A method for preparing a kit for simultaneously detecting multiple nafil-like substances based on a novel multi-quantum dot-protein polymer probe and its application

Abstract

The present invention discloses a method for preparing a kit for simultaneously detecting a variety of nafil-like substances based on a novel multi-quantum dot-protein polymer probe and its application. The present invention is based on a novel solvent-induced effect strategy, and a one-step method is used to prepare a multi-quantum dot-protein immune probe. By mixing quantum dots, BSA and antibodies, and utilizing hydrophobic interaction and π-π stacking, a fluorescent immune probe with a large number of fluorescent quantum dots is prepared, thereby increasing the loading capacity of quantum dots in the probe and simplifying the probe preparation method. Sildenafil and tadalafil and their derivatives are used as detection objects, and sildenafil and tadalafil antibodies and antigens are used. In combination with the above method, probes and lateral flow immunochromatography test strips for specific detection of sildenafil and tadalafil and their derivatives are prepared, and a solid phase extraction microfluidic chip is combined to construct a system for detecting nafil-like substances, thereby realizing qualitative and quantitative detection of 12 nafil-like drugs, with strong specificity, high sensitivity, good accuracy, and reliable detection results.

Description

Technical Field

[0001] The present invention relates to the technical field of food and drug safety detection, and in particular to a method for preparing a kit for simultaneously detecting a variety of nafil-like substances based on a novel multi-quantum dot-protein polymer probe and its application. Background Art

[0002] Compared with traditional probes such as colloidal gold, latex microspheres and fluorescent dyes, fluorescent quantum dots (QDs) have become a popular material for constructing highly sensitive immunochromatography due to their advantages such as high fluorescence intensity, long fluorescence lifetime, resistance to photobleaching and diverse luminescent colors. However, most high-quality fluorescent quantum dots are fat-soluble and require tedious surface chemical modification to modify antibodies; and due to the small size of quantum dots, the coupling efficiency of quantum dots and antibodies is low, and the loading of quantum dots on antibodies is also low (https: / / doi.org / 10.1016 / j.bios.2016.04.091). In order to solve these problems, Zou et al. (https: / / doi.org / 10.1021 / acs.analchem.6b01941) used covalent bonding to modify fat-soluble quantum dots on the surface of silica spheres, and then coupled biorecognition materials to obtain fluorescent silica sphere probes with a high quantum dot loading. However, this method requires more synthesis steps and is not convenient enough.

[0003] Establishing a highly sensitive and quantitative immunochromatographic method is of great significance for on-site screening of food safety. Although the lateral flow immunochromatographic method currently used for on-site screening of narcofil-like drugs that may be illegally added to food has high sensitivity, such as patent CN114308153A, a solid phase extraction microfluidic chip for detecting narcofil-like substances and a system for detecting narcofil-like substances, there is still room for further improvement in sensitivity and ease of probe preparation. Therefore, a simple method for preparing a probe with high quantum dot loading is urgently needed. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and to provide a method for preparing a kit for simultaneously detecting a variety of nafil-like substances based on a novel multi-quantum dot-protein polymer probe and its application, which has a convenient, efficient and highly sensitive detection effect.

[0005] The first object of the present invention is to provide a method for preparing a fluorescent immunoprobe.

[0006] The second object of the present invention is to provide a fluorescent immunoprobe.

[0007] The third object of the present invention is to provide the use of the fluorescent immunoprobe in the preparation of products for detecting phenotype substances and / or phenotype substance derivatives.

[0008] The fourth object of the present invention is to provide a kit for detecting narcolepsy substances and / or narcolepsy substance derivatives.

[0009] The fifth object of the present invention is to provide a method for using the kit for detecting narcolepsy substances and / or narcolepsy substance derivatives.

[0010] In order to achieve the above object, the present invention is implemented by the following scheme:

[0011] A method for preparing a fluorescent immunoprobe, the steps of the preparation method are as follows:

[0012] S1: mixing fluorescent quantum dots and n-hexane to obtain a fluorescent quantum dot solution, wherein the concentration of the fluorescent quantum dots is 1 to 3 mg / mL;

[0013] S2: mixing the fluorescent quantum dot solution of step S1 with a PBS solution, stirring for 2 to 4 hours, wherein the volume of the PBS to n-hexane is 4 to 12:1, the PBS solution contains BSA and antibodies to nafil-like substances, and the concentration of the BSA is 0.25 to 2 mg / mL;

[0014] S3: Centrifuge, remove the supernatant, and obtain the fluorescent immunoprobe.

[0015] Preferably, in step S1, the concentration of the fluorescent quantum dots is 1.5-2.5 mg / mL.

[0016] More preferably, the fluorescent quantum dots are ZnCdSe / ZnS QDs.

[0017] More preferably, the concentration of the fluorescent quantum dots is 2 mg / mL.

[0018] Preferably, in step S2, the volume ratio of PBS to n-hexane is 6-10:1, the stirring time is 2.5-3.5 h, and the BSA concentration is 0.5-1.5 mg / mL.

[0019] More preferably, the volume ratio of PBS to n-hexane is 8:1, the concentration of BSA is 1 mg / mL, and the stirring time is 3 h.

[0020] Preferably, in step S2, the fil-like substance is sildenafil and / or tadalafil, the concentration of the sildenafil antibody is 0.15-0.45 mg / mL, and the concentration of the tadalafil antibody is 0.2-0.4 mg / mL.

[0021] More preferably, in step S2, the nafil-like substance is sildenafil and / or tadalafil, the concentration of the sildenafil antibody is 0.23-0.37 mg / mL, and the concentration of the tadalafil antibody is 0.27-0.40 mg / mL.

[0022] More preferably, the concentration of the antibody against sildenafil is 0.3 mg / mL, and the concentration of the antibody against tadalafil is 0.34 mg / mL.

[0023] Preferably, the antibody against sildenafil can recognize sildenafil in a combined sample and sildenafil fixed on a test strip; the antibody against tadalafil can recognize tadalafil in a combined sample and tadalafil fixed on a test strip.

[0024] More preferably, the antibody to sildenafil is Ab Sildenafil , the antibody to tadalafil is Ab Tadalafil .

[0025] A fluorescent immunoprobe is prepared by the preparation method.

[0026] The fluorescent immunoprobe is used in preparing products for detecting narcolepsy substances and / or narcolepsy substance derivatives.

[0027] Preferably, the nafil-like substance is sildenafil and / or tadalafil.

[0028] Preferably, the sildenafil derivative is desmethylsildenafil, erythroidenafil, hydroxyerythroidenafil, pseudovardenafil, narmosildenafil, udenafil, homosildenafil, thiohomosildenafil and / or thioaldenafil.

[0029] More preferably, the sildenafil derivative is desmethylsildenafil, erythroidenafil, hydroxyerythroidenafil, pseudovardenafil, udenafil, homosildenafil and / or thiosildenafil.

[0030] Preferably, the tadalafil derivative is N-desmethyl tadalafil, N-butyl tadalafil, N-desmethlent tadalafil, N-octyl demethyl tadalafil, acetamido tadalafil and / or amino tadalafil.

[0031] More preferably, the tadalafil derivative is N-desmethyl tadalafil and / or N-butyl tadalafil and acetamido tadalafil.

[0032] A kit for detecting nalidixic substances and / or nalidixic substance derivatives, the kit containing the fluorescent immunoprobe and a lateral flow immunochromatography test strip; the lateral flow immunochromatography test strip consists of a sample pad, a nitrocellulose membrane, absorbent paper and a PVC bottom plate, the detection line of the lateral flow immunochromatography test strip is coated with nalidixic substance antigens, and the quality control line is coated with goat anti-rabbit IgG.

[0033] Preferably, the treatment solution of the sample pad contains 4.5-5.5 mg / mL PVP, 4.5-5.5 mg / mL sucrose, 4.5-5.5 mg / mL BSA, 0.5%-1.5% Tween-20 (v / v) and 0.04-0.06 mol / L PB solution.

[0034] More preferably, the treatment solution of the sample pad contains 5 mg / mL PVP, 5 mg / mL sucrose, 5 mg / mL BSA, 1% Tween-20 (v / v) and 0.05 mol / L PB solution.

[0035] Preferably, the nafil-like substance antigen is an antigen of sildenafil and / or tadalafil, the concentration of the sildenafil antigen is 0.095-1.5 mg / mL, and the concentration of the tadalafil antigen is 0.10-0.75 mg / mL.

[0036] More preferably, the concentration of the sildenafil antigen is 0.19-0.75 mg / mL, and the concentration of the tadalafil antigen is 0.10-0.38 mg / mL.

[0037] Most preferably, the concentration of the sildenafil antigen is 0.38 mg / mL, and the concentration of the tadalafil antigen is 0.19 mg / mL.

[0038] Preferably, the sildenafil antigen is fixed on the test strip, and when the immune probe passes by, it can identify the antibody on the capture probe; the tadalafil antigen is fixed on the test strip, and when the immune probe passes by, it can identify the antibody on the capture probe.

[0039] More preferably, the antigen of sildenafil is Ag Sildenafil The antigen of tadalafil is Ag Tadalafil .

[0040] The method for using the kit for detecting nafil-like substances and / or nafil-like substance derivatives is a method for non-disease diagnosis and treatment, and the steps are as follows:

[0041] S12: loading a sample extract containing narcolepsy substances and / or narcolepsy substance derivatives using a solid phase extraction microfluidic chip;

[0042] S22: using a methanol solution containing 0.05% to 0.15% acetic acid (v / v) as an eluent to elute the sample extract containing pheniramine-like substances and / or pheniramine-like substance derivatives in step S21;

[0043] S23: using 0.024-0.026 mol / L PB solution as a chromatography solution, collecting the narcolepsy substances and / or narcolepsy substance derivatives in step S22, to obtain a PB solution containing narcolepsy substances and / or narcolepsy substance derivatives;

[0044] S24: Dispense the PB solution containing pheniramine-like substances and / or pheniramine-like substance derivatives described in step S23 into a reaction microtiter plate, 99-101 μL per well, mix with the fluorescent immunoprobes, 1.5-3.5 μL of each probe per well, and then react with the lateral flow immunochromatography test strip for 4-8 minutes, take out the lateral flow immunochromatography test strip after the reaction, and collect the fluorescent signal.

[0045] Preferably, in step S22, a methanol solution (v / v) containing 0.1% acetic acid is used as the eluent.

[0046] Preferably, in step S23, 0.025 mol / L PB solution is used as the chromatography liquid.

[0047] Preferably, in step S24, the PB solution containing pheniramine-like substances and / or pheniramine-like substance derivatives described in step S23 is dispensed into a reaction microplate, 100 μL per well, mixed with the fluorescent immunoprobes, 0.48 to 0.72 μg of each probe per well, and then reacted with the lateral flow immunochromatography test strip for 5 to 7 minutes.

[0048] More preferably, each probe is present at 0.6 μg per well.

[0049] More preferably, the reaction with the lateral flow immunochromatography test strip is for 6 minutes.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The present invention discloses a method for preparing a kit for simultaneously detecting a variety of nafide substances based on a novel multi-quantum dot-protein polymer probe and its application. The present invention proposes a strategy based on solvent-induced effect to prepare a multi-quantum dot-antibody immune probe in one step. By adding oil-soluble red quantum dots (Quantum dots, QDs) into an aqueous solution containing BSA and antibodies and stirring, a fluorescent immune probe with a large number of fluorescent quantum dots is prepared by utilizing hydrophobic interaction and π-π stacking, namely, a multi-quantum dot-bovine serum albumin-antibody nanospheres (Multiple QDs-Bovine Serum Albumin-AntibodyNanospheres, MQDs-BSA-Ab NSs) probe. On the one hand, the loading capacity of quantum dots in the probe is increased to obtain a probe with ultra-high fluorescence intensity; on the other hand, the preparation method of the probe is simplified, and antibody labeling and blocking of non-specific sites can be achieved.

[0052] The present invention uses sildenafil and tadalafil and their derivatives as detection objects, and uses sildenafil and tadalafil antibodies and antigens (Ab Sildenafil ,Ab Tadalafil 、Ag Sildenafil and Ag Tadalafil ), combined with the above method, a fluorescent immunoprobe and lateral flow immunochromatographic test strip with a large number of fluorescent quantum dots that can specifically detect sildenafil, tadalafil and their derivatives were prepared, and then combined with the solid phase extraction microfluidic chip prepared in the laboratory in the early stage, a detection system for detecting sildenafil-like substances was constructed, which realized the qualitative and quantitative detection of 12 sildenafil-like drugs, with a qualitative cut-off value of 0.25-3.0 ng / mL and a quantitative detection limit of 0.008-0.12 ng / mL. Among them, the cut-off values ​​for desmethylsildenafil, erythroidenafil, hydroxyerythroidenafil, pseudovardenafil, udenafil, homosildenafil and thiosildenafil were 1.0 ng / mL, 2.0 ng / mL, 0.5 ng / mL, 0.5 ng / mL, 0.25 ng / mL, 0.5 ng / mL and 2.0 ng / mL, respectively, and the cut-off values ​​for N-desmethyltadalafil, N-butyltadalafil and acetamidotadalafil were 3 ng / mL, 1.5 ng / mL and 1.5 ng / mL, respectively.

[0053] The detection system of the present invention has strong specificity, high sensitivity and good accuracy. The actual detection results of nafil-like substances in 21 kinds of health foods purchased on the market are highly correlated with the results of the LC-MS / MS method, and the detection results are reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 TEM characterization of the self-assembly process of MQDs-BSA-Ab, where A is 0.5 h self-assembly time; B is 1.5 h self-assembly time; C is 3 h self-assembly time.

[0055] Figure 2 Characterization of MQDs-BSA-Ab, where A is a cold field scanning electron microscope image; B is a hot field scanning electron microscope image; C is an infrared spectrum of QDNPs and MQDs-BSA-Ab NPs; D is a UV-visible spectrum and fluorescence spectrum of MQDs-BSA-Ab NPs. Figure 2 The inset of D is a picture of MQDs-BSA-Ab NPs under 365 nm UV light irradiation.

[0056] Figure 3 These are electron images of quantum dot polymer solutions, where 1 is MQDs NPs; 2 is MQDs-BSA NPs; 3 is MQDs-BSA-Ab NPs; and 4 is MQDs-Ab NPs.

[0057] Figure 4 This is a structural diagram of the immunochromatographic test strip.

[0058] Figure 5 The figure shows the detection principle, where A is the preparation method of quantum dot-protein polymer probe; B is the design diagram of the chromatography test strip; C is the immunochromatography effect diagram of negative samples; D is the immunochromatography effect diagram of positive samples; and E is the quantification of the test strip using a small detection device based on a smartphone.

[0059] Figure 6 These are the quantitative standard curves and qualitative test results of sildenafil and tadalafil, where A is the quantitative standard curve and B is the qualitative test result.

[0060] Figure 7 Comparison of the immunoprobes synthesized using the two technologies, where A is the standard curve of immunochromatography based on traditional probes; B is the TEM characterization of the MQDs-BSA-Ab immunoprobe; and C is the TEM characterization of the QD-Ab immunoprobe.

[0061] Figure 8 MQDs-BSA-Ab Sildenafil and MQDs-BSA-Ab Tadalafil Specificity, where A is MQDs-BSA-Ab Sildenafil ;B is MQDs-BSA-Ab Tadalafil .

[0062] Fig. 9 The figures are the quantitative standard curves and qualitative detection of desmethylsildenafil, erythroidenafil, hydroxyerythroidenafil and pseudovardenafil, wherein A is the quantitative standard curve of desmethylsildenafil; B is the qualitative detection result of desmethylsildenafil; C is the quantitative standard curve of erythroidenafil; D is the qualitative detection result of erythroidenafil; E is the quantitative standard curve of hydroxyerythroidenafil; F is the qualitative detection result of hydroxyerythroidenafil; G is the quantitative standard curve of pseudovardenafil; and H is the qualitative detection result of pseudovardenafil.

[0063] Fig.10 The quantitative standard curves and qualitative detection of udenafil, homosildenafil, and thio-edenafil, wherein A is the quantitative standard curve of udenafil; B is the qualitative detection result of udenafil; C is the quantitative standard curve of homosildenafil; D is the qualitative detection result of homosildenafil; E is the quantitative standard curve of thio-edenafil; and F is the qualitative detection result of thio-edenafil.

[0064] Fig.11The quantitative standard curves and qualitative detection of N-desmethyl tadalafil, N-butyl tadalafil and acetamido tadalafil are shown in Figure 1, wherein A is the quantitative standard curve of N-desmethyl tadalafil; B is the qualitative detection result of N-desmethyl tadalafil; C is the quantitative standard curve of N-butyl tadalafil; D is the qualitative detection result of N-butyl tadalafil; E is the quantitative standard curve of acetamido tadalafil; and F is the qualitative detection result of acetamido tadalafil.

[0065] Fig.12 It represents the method reproducibility and storage stability, where A represents the method reproducibility and B represents the storage stability.

[0066] Fig.13 is the effect of probe synthesis conditions on the probe synthesis effect, where A is the quantum dot concentration; B is the BSA concentration; C is the Ab Sildenafil concentration; D is Ab Tadalafil concentration; E is the optimized PBS / n-hexane ratio; F is the synthesis time.

[0067] Fig.14 These are pictures of the probe solutions after centrifugation at different synthesis times, where A is 0.5 h, B is 1.0 h, C is 1.5 h, D is 2.0 h, E is 2.5 h, F is 3.0 h, G is 3.5 h, and H is 4.0 h.

[0068] Fig.15 is the effect of lateral flow immunochromatography test conditions on the test results of lateral flow immunochromatography, where A is Ag Sildenafil concentration; B is Ag Tadalafil concentration; C is the amount of probe used; D is the sample running time; E is the Tween concentration of the sample pad treatment solution; F is the salt ion concentration of the chromatography solution. DETAILED DESCRIPTION

[0069] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0070] The following are the materials used in the examples:

[0071] Main reagents: Tadalafil (98%) (Shanghai Sane Chemical Technology Co., Ltd.); N-desmethyl tadalafil (98%) (Shanghai Aladdin Reagent Co., Ltd.); N-Desmethyl ent-Tadalafil (98%) (Shanghai Aladdin Reagent Co., Ltd.); N-Butyl Nortadalafil (98%) (Shanghai Aladdin Reagent Co., Ltd.); N-Octyl Nortadalafil (98%) (Shanghai Aladdin Reagent Co., Ltd.); Acetamino Tadalafil (Shanghai Aladdin Reagent Co., Ltd.); Amino Tadalafil (Shanghai Aladdin Reagent Company); sildenafil (99%) (Beijing Bailingwei Technology Co., Ltd.); udenafil (98%) (Shanghai Sane Chemical Technology Co., Ltd.); thio-edenafil (98%) (Shanghai Sane Chemical Technology Co., Ltd.); thio-homosildenafil (98%) (Beijing Bailingwei Technology Co., Ltd.); pseudo-vardenafil (98%) (Beijing Bailingwei Technology Co., Ltd.); hydroxy-red denafil (98%) (Beijing Bailingwei Technology Co., Ltd.); hydroxy-thio-homosildenafil (98%) (Beijing Bailingwei Technology Co., Ltd.); desmethylsildenafil (98%) (Beijing Bailingwei Technology Co., Ltd.); homosildenafil (98%) (Beijing Bailingwei Technology Co., Ltd.); disodium hydrogen phosphate dodecahydrate (Sigma, USA); sodium chloride (Sigma, USA); potassium chloride (Tianjin Damao Chemical Reagent Factory); ammonium sulfate (Tianjin Damao Chemical Reagent Factory); sodium dodecyl sulfonate (SDS) (Guangzhou Jianyang Sheng Technology Co., Ltd.); sodium periodate (Guangzhou Jianyang Biotechnology Co., Ltd.); tris(hydroxymethylaminomethane) (Tris) (Guangzhou Weijia Technology Co., Ltd.); dimethyl sulfoxide (DMF) (Shanghai Aladdin Biotechnology); glycine (Sigma, USA); polyvinyl pyrrolidone (PVP) (Sinopharm Chemical Reagent Co., Ltd.); fat-soluble quantum dots (625nm) (Wuhan Jiayuan Quantum Dot Co., Ltd.); Tween-20 (Tween-20) (Sinopharm Chemical Reagent Co., Ltd.); sucrose (Sinopharm Chemical Reagent Co., Ltd.); standard protein marker (Bio-Rad, USA); bovine serum albumin (BSA) (Sigma, USA); MCX solid phase extraction filler (Qingdao Bangkai); sample pad (SB08) (Shanghai Liangxin Technology Co., Ltd.); PVC base plate (SMA31-40) (Shanghai Liangxin Technology Co., Ltd.); absorbent paper (CH37K) (Shanghai Liangxin Technology Co., Ltd.); nitrocellulose membrane (UniSart CN 140) (Sartorius, Germany); 96-well ELISA plate (Shenzhen Jincanhua Industrial Co., Ltd.); other reagents were of analytical grade

[0072] Main instruments and equipment: LC-MS / MS (Shimadzu, Japan - AB SCIEX, USA); Mettler ToleDo electronic balance (Mettler-Toledo Electronic Instrument (Shanghai) Co., Ltd.); QL-861 oscillator (Qilin Bell Instrument Manufacturing Co., Ltd., Haimen); MS1 vortex mixer (Qilin Bell Instrument Manufacturing Co., Ltd., Haimen); ZW-A micro-oscillator (Jintan Fuhua Instrument Co., Ltd.); magnetic stirrer (IKA, Germany); CNC ultrasonic cleaner (KQ-100DE) (Kunshan Ultrasonic Instrument Co., Ltd.); BCD-191W / H adjustable digital display refrigerator (Ke, Japan); lon company); pure water equipment (18.2Ω) (Millipore company, USA); constant temperature water bath (Shanghai Yuefeng Instrument Representative Co., Ltd.); single-channel pipette (Ependorf company, USA); SPE extraction column (Agilent company, USA); scanning electron microscope (ZEISS company, Germany); liquid phase vial (Agilent company, USA); dialysis bag (molecular weight cutoff 8000-10000D) (Sigma company); 0.22μM filter membrane (Tianjin Jinteng Experimental Equipment Co., Ltd.); ODS-SP chromatographic column (Shimadzu Corporation, Japan); other glassware were all domestically produced.

[0073] Example 1 Preparation method of self-assembled quantum dot-protein complex fluorescent probe

[0074] 1. Experimental Methods

[0075] Using the emulsification method, red oil-soluble quantum dots were added to a PBS solution containing BSA and antibodies under ultrasound, and then emulsification was continued to obtain a uniform product. The specific preparation steps are as follows:

[0076] 1. Dissolve red oil-soluble quantum dots ZnCdSe / ZnS QDs (Q1625, Wuhan Jiayuan Quantum Dot Co., Ltd.) in n-hexane to make the concentration of the quantum dot solution 2 mg / mL.

[0077] 2. Take 12 μL of the 2 mg / mL quantum dot solution prepared in step 1 and add it to 100 μL of the stirring PBS solution (containing 1 mg / mL BSA, 0.3 mg / mL Ab Sildenafil Antibody 1 μL and 0.34 mg / mL Ab Tadalafil antibody 1 μL), stirred at 500 rpm for 3 h at 25-30° C. to obtain stirred quantum dot polymer MQDs-BSA-Ab NPs. Sildenafil The preparation method is derived from document 1, the DOI number of which is 10.1016 / j.aca.2020.10.032; the Ab TadalafilThe preparation method is derived from document 2, and the DOI number of document 2 is DOI: 10.1080 / 09540105.2019.1585417.

[0078] 3. Centrifuge the stirred quantum dot polymer obtained in step 2 at 12000 rpm for 30 min, remove the supernatant, and obtain the quantum dot polymer after centrifugation.

[0079] 4. Storage. Add 100 μL of storage solution (PBS solution containing 0.05% sodium azide) to the centrifuged quantum dot polymer and vortex for 15 seconds to resuspend the product to obtain MQDs-BSA-Ab. Sildenafil NSs and MQDs-BSA-Ab Tadalafil NSs probe, stored at 4℃ for future use, the obtained MQDs-BSA-Ab Sildenafil NSs and MQDs-BSA-Ab Tadalafil The concentration of NSs probes was 0.24 mg / mL.

[0080] 5. Characterization: Characterize the morphology and spectral information of the probe obtained in step 4.

[0081] 2. Experimental Results

[0082] TEM characterization of the complex probes at the early, middle and late stages of self-assembly (corresponding to stirring times of 0.5 h, 1.5 h and 3 h, respectively) showed that in the early stage of self-assembly, the fat-soluble quantum dots first aggregated into denser nanospheres in PBS due to hydrophobic interaction and π-π stacking effect ( Figure 1 A). As the assembly time increases, the quantum dot nanospheres gradually bind to BSA and antibodies due to hydrogen bonds and van der Waals forces, and the interior of the spheres begins to loosen ( Figure 1 B), encapsulating part of the protein (BSA and antibody mixture). When the assembly time reaches 3 hours, the protein and quantum dot nanospheres are more tightly bound, and the interior of the quantum dot nanospheres is gradually occupied by proteins, so that more quantum dots are squeezed to the edge of the sphere, gradually becoming hollow ( Figure 1 C). In order to better confirm the inference of the protein core-quantum dot shell structure, the quantum dot-protein complex fluorescent probe assembled for 3 hours was characterized by cold field scanning electron microscopy (SEM) ( Figure 2 A), it was found that a large number of quantum dots were aggregated into spheres to form a single probe. However, most of the spheres were incomplete, hollow or missing corners. It was speculated that these black hollow / missing corner areas were protein bonding areas. In order to clearly capture the protein, a thermal field scanning electron microscopy characterization image was added. The results showed that ( Figure 2B), quantum dots do aggregate into clusters, adhere to the surface of proteins or embed into proteins. These results all indicate that the synthesized probes are loaded with a large number of quantum dots, providing immunoprobes with high fluorescence loading for the construction of highly sensitive immunochromatography.

[0083] Quantum dot polymers (MQDs NPs) without BSA and antibodies could not disperse in PBS solution, but precipitated at the bottom of the centrifuge tube ( Figure 3 ), after adding BSA or a mixture of BSA and antibodies, the quantum dot polymers (MQDs-BSA NPs, MQDs-BSA-Ab NPs) formed can be well dispersed in PBS solution to form a stable solution. The stability of the polymer assembled from the quantum dot solution with only antibodies is better than that of MQDs NPs, but worse than that of MQDs-BSA NPs and MQDs-BSA-Ab NPs. This is because the amount of antibodies required to recognize the antigen is low and is not enough to completely stabilize the polymer in the solution. The role of BSA in the synthesis of the self-assembled fluorescent complex probe is to block nonspecific sites and use its own hydrophilicity to improve the stability of the complex in the solution. Figure 2 C is the infrared spectrum showing that after the probe is synthesized, a C=O bond appears, while the original quantum dots do not have this bond, indicating that the protein is combined with the quantum dots. At the same time, the fluorescence spectrum scanning results show that the optimal excitation wavelength of MultiQDs-BSA-Ab NPs is 370nm, and the optimal absorption wavelength is 630nm ( Figure 2 D). When excited by UV LED light, it emits red light ( Figure 2 D illustration).

[0084] Example 2 Kit for detecting nafil-like substances

[0085] 1. Preparation method

[0086] 1. Preparation of immunochromatographic test strips

[0087] like Figure 4 As shown, the immunochromatographic test strip for detecting sildenafil and tadalafil consists of a sample pad, a nitrocellulose membrane (NC membrane), absorbent paper and a PVC bottom plate. A 30 cm long NC membrane is placed on a film stripper, and three 1 mm vertical lines are drawn from left to right with the long side of the membrane as the horizontal side and the short side as the vertical side. The first two lines are the test lines, and the third line is the quality control line. The first test line is 7 mm away from the short side on the left, and the two test lines are 6 mm apart. The quality control line is 6 mm away from the second test line.

[0088] Use a three-dimensional film scribing instrument to spray 20 μL of 0.19 mg / mL and 0.38 mg / mL tadalafil antigen and 0.38 mg / mL sildenafil antigen onto the first and second test lines, respectively, at a rate of 0.8 μL / min. At the same time, 20 μL of 0.02 mg / mL goat anti-rabbit IgG was scribed onto the quality control line. The NC membrane was then transferred to a 37°C oven for drying and taken out after drying for 6 hours. Sildenafil The preparation method is derived from document 3, the DOI number of which is 10.1016 / j.aca.2020.10.032; the Ag Tadalafil The preparation method is derived from document 4, and the DOI number of document 4 is 10.1080 / 09540105.2019.1585417.

[0089] The sample pad is a glass cellulose membrane with a length of 30 cm and a width of 25 cm. After the sample pad is fully infiltrated with the sample pad treatment solution, it is placed in a 37°C oven for drying for 12 hours and then taken out, and the sample pad is cut into 15 mm lengths using a strip cutter. The sample pad treatment solution consists of 5 mg / mL PVP, 5 mg / mL sucrose, 5 mg / mL BSA, 1% Tween-20 (v / v) and 0.05 mol / L PB solution. The preparation method of the sample pad treatment solution is: 0.2g PVP, 0.2g sucrose and 0.2g BSA are dissolved in 30mL ultrapure water, and then 6mL of 6.67% Tween-20 solution and 4mL PB solution (0.5mol / L) are added. The preparation method of the 0.5mol / LPB solution is: 13.5g KH2PO4, 143g Na2HPO4·12H2O and 10g KCl, and water is added to make the volume to 1000mL.

[0090] The prepared sample pad, NC membrane and absorbent paper were placed in Figure 4 Finally, the assembled test strips were cut into 3.05 mm wide strips using a chopper, placed in a sealed bag, and stored in a dry and cool place for later use.

[0091] 2. Preparation of reaction microplate

[0092] The MQDs-BSA-Ab prepared in Example 1 Sildenafil and MQDs-BSA-Ab Tadalafil 2.5 μL of each probe was added to the microwells of a 96-well plate, i.e., each well contained MQDs-BSA-Ab Sildenafil and MQDs-BSA-Ab Tadalafil The probes were 0.6 μg each.

[0093] 2. Composition

[0094] The kit for detecting phenotype substances is prepared by the preparation method of the kit for detecting phenotype substances.

[0095] Example 3 Lateral flow immunochromatography detection process

[0096] Detection principle see Figure 5 , the specific steps are as follows:

[0097] 1. Use a small syringe pump to inject 1 mL of methanol and 1 mL of ultrapure water into the solid phase extraction microfluidic chip to activate and clean the MCX filler in the chip.

[0098] 2. Use a small syringe pump to inject the sample extract to be tested into the chip at a flow rate of 80 μL / min.

[0099] 3. After the sample extract has flowed away, use a small syringe pump to inject a methanol solution containing 0.1% acetic acid into the chip at a flow rate of 20 μL / min to elute the analytes on the chip. At the same time, place a ceramic heating plate under the solvent evaporation area of ​​the chip and set the temperature of the ceramic heating plate to 60°C to evaporate the methanol.

[0100] 4. After the eluent has run out, use a small syringe pump to inject the chromatographic solution into the chip at a flow rate of 80 μL / min to obtain a PB solution containing the substance to be tested. The chromatographic solution is a 0.025 mol / L PB solution, which is prepared by: 0.675 g KH2PO4, 7.15 g Na2HPO4·12H2O and 0.5 g KCl, add water to make the volume 1000 mL.

[0101] 5. Collect the PB solution containing the test substance in step 4 into the microwells of a 96-well plate, 100 μL per well, and then add the MQDs-BSA-Ab prepared in Example 1. Sildenafil and MQDs-BSA-Ab Tadalafil 2.5 μL of each probe, i.e. each well contains MQDs-BSA-Ab Sildenafil and MQDs-BSA-Ab Tadalafil 0.6 μg of each probe was added and mixed to obtain a reaction solution. The immunochromatographic test strip prepared in Example 2 was inserted into the reaction solution and fully reacted for 6 minutes, then the test strip was taken out and placed on the 3D printed test strip slot, and pushed into a homemade portable fluorescence detection device to collect the fluorescence signal. The chip and the homemade portable fluorescence detection device are described in another patent of the inventor, application number: 2021115566638.

[0102] Example 4 Method Sensitivity

[0103] Preparation of nafil standard: dissolve 1 mg nafil in 1 mL DMF solution to prepare a mother solution, and then dilute with 0.025 mol / L PB solution to prepare the test solution. The preparation method of 0.025 mol / L PB solution is shown in Example 3.

[0104] A mixed solution containing tadalafil and sildenafil was prepared, wherein the concentrations of the tadalafil solution in the mixed solution were 0, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 ng / mL, respectively, and the sildenafil concentration was the same as that of tadalafil. The prepared mixed solution was injected into a solid phase extraction microfluidic chip by a small syringe pump, and other detection processes were the same as those in Example 3. Each concentration was fitted to the corresponding grayscale value using a mobile phone detection applet to obtain a standard curve.

[0105] MQDs-BSA-Ab Sildenafil and MQDs-BSA-Ab Tadalafil After incubating with the unknown concentration of free antigen for a certain period of time, the sample flows to the sildenafil antigen and tadalafil antigen fixed on the double detection lines (T lines) of the test strip under the action of chromatography. When the sample solution does not contain free antigen or the concentration is low, the probe is captured by the fixed antigen, making the T line fluorescence stronger; conversely, only a small amount of probe is captured by the antigen on the T line, so the T line presents a weaker fluorescence signal. Figure 6 A illustration and Figure 6 As shown in Figure B, as the content of sildenafil antigen and tadalafil in the solution increases, the fluorescence of the two T lines of the test strip gradually weakens. When the content of sildenafil is 1 ng / mL and the content of tadalafil is 1.25 ng / mL, the corresponding T lines just have no fluorescence. Therefore, the cut-off values ​​of this method for detecting sildenafil and tadalafil are 1 ng / mL and 1.25 ng / mL, respectively.

[0106] After fitting with the detection applet, the standard curve equation of sildenafil can be obtained as follows: G = (87.12-55.56) / [1+(X / 0.15) 1.98 ]+55.56, LOD value was 0.05ng / mL, and the half inhibition concentration (IC 50 ) is 0.15 ng / mL;

[0107] The standard curve equation of tadalafil is G = (89.02-55.15) / [1+(X / 0.18) 1.97 ]+55.15, LOD value was 0.057ng / mL, and the half inhibition concentration (IC 50 ) is 0.18ng / mL.

[0108] Compared with the sildenafil and tadalafil immunochromatographic method developed earlier in this experiment (source: https: / / doi.org / 10.1016 / j.snb.2022.131450), the sensitivity of this chapter has been significantly improved. The reason is speculated to be that the probe MQDs-BSA-Ab prepared in Example 1 Sildenafil and MQDs-BSA-Ab Tadalafil With ultra-high QDs loading capacity.

[0109] To prove this conclusion, we used the same QDs to label Ab according to conventional modification methods (DOI: 10.1002 / bio.1029). Sildenafil and Ab Tadalafil , that is, first modify the QDs surface to be hydrophilic, then couple the antibody. Then construct the immunochromatographic test strip. TEM characterization shows that the probe prepared by the traditional method contains a single QD ( Figure 7 C), while the single self-assembled probe prepared by this method contains dozens of QDs ( Figure 7 B), which greatly enhances the fluorescence loading of the unit probe. The chromatography standard curve constructed by the traditional QD-Ab probe is G Sildenafil =(69.65-34.5) / [1+(X / 2.36) 1.71 ]+34.5; G Tadalafil =(70.62-34.55) / [1+(X / 5.20) 1.75 ]+34.55, and the LOD values ​​for sildenafil and tadalafil were 0.7ng / mL and 1.6ng / mL, respectively. It was calculated that the sensitivity of the self-assembled probe method was 14 to 26.6 times higher than that of the traditional probe method.

[0110] Example 5 Method Specificity

[0111] 1. Experimental Methods

[0112] Nine sildenafil derivatives (desmethylsildenafil, erythroidenafil, hydroxyerythroidenafil, pseudovardenafil, narmosildenafil, udenafil, homosildenafil, thiohomosildenafil, thioedenafil) and six tadalafil derivatives (N-desmethyltadalafil, N-butyltadalafil, N-desmethlenttadalafil, N-octylnortadalafil, acetamidotadalafil, aminotadalafil) were selected for testing.

[0113] 1. MQDs-BSA-Ab Sildenafil Specificity

[0114] PB solutions (0.025 mol / L) containing 100 ng / mL desmethylsildenafil, erythroidenafil, hydroxyerythroidenafil, pseudovardenafil, nalmosildenafil, udenafil, homosildenafil, thiohomosildenafil, and thioidenafil were prepared respectively, and 100 μL of each of the above 9 solutions was added to different microwells, and then 2.5 μL of MQDs-BSA-Ab prepared in Example 1 was added respectively. Sildenafil Probe, i.e. each well contains MQDs-BSA-Ab Sildenafil The probe was 0.6 μg. The immunochromatographic test strip prepared in Example 2 was inserted into the reaction solution and fully reacted for 6 minutes. The test strip was then taken out and placed on the 3D printed test strip slot, and pushed into a homemade portable fluorescence detection device to collect the fluorescence signal. The chip and the homemade portable fluorescence detection device are described in another patent of the inventor, application number: 2021115566638. The preparation method of 0.025 mol / LPB solution is described in Example 3.

[0115] 2. MQDs-BSA-Ab Tadalafil Specificity

[0116] PB solutions (0.025 mol / L) containing 100 ng / mL N-desmethyl tadalafil, N-butyl tadalafil, N-desmethlent tadalafil, N-octyl demethyl tadalafil, acetamido tadalafil, and amino tadalafil were prepared respectively, and 100 μL of each of the above 6 solutions was added to different microwells, and then 2.5 μL of MQDs-BSA-Ab prepared in Example 1 was added respectively. Tadalafil Probe, i.e. each well contains MQDs-BSA-Ab Tadalafil The probe is 0.6 μg and mixed. Other detection procedures are the same as MQDs-BSA-Ab Sildenafil The specific detection is the same, and the preparation method of 0.025 mol / LPB solution is shown in Example 3.

[0117] 2. Experimental Results

[0118] MQDs-BSA-Ab Sildenafil In addition to identifying sildenafil, it can also identify desmethylsildenafil, erythridine, hydroxyerythridine, pseudovardenafil, udenafil, homosildenafil, and thiosildenafil ( Figure 8 A). Although it also has a weak recognition effect on nalmosildenafil and thiohomosildenafil, the concentration of 100ng / mL has only a weak inhibitory effect, indicating that the cut-off value of this probe for nalmosildenafil and thiohomosildenafil is much higher than 100ng / mL, so it can be regarded as a substance with less interference. MQDs-BSA-Ab TadalafilIn addition to identifying tadalafil, it can also identify N-desmethyl tadalafil, N-butyl tadalafil, and acetamido tadalafil ( Figure 8 B).

[0119] Based on the above situation, quantitative and qualitative detection methods were established for seven sildenafil derivatives (desmethylsildenafil, erythroidenafil, hydroxyerythroidenafil, pseudovardenafil, udenafil, homosildenafil, thio-edenafil) and three tadalafil derivatives (N-desmethyltadalafil, N-butyltadalafil and acetamidotadalafil). Fig. 9 As shown, the cut-off values ​​of desmethylsildenafil, erythrodenafil, hydroxyerythrodenafil, and pseudovardenafil are 1.0 ng / mL, 2.0 ng / mL, 0.5 ng / mL, and 0.5 ng / mL, respectively.

[0120] The quantitative equation of desmethylsildenafil is G = (89.39 + 20.08 / [1 + (X / 0.18) 1.50 ]–20.08, LOD was 0.04 ng / mL, IC 50 0.18 ng / mL;

[0121] The quantitative equation of red denafil is G = (87.94-32.73) / [1+(X / 0.32) 1.30 ]+32.73, LOD was 0.058 ng / mL, IC 50 0.32 ng / mL;

[0122] The quantitative equation of hydroxy red denafil is G = (87.47-33.71) / [1+(X / 0.04) 1.43 ]+33.71, LOD is 0.01ng / mL, IC 50 0.04 ng / mL;

[0123] The quantitative equation of pseudovardenafil is G = (87.87-33.96) / [1+(X / 0.07) 1.18 ]+33.96, LOD is 0.01ng / mL, IC 50 It is 0.07ng / mL.

[0124] like Fig.10 As shown, the cut-off values ​​of udenafil, homosildenafil, and thiosildenafil were 0.25, 0.5, and 2.0 ng / mL, respectively.

[0125] The quantitative equation of Udenafil is G = (85.61-31.41) / [1+(X / 0.07) 1.02 ]+33.95, LOD is 0.01ng / mL, IC 500.07 ng / mL;

[0126] The quantitative equation of homosildenafil is G = (86.65-31.80) / [1+(X / 0.14) 1.27 ]+31.80, LOD is 0.03ng / mL, IC 50 It is 0.14ng / mL.

[0127] The quantitative equation of thiodenafil is G = (86.82-34.14) / [1+(X / 0.31) 1.07 ]+33.95, LOD was 0.04ng / mL, IC 50 It is 0.31ng / mL.

[0128] like Fig.11 As shown, the cut-off values ​​of N-desmethyl tadalafil, N-butyl tadalafil, and acetamido tadalafil are 3 ng / mL, 1.5 ng / mL, and 1.5 ng / mL, respectively.

[0129] The quantitative equation of N-desmethyl tadalafil is G = (89.88-36.07) / [1+(X / 0.24) 0.82 ]+36.07, LOD was 0.02ng / mL, IC 50 0.24 ng / mL;

[0130] The quantitative equation of N-butyl tadalafil is G = (89.40-37.68) / [1+(X / 0.23) 0.73 ]+37.68, LOD was 0.02ng / mL, IC 50 0.23 ng / mL;

[0131] The quantitative equation of O-acetamido tadalafil is G = (88.66-38.59) / [1+(X / 0.34) 1.06 ]+38.59, LOD was 0.04ng / mL, IC 50 It is 0.34ng / mL.

[0132] As shown in Table 1, compared with other immunochromatographic methods, this method has a significantly lower cut-off value and a larger number of targets.

[0133] Table 1 Comparison of the performance of the method in Example 3 with other immunochromatographic methods of phenafil drugs

[0134]

[0135] Example 6 Method Reproducibility

[0136] 1. Experimental Methods

[0137] Preparation of nafil standard: 1 mg of nafil drug was dissolved in 1 mL of DMF solution to prepare a mother solution. Then, 0.025 mol / L PB solution was used to dilute the solution to be tested. The preparation method of 0.025 mol / L PB solution is shown in Example 3. Three concentrations of sildenafil solution (0, 0.05 and 0.5 ng / mL) and tadalafil solution (0, 0.25 and 1 ng / mL) were set up. Each concentration was repeated 5 times, and the deviation between each actual detection concentration was finally calculated. The specific method is as follows:

[0138] 100 μL of PB solution (0.025 mol / L) containing 0 ng / mL sildenafil and 0 ng / mL tadalafil, 0.05 ng / mL sildenafil and 0.25 ng / mL tadalafil, 0.5 ng / mL sildenafil and 1 ng / mL tadalafil were respectively taken and injected into the solid phase extraction microfluidic chip through a small syringe pump. The other detection processes were the same as those in Example 3.

[0139] 2. Experimental Results

[0140] The results are as follows Fig.12 As shown in A, the coefficient of variation between the actual measured concentrations of sildenafil is between 5.34% and 7.86%, and the coefficient of variation between the actual detected concentrations of tadalafil is between 6.92% and 9.34%, both of which are less than 15%, indicating that the precision of this method is relatively high.

[0141] Example 7 Storage stability of the method

[0142] 1. Experimental Methods

[0143] In order to investigate the stability of the self-assembled fluorescent probe under 4°C refrigerator storage conditions and the storage stability of the test strip under 37°C dry environment, 100 μL of PB solution (0.025 mol / L) was taken into the microwells of the ELISA plate, and the MQDs-BSA-Ab prepared in Example 1 after being stored for 2 weeks, 4 weeks, 6 weeks, 8 weeks and 10 weeks were added respectively. Sildenafil and MQDs-BSA-Ab Tadalafil 2.5 μL of each probe, i.e. each well contains MQDs-BSA-Ab Sildenafil and MQDs-BSA-Ab Tadalafil 0.6 μg of each probe was mixed. Then the immunochromatographic test strip prepared in Example 2 after being stored for 2 weeks, 4 weeks, 6 weeks, 8 weeks and 10 weeks was inserted into the solution for reaction. The other detection procedures were the same as those in Example 3. The preparation method of 0.025 mol / L PB solution is shown in Example 3.

[0144] 2. Experimental Results

[0145] Storage stability is also an important indicator for evaluating the quality of an analytical method. The results show that ( Fig.12 B) MQDs-BSA-Ab prepared in Example 1 after being stored at 4°C for 6 weeks Sildenafil and MQDs-BSA-Ab Tadalafil The probe and the immunochromatographic test strip prepared in Example 2 were stored at 4°C for 6 weeks, and the signal still had a response of 92.0%. After 8 weeks of storage, the signal response dropped to 83.0%; and after 10 weeks of storage, the signal response dropped significantly, retaining only about 76.0%. The above results show that the MQDs-BSA-Ab prepared in Example 1 of the present invention Sildenafil and MQDs-BSA-Ab Tadalafil The probe and the immunochromatographic test strip prepared in Example 2 had high stability within 6 weeks.

[0146] Example 8 Method Accuracy

[0147] 1. Experimental Methods

[0148] The accuracy of the established method was preliminarily evaluated by adding recovery experiments. Negative kidney-tonifying pills and kidney-tonifying medicinal wine were purchased from supermarkets or pharmacies around South China Agricultural University as sample matrices, and sildenafil and tadalafil standards were added to perform addition recovery experiments.

[0149] 1. Addition and recovery experiment of solid samples

[0150] Weigh 15 portions of Bushen Pills, 1 g each, and add 100 μL of a methanol mixture of sildenafil and tadalafil respectively, so that the samples contain 0.1 μg / g sildenafil and 0.2 μg / g tadalafil (5 parallel samples), 0.2 μg / g sildenafil and 0.4 μg / g tadalafil (5 parallel samples), 0.3 μg / g sildenafil and 0.6 μg / g tadalafil (5 parallel samples). The preparation method of sildenafil and tadalafil mother solutions is shown in Example 6, and they are diluted with methanol when used.

[0151] (1) Immunochromatographic testing

[0152] The Bushen Pills with sildenafil and tadalafil added were ground into powder, and then 3 mL of methanol was added. After ultrasonication for 5 min, the supernatant was retained to obtain the Bushen Pills sample extract. Then, the sample was processed according to Implementation Case 3. It should be noted that since the standard addition concentration is higher than the response analysis range of the lateral flow immunochromatography, the sample solution after chip processing needs to be diluted 1000 times with 0.025 mol / L PB solution before mixing with the probe and inserting the test strip for detection. The actual addition concentrations of the diluted sample solution are: 0.1, 0.2 and 0.3 ng / mL for sildenafil, and 0.2, 0.4 and 0.6 ng / mL for tadalafil.

[0153] (2) LC-MS / MS detection

[0154] First, the solid samples with the above added concentrations were pretreated according to the standard method SNT4054-2014. That is, 1.0 g of solid sample (ground into powder) was placed in a 100 mL stoppered conical flask, 80 mL of methanol was added for ultrasonic extraction for 30 minutes, the sample was taken out and cooled to room temperature, the extract was rotary evaporated to remove the solvent (a small amount remained, not completely dry), the flask was rinsed with a 0.1% acetic acid-methanol mixed solution, and transferred to a 10 mL volumetric flask, the flask was washed with a 0.1% acetic acid-methanol mixed solution several times, the washing solution was combined into a 10 mL volumetric flask, and the 0.1% acetic acid-methanol mixed solution was used to make up to the scale, and the solution was shaken well. After the solution passed through a 0.45 μm microporous organic filter membrane, it was subjected to LC-MS / MS determination.

[0155] The chromatographic conditions for LC-MS / MS detection are as follows: (1) chromatographic column: Agilent Eclipse Plus C18 (3.0×150 mm, 1.8 μm), or equivalent; (2) mobile phase: A is 0.1% formic acid in water by volume, B is methanol, and gradient elution is performed; (3) flow rate: 400 μL / min; (4) column temperature: 35°C; (5) injection volume: 5 μL. The gradient elution program is shown in Table 2.

[0156] Table 2 Gradient elution program

[0157] Gradient time / min Mobile phase A / % Mobile phase B / % 0 90 10 1 50 50 16 35 65 19 2 98 22 2 98 22.1 90 10 25 90 10

[0158] The mass spectrometry conditions for LC-MS / MS detection are: positive ion scanning, multiple reaction monitoring (MRM), curtain gas 138 kPa (20 psi), nebulizer gas 276 kPa (40 psi), auxiliary gas: 310 kPa (45 psi), collision gas: 55 kPa (8 psi), electrospray voltage: 5000 V, ion source temperature: 550.0° C. Table 3 shows the qualitative and quantitative ion and mass spectrometry analysis parameters of the analytes.

[0159] Table 3 Qualitative and quantitative ions and mass spectrometry analysis parameters of compounds

[0160]

[0161]

[0162] 2. Liquid sample addition recovery experiment

[0163] Weigh 15 portions of kidney-tonifying medicinal wine, 1 mL each, and add 100 μL of a mixture of sildenafil and tadalafil respectively, so that the samples contain 0.1 μg / g sildenafil and 0.2 μg / g tadalafil (5 parallel samples), 0.2 μg / g sildenafil and 0.4 μg / g tadalafil (5 parallel samples), and 0.3 μg / g sildenafil and 0.6 μg / g tadalafil (5 parallel samples).

[0164] (1) Immunochromatography

[0165] The medicinal liquor with added sildenafil and tadalafil was directly taken and processed according to Implementation Case 3. It should be noted that since the standard addition concentration is higher than the response analysis range of the lateral flow immunochromatography, the sample solution after chip processing needs to be diluted 1000 times with 0.025 mol / L PB solution before it can be mixed with the probe and inserted into the test strip for detection.

[0166] (2) LC-MS / MS method

[0167] First, the solid samples with the above added concentrations were pretreated according to the standard method SNT4054-2014. That is, 1.0 mL of liquid sample was taken into a 100 mL stoppered conical flask, 80 mL of methanol was added for ultrasonic extraction for 30 min, the sample was taken out and cooled to room temperature, the extract was rotary evaporated to remove the solvent (a small amount remained, not completely dry), the flask was rinsed with a 0.1% acetic acid-methanol mixed solution, and transferred to a 10 mL volumetric flask, the flask was washed with a 0.1% acetic acid-methanol mixed solution several times, the washing solution was combined into a 10 mL volumetric flask, and the 0.1% acetic acid-methanol mixed solution was used to make up to the scale, and the solution was shaken well, and the solution was passed through a 0.45 μm microporous organic filter membrane before LC-MS / MS determination.

[0168] The LC-MS / MS determination method is consistent with the LC-MS / MS method of the solid sample mentioned above.

[0169] 2. Experimental Results

[0170] The results are shown in Table 4. The detection recovery rate of sildenafil by the lateral flow immunochromatography method of the present invention is between 91.5% and 103%, and the coefficient of variation is 4.39% to 9.58%; the detection recovery rate of tadalafil is between 90.0% and 99.8%, and the coefficient of variation is 3.50% to 11.0%. The detection recovery rate of sildenafil by the LC-MS / MS method is 90.9% to 101%, and the coefficient of variation is 2.77% to 5.79%; the detection recovery rate of tadalafil is 92.8% to 96.7%, and the coefficient of variation is 2.62% to 6.52%. The results measured by this method are highly correlated with the results of the LC-MS / MS method, indicating that this method has high accuracy and good precision.

[0171] Table 4 Addition recovery experiment

[0172]

[0173] Note: After sample pretreatment, the extract needs to be diluted 1000 times before being used for lateral flow immunochromatography detection. Therefore, the spike concentrations for LC-MS / MS are the original spike concentrations: sildenafil (0.1, 0.2, 0.3 μg / mL), tadalafil (0.2, 0.4, 0.6 μg / mL). The solutions used for lateral flow immunochromatography detection are sildenafil (0.1, 0.2, 0.3 ng / mL), tadalafil (0.2, 0.4, 0.6 ng / mL)

[0174] Example 9 Practicality of the method

[0175] 1. Experimental Methods

[0176] 21 kinds of kidney-tonifying pills, teas, coffees and wines commonly found on the market were tested and the results were compared with those of LC-MS / MS to evaluate the accuracy and precision of the method.

[0177] 1. Detection of solid samples

[0178] (1) Weigh 3 portions of each solid sample of Bushen Pills, Bushen Tea and Energy Coffee, 1 g each, grind into powder, add 3 mL of methanol, sonicate for 5 min, retain the supernatant and obtain the sample extract.

[0179] The lateral flow immunochromatography detection method and LC-MS / MS detection method and steps are the same as those in Example 8.

[0180] 2. Detection of liquid samples

[0181] The pretreatment method, lateral flow immunochromatography detection method and LC-MS / MS detection method and steps of the kidney-tonifying medicinal wine are the same as those in Example 8.

[0182] 2. Experimental Results

[0183] The results are shown in Table 5. Three products were found to contain sildenafil, namely, No. 7 kidney-tonifying tea (118±3.3ng / g), No. 13 energy coffee powder (112.7±6.6ng / g), and No. 15 energy coffee powder (7040±222ng / g). One product (No. 14 energy coffee powder) was found to contain tadalafil, with a content of 5290±151ng / g. These test results are highly correlated with the test results of LC-MS / MS, indicating that this method has high accuracy and strong practical applicability, and can be used for the detection of tadalafil drugs in actual samples.

[0184] Table 5 Blind sample test

[0185]

[0186] Example 10 Quantum dot concentration, BSA concentration, Ab Sildenafil Concentration, Ab Tadalafil Effects of concentration, PBS / n-hexane ratio and synthesis time on the synthesis of probes

[0187] 1. Experimental Methods

[0188] The probe was synthesized under the following conditions, and the immunochromatographic test strips assembled by Example 2 were prepared. The lateral flow immunochromatographic detection process of Example 3 was used to compare the color difference between the negative group (no sildenafil and tadalafil) and the positive group (the concentration of sildenafil and tadalafil was 100 ng / mL) to determine the optimal experimental conditions.

[0189] 1. Effect of quantum dot concentration on probe synthesis effect

[0190] The concentrations of ZnCdSe / ZnS QDs were set to 0.5, 1, 1.5, 2, 2.5, and 3 mg / mL, and the antibody was 1 μL Ab. Sildenafil , other probe synthesis conditions are the same as those in Example 1.

[0191] 2. Effect of BSA concentration on probe synthesis

[0192] The PBS concentrations were set to 0.25, 0.5, 1, 1.5, 2, 2.5 and 3 mg / mL, and the antibody was 1 μL Ab Sildenafil , other probe synthesis conditions are the same as those in Example 1.

[0193] 3. Ab Sildenafil Effect of concentration on probe synthesis

[0194] Setting Ab Sildenafil The concentrations were 0.04, 0.08, 0.15, 0.23, 0.30, 0.37, 0.45 and 0.53 mg / mL, respectively. Other probe synthesis conditions were the same as in Example 1.

[0195] 4. Ab Tadalafil Effect of concentration on probe synthesis

[0196] Setting Ab Tadalafil The concentrations were 0.07, 0.14, 0.2, 0.27, 0.34 and 0.4 mg / mL, respectively. Other probe synthesis conditions were the same as in Example 1.

[0197] 5. Effect of PBS / n-hexane ratio on probe synthesis

[0198] The concentration of quantum dots added to 100 μL PBS solution was set to 0.48 mg / mL, and the added amount was 50 μL. At this time, the volume ratio of PBS solution to n-hexane containing quantum dots was 2:1;

[0199] The concentration of quantum dots added to 100 μL of PBS solution was 0.96 mg / mL, and the amount added was 25 μL. At this time, the volume ratio of PBS solution to n-hexane containing quantum dots was 4:1;

[0200] The concentration of quantum dots added to 100 μL of PBS solution was 1.44 mg / mL, and the added amount was 16.67 μL. At this time, the volume ratio of PBS solution to n-hexane containing quantum dots was 6:1;

[0201] The concentration of quantum dots added to 100 μL of PBS solution was 2.4 mg / mL, and the amount added was 12.5 μL. At this time, the volume ratio of PBS solution to n-hexane containing quantum dots was 8:1;

[0202] The concentration of quantum dots added to 100 μL of PBS solution was 2.88 mg / mL, and the amount added was 10 μL. At this time, the volume ratio of PBS solution to n-hexane containing quantum dots was 10:1;

[0203] The concentration of quantum dots added to 100 μL of PBS solution was 3.36 mg / mL, and the amount added was 8.33 μL. At this time, the volume ratio of PBS solution to n-hexane containing quantum dots was 12:1;

[0204] The concentration of quantum dots added to 100 μL of PBS solution was 0.48 mg / mL, and the amount added was 7.14 μL. At this time, the volume ratio of PBS solution to n-hexane containing quantum dots was 14:1;

[0205] The concentration of quantum dots added to 100 μL of PBS solution was 0.48 mg / mL, and the amount added was 6.25 μL. At this time, the volume ratio of PBS solution to n-hexane containing quantum dots was 16:1;

[0206] Antibody is 0.3mg / mLAb Sildenafil , other probe synthesis conditions are the same as those in Example 1.

[0207] 6. Effect of synthesis time on probe synthesis effect

[0208] The stirring time of the quantum dot solution, PBS solution and antibody was set to 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 h respectively, and the antibody was 0.3 mg / mL Ab Sildenafil , other probe synthesis conditions are the same as those in Example 1.

[0209] 2. Experimental Results

[0210] 1. Effect of quantum dot concentration on probe synthesis effect

[0211] When fat-soluble quantum dots are added to the PBS solution, the quantum dots will instantly aggregate into nanospheres due to the hydrophobic effect and π-π stacking effect. Then, during the continued stirring process, the quantum dot nanospheres will gradually combine with bovine serum albumin and antibodies under the action of hydrogen bonds and van der Waals forces to form immune probes with ultra-high quantum dot loading. The concentration of quantum dots will affect the density of the formed nanospheres and the quantum dot loading of the polymer probe. The higher the concentration of quantum dots, the stronger the generated probe fluorescence ( Fig.13 A). However, when the concentration of quantum dots exceeded 2 mg / mL, the fluorescence intensity of the probe no longer increased, indicating that 2 mg / mL was the optimal concentration of quantum dots.

[0212] 2. Effect of BSA concentration on probe synthesis

[0213] BSA is an important component of the polymer probe. It not only serves as a skeleton to embed quantum dot nanospheres, but also can block non-specific binding sites. When the BSA concentration is low, the probe quantum dot load formed is low and the fluorescence is weak; as the BSA concentration increases, the fluorescence intensity of the negative test strip gradually increases, and the inhibitory effect of the positive test strip also gradually increases. When the BSA concentration reaches 1 mg / mL, the fluorescence intensity of the negative test strip is basically the strongest, and the positive test strip has no fluorescence ( Fig.13 B), therefore, 1 mg / mL was selected as the optimal BSA concentration.

[0214] 3. Ab Sildenafil and Ab Tadalafil Effect of concentration on probe synthesis

[0215] Antibodies can not only support some quantum dot nanospheres and improve their stability in solution, but more importantly, they can also play a role in recognizing antigens. Sildenafil and Ab Tadalafil When the concentration is low, only a small number of probes can recognize the antigen on the T line, so the fluorescence of the test strip is very weak; as the antibody concentration increases, more and more probes are captured by the antigen on the T line, and the signal is enhanced; but when Ab Sildenafil The concentration of Ab was higher than 0.3 mg / mL. Tadalafil When the concentration is higher than 0.34 mg / mL, the T line of the positive test strip also shows a fluorescent signal ( Fig.13 C and Fig.13 D), indicating that the antibody concentration is too high, causing the test strip to have a low sensitivity. Sildenafil The concentration is 0.3 mg / mL, and the best Ab Tadalafil The concentration is 0.34mg / mL.

[0216] 4. Effect of PBS / n-hexane ratio on probe synthesis

[0217] The self-assembly process of the probe is actually the emulsification process of fat-soluble quantum dots and water-soluble protein solution. The appropriate water / oil ratio (PBS / n-hexane) plays a vital role in forming a stable probe solution. If the PBS / n-hexane ratio is low, that is, the n-hexane content in the system is high, it will damage the biological activity of the protein, reduce the recognition of antibodies and antigens, and make the fluorescence signal of the T line of the test strip weaker; when the PBS / n-hexane ratio is 8:1, the number and stability of the obtained probes are both high, which is reflected in the strong fluorescence of the T line of the negative test strip and the lack of fluorescence of the T line of the positive group; when the PBS / n-hexane ratio continues to increase, the emulsification effect becomes worse, and the number of fluorescent probes obtained becomes less, which is reflected in the significant weakening of the fluorescence on the T line of the negative test strip ( Fig.13 E). Fig.13 F is the optimization of the probe synthesis time, specifically the stirring time after the quantum dots are added to the protein solution. Therefore, the optimal water / oil ratio is 8:1. For the convenience of calculation, when preparing the probe, the concentration of the quantum dots added to 100 μL PBS solution is 2 mg / mL, and the addition amount is 12 μL.

[0218] 5. Effect of synthesis time on probe synthesis effect

[0219] After the quantum dots are polymerized into compact nanospheres, continued stirring will cause the protein molecules to gradually embed into the nanospheres to form copolymers. If the polymerization time is too short, most of the nanospheres are not attached to the protein molecules, so the hydrophilicity is poor, so there is a large amount of floating oil in the supernatant of the solution after centrifugation ( Fig.14 A and B); as the polymerization continues, more and more protein molecules are embedded in the interior of the quantum dot nanospheres, improving the hydrophilicity of the polymer, so the supernatant after centrifugation gradually becomes clear ( Fig.14 C~E). When the polymerization time reaches more than 2.5h, the hydrophilicity of the polymer basically reaches the best, and there is no residual polymer in the supernatant ( Fig.14 F~H). At the same time, according to the results of the immunochromatography test, it can be seen that when the polymerization time is 3h, the number of synthesized probes has basically reached the highest, so the optimal synthesis time of the probe is 3h.

[0220] Example 11Ag Tadalafil Film concentration, Ag Sildenafil Effects of stripping concentration, probe dosage, sample running time, Tween-20 concentration in sample pad treatment solution, and phosphate concentration in chromatography solution on lateral flow immunochromatography

[0221] 1. Experimental Methods

[0222] 1. Ag Tadalafil Effect of stripping concentration on lateral flow immunochromatography

[0223] Ag Tadalafil Dilute to 0.10, 0.19, 0.38, 0.75 and 1.5 mg / mL, and dilute the goat anti-mouse secondary antibody to 0.1 mg / mL. The preparation method of other immunochromatographic test strips is the same as that of Example 2. The preparation method of 0.02 mol / LPB solution is: 0.54 g KH2PO4, 5.72 g Na2HPO4·12H2O, 0.4 g KCl, and add water to make up to 1000 mL.

[0224] Negative group and positive group were set up. Negative group: 100 μL PB solution (0.025 mol / L) was taken into the microwells of the ELISA plate, and the MSNs-PDA-Ab prepared in Example 1 was added. Tadalafil 2.5 μL of probe, i.e. each well contains MQDs-BSA-Ab Tadalafil Probe 0.6μg, mix well.

[0225] Positive group: 100 μL of PB solution (0.025 mol / L) containing 100 ng / mL tadalafil was taken into the microwells of the ELISA plate, and the MSNs-PDA-Ab prepared in Example 1 was added. Tadalafil 2.5 μL of probe, i.e. each well contains MQDs-BSA-Ab Tadalafil Probe 0.6μg, mix well.

[0226] The other detection processes are the same as those in Example 3.

[0227] 2. Ag Sildenafil Effect of stripping concentration on lateral flow immunochromatography

[0228] Ag Sildenafil Dilute to 0.048, 0.095, 0.19, 0.38, 0.75 and 1.5 mg / mL, and dilute the goat anti-mouse secondary antibody to 0.1 mg / mL. The preparation method of other immunochromatographic test strips is the same as that of Example 2.

[0229] Negative group and positive group were set up. Negative group: 100 μL PB solution (0.025 mol / L) was taken into the microwells of the ELISA plate, and the MSNs-PDA-Ab prepared in Example 1 was added. Sildenafil 2.5 μL of probe, i.e. each well contains MQDs-BSA-Ab Sildenafil Probe 0.6μg, mix well.

[0230] Positive group: 100 μL of PB solution (0.025 mol / L) containing 100 ng / mL sildenafil was taken into the microwells of the ELISA plate, and the MSNs-PDA-Ab prepared in Example 1 was added.Sildenafil 2.5 μL of probe, i.e. each well contains MQDs-BSA-Ab Sildenafil Add 0.6 μg and mix well.

[0231] The other detection processes are the same as those in Example 3.

[0232] 3. Effect of probe dosage on lateral flow immunochromatography

[0233] Negative group and positive group were set up. Negative group: 100 μL PB solution (0.025 mol / L) was taken into the microwells of the ELISA plate, and the MQDs-BSA-Ab prepared in Example 1 was added respectively. Sildenafil and MQDs-BSA-Ab Tadalafil Probes 1, 1.5, 2, 2.5, 3 and 3.5 μL, mix well.

[0234] Positive group: 100 μL of PB solution (0.025 mol / L) containing 100 ng / mL sildenafil and tadalafil was taken into the microwells of the ELISA plate, and the MQDs-BSA-Ab prepared in Example 1 was added respectively. Sildenafil and MQDs-BSA-Ab Tadalafil Probes 1, 1.5, 2, 2.5, 3 and 3.5 μL, mix well.

[0235] The preparation method of the immunochromatographic test strip is the same as that of Example 2, and the other detection processes are the same as those of Example 3.

[0236] 4. Effect of running time on lateral flow immunochromatography

[0237] Negative group and positive group were set up. Negative group: 100 μL PB solution (0.025 mol / L) was taken into the microwells of the ELISA plate, and the MQDs-BSA-Ab prepared in Example 1 was added respectively. Sildenafil and MQDs-BSA-Ab Tadalafil 2.5 μL of each probe, i.e. each well contains MQDs-BSA-Ab Sildenafil and MQDs-BSA-Ab Tadalafil 0.6 μg of each probe was added and mixed well.

[0238] Positive group: 100 μL of PB solution (0.025 mol / L) containing 100 ng / mL sildenafil and tadalafil was taken into the microwells of the ELISA plate, and the MQDs-BSA-Ab prepared in Example 1 was added respectively. Sildenafil and MQDs-BSA-Ab Tadalafil 2.5 μL of each probe, i.e. each well contains MQDs-BSA-Ab Sildenafil and MQDs-BSA-Ab Tadalafil 0.6 μg of each probe was added and mixed well.

[0239] Then the immunochromatographic test strip prepared in Example 2 was inserted into the solution and reacted for 3, 4, 5, 6, 7 and 8 minutes respectively. The other detection procedures were the same as in Example 3.

[0240] 5. Effect of Tween-20 concentