4 °C, and resuspend the precipitate with PB buffer (0.01 M).

[0072] 3. Preparation of quantum dot marker QDs-BSA

[0073] For the preparation of QDs-BSA, PB (0.01 M) and 100-200 μL BSA (100 mg / mL) were added after 30 min of dark incubation, and the other conditions were the same as those of QDs-BSA-MET.

[0074] 4. Preparation of fluorescence quenching test strips

[0075] The test strip consists of a sample pad, glass fiber filter paper, a gold label pad, an NC membrane, and a water absorbent pad.

[0076] The preparation steps are:

[0077] Line C: QDs-BSA solution with a concentration of 0.1-2 μmol / L was coated on the NC membrane at a rate of 1 μL / cm.

[0078] T line: QDs-MET-BSA solution with a concentration of 0.1-2 μmol / L was coated on the NC membrane at a rate of 1 μL / cm.

[0079] Preparation of gold label pad: soak in phosphate buffered saline (PBS, 0.01 M, pH 7.4) containing 1% BSA, 5% sucrose and 0.6% Tween-20 for 5 min and then dry at 60°C. Spray AuNPs-mAb quencher with a concentration of 0.1-2 mg / mL on the gold label pad at 6 μL / cm and dry at 37°C for 3 h.

[0080] The sample pad and glass fiber filter paper were pretreated by soaking them in phosphate buffered saline (PBS, 0.01 M, pH 7.4) containing 1% BSA, 5% sucrose and 0.6% Tween-20 for 5 min and then drying them at 60°C.

[0081] Sample diluent: PB solution (0.01 M, pH 5.5) containing 1% Tween-80.

[0082] Example 3

[0083] This embodiment provides an application of a fluorescence quenching immunochromatographic test strip in screening and quantification of methamphetamine in saliva.

[0084] Without dilution or other pre-treatment steps, the saliva sample is directly dropped into the sample pad of the test strip, and the fluorescence intensity of the C and T lines is observed by naked eyes under ultraviolet light for semi-quantification, and the signal is collected by a fluorescent immunoassay analyzer for quantification.

[0085] The saliva sample was dropped onto the sample pad, and the fluorescence signal became stable after 1 minute ( Figure 2 ), you can interpret the result.

[0086] As the concentration of methamphetamine in the sample increases, fluorescence appears on the T line and the fluorescence intensity gradually increases. Obvious fluorescence appears on the 1ng / mL T line. Figure 3 As shown, the concentrations from left to right are 0, 1, 2, 4, 6, 8, 10, 12, 16, 20, 25, 50, and 100 ng / mL.

[0087] 1. Qualitative screening

[0088] Take 70-100 μL of saliva sample and drop it on the sample pad of the test strip. After 1 minute, observe the intensity of fluorescence at the T line and C line under ultraviolet light (365nm), and compare it with the standard color card to preliminarily judge the residual level of methamphetamine.

[0089] 2. Quantitative detection

[0090] (1) Draw a standard curve: Prepare a series of different concentrations of methamphetamine standard solutions (6-20 ng mL -1 ), take 80 μL and drop it on the sample pad of the test strip. After 1 minute, use the fluorescence immunoassay analyzer to collect the fluorescence signals at the T line and C line to obtain the corresponding fluorescence intensity ratio of the T line to the C line (FI T / FI C ). The Log value of the standard solution concentration is the horizontal axis x, FI T / FI C With y as the ordinate, draw the standard curve y=ax+b.

[0091] (2) Test strip test: Take a saliva sample and drop it onto the sample pad of the test strip. After 1 minute, insert it into the fluorescence detector for detection and obtain the result FI T样 / FI C样 ;

[0092] (3) Result processing: Obtain data FI T样 / FI C样 Substitute into the formula to obtain the methamphetamine concentration in the sample to be tested.

[0093] In a specific embodiment, the concentration of methamphetamine is linear between 6 ng / mL and 20 ng / mL, and the concentration of methamphetamine in the sample can be quantitatively determined according to the linear equation ( Figure 4 ).

[0094] Blank saliva was used to prepare three concentrations of methamphetamine standard solutions, low (6 ng / mL), medium (12 ng / mL), and high (20 ng / mL). Each concentration was tested 6 times. The fluorescence signals at the T line and C line were collected by a fluorescence immunoassay analyzer to obtain the FI T / FI C The values ​​were substituted into the standard curve to calculate the concentration values. The average recoveries were 98.04%, 105.89%, 100.58%, and the RSDs were 2.57%, 6.81%, and 2.09% ( Figure 5 ), which proves that the fluorescence quenching test paper has high accuracy in the quantification of methamphetamine.

[0095] The test strips of the same batch were sealed and stored at 4°C for 1, 3, 5, 9, 15 and 30 days, and then validated with saliva samples containing 16 ng / mL of methamphetamine. After 30 days of storage, there was no significant degradation in the fluorescence intensity on the test strips, and the RSD was 2.60% ( Figure 6 ), indicating that the test paper has good stability.

[0096] Example 4

[0097] For the detection of methamphetamine in saliva samples

[0098] Saliva of four people (numbered ①, ②, ③, and ④) was collected by chewing absorbent cotton strips, and different concentrations of methamphetamine standards were added. 80 μL was taken and dropped onto the sample pad of the test strip. After 1 minute, the intensity of fluorescence at the T line and C line was observed under ultraviolet light, and the fluorescence signal at the T line and C line was collected by a fluorescence immunoassay analyzer to obtain the corresponding fluorescence intensity ratio (FI T / FI C ), and the methamphetamine concentration in the sample was calculated based on the standard curve.

[0099] The results are shown in Table 1. The semi-quantitative results of the inspection graph and the FI T / FI C The calculated results of the values ​​have good consistency and are close to the added concentration, indicating that the test paper has good applicability in saliva samples.

[0100] Table 1 Application of fluorescence quenching test paper in saliva samples

[0101]

[0102]

[0103] The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, modification, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fluorescence quenching immunochromatographic test paper for rapid detection of methamphetamine in saliva, characterized in that: The invention comprises a sample pad, a glass fiber filter paper, a gold label pad, a NC membrane and a water absorbent pad. A detection line formed by quantum dots coupling to methamphetamine antigen and a quality control line formed by quantum dots coupling to bovine serum albumin are arranged in sequence on the NC membrane. An AuNPs-mAb quencher is coated on the gold label pad. The AuNPs-mAb quencher is prepared by coupling colloidal gold with a methamphetamine antibody.

2. The fluorescence quenching immunochromatographic test paper according to claim 1, characterized in that: The quantum dots serve as fluorescence donors, and the colloidal gold serves as fluorescence acceptors.

3. The method for preparing the fluorescence quenching immunochromatographic test paper according to claim 1 or 2, characterized in that: The following steps are involved: (1) Preparation of AuNPs-mAb quencher: The precipitate obtained by centrifugation of colloidal gold was re-dissolved in PB buffer, the pH was adjusted to 7-8, and then methamphetamine antibody was added and incubated in the dark for 30-60 min; BSA solution was added to block the blank sites of colloidal gold and incubated for 30-60 min. The blocked solution was centrifuged at 4°C, the supernatant was discarded, and the precipitate was re-dissolved in resuspension buffer and stored at 4°C in the dark; (2) Preparation of QDs-MET-BSA: The concentration of QDs was adjusted to 1-2 μmol / L with ultrapure water, and a 100 mg / mL EDC / NHS mixture was added, and the mixture was incubated in the dark for 20-30 min; methamphetamine antigen was added, and the mixture was incubated in the dark for 40-60 min; 100 mg / mL BSA solution was added, and the mixture was incubated in the dark for 40-60 min; the blocked solution was centrifuged, the supernatant was discarded, and the precipitate was dispersed in PB buffer and stored at 4°C in the dark. (3) Preparation of QDs-BSA: QDs were added to ultrapure water to a concentration of 1-2 μmol / L, and then a 100 mg / mL EDC / NHS mixture was added and incubated in the dark for 20-30 min; BSA was added and incubated in the dark for 40-60 min, centrifuged, the supernatant was discarded, the precipitate was dispersed in PB buffer, and stored at 4°C in the dark; (4) Preparation of fluorescence quenching immunochromatographic test strips: The prepared QDs-MET-BSA and QDs-BSA were coated on the NC membrane at a rate of 1 μL / cm, respectively, and designated as the detection line and quality control line; the AuNPs-mAb quencher was sprayed on the gold label pad at 6 μL / cm and dried at 37°C; the sample pad and glass fiber filter paper were treated with phosphate buffer containing 1% BSA, 5% sucrose and 0.6% Tween-20, and dried at 60°C; then the sample pad, glass fiber filter paper, gold label pad, NC membrane and absorbent pad were overlapped by 2 mm in sequence and combined on a PVC plate.

4. The preparation method according to claim 3, characterized in that: The PB buffer solution was prepared by dissolving 0.01 M Na2HPO4, 0.72 g Na2HPO4 and 0.12 g KH2PO4 in 500 mL water.

5. The preparation method according to claim 3, characterized in that: In the step (1), the colloidal gold is centrifuged at 10000 rpm for 30 min, and the precipitate is redissolved with PB buffer, the pH of the colloidal gold is adjusted to 7-8 with 0.2 mol / L K2CO3, methamphetamine antibody is added, and the mixture is gently stirred and incubated in the dark for 30-60 min; BSA solution (10%, w / v) is added to block the blank sites of the colloidal gold, and the mixture is incubated for 30-60 min. The blocked solution is centrifuged at 4°C and 12000 rpm, the supernatant is discarded, the precipitate is redissolved with resuspension buffer, and stored at 4°C in the dark.

6. The preparation method according to claim 3, characterized in that: In the step (2), 0.5-1 nmol QDs are taken, the concentration is adjusted to 1-2 μmol / L with ultrapure water, and then 60-100 μL of 100 mg / mL EDC / NHS mixed solution is added, and incubated in the dark for 20-30 min; 300-400 μg of methamphetamine antigen is added, and incubated in the dark for 40-60 min; then 100-200 μL of 100 mg / mL BSA solution is added, and incubated in the dark for 40-60 min; the blocked solution is centrifuged at 9000 rpm for 15-20 minutes, the supernatant is discarded, the precipitate is dispersed in PB buffer, and stored at 4°C in the dark for later use.

7. The preparation method according to claim 3, characterized in that: In the step (3), 0.5-1 nmol QDs are added to ultrapure water to a concentration of 1-2 μmol / L, and then 60-100 μL of 100 mg / mL EDC / NHS mixed solution is added, and incubated in the dark for 20-30 min; 10-20 mg BSA is added, and incubated in the dark for 40-60 min; the solution is centrifuged at 9000 rpm for 15-20 minutes, the supernatant is discarded, and the precipitate is dispersed in PB buffer and stored at 4°C in the dark for later use.

8. Use of the fluorescence quenching immunochromatographic test paper according to claim 1 or 2 or the fluorescence quenching immunochromatographic test paper prepared by the preparation method according to any one of claims 3 to 7 in the rapid detection of methamphetamine in saliva.

9. The use according to claim 8, characterized in that: The fluorescence quenching immunochromatographic test paper is used for qualitative screening of methamphetamine in saliva, and the detection steps include: Drop the saliva sample onto the sample pad of the test strip. After 1 minute, observe the intensity of fluorescence at the T line and C line under ultraviolet light, compare it with the standard color card, and preliminarily judge the residual level of methamphetamine.

10. The use according to claim 8, characterized in that: The fluorescent quenching immunochromatographic test paper is used for quantitative detection of methamphetamine in saliva, and the detection method comprises: (1) Drawing a standard curve: a series of different concentrations of methamphetamine standard solutions were prepared with artificial saliva and dropped onto the sample pad of the test strip. After 1 min, the fluorescence signals at the T line and the C line were collected using a fluorescence immunoassay analyzer to obtain the corresponding fluorescence intensity ratio of the T line to the C line. The Log value of the concentration of the standard solution was used as the horizontal coordinate x, and the fluorescence intensity ratio of the T line to the C line was used as the vertical coordinate y to draw a standard curve y=ax+b; (2) Test strip test: Take a saliva sample and drop it onto the sample pad of the test strip. After 1 minute, insert it into the fluorescence detector for detection to obtain the fluorescence intensity ratio of the T line to the C line of the saliva sample; (3) Result processing: Substitute the fluorescence intensity ratio of the T line to the C line of the saliva sample into the formula to obtain the methamphetamine concentration in the sample to be tested.

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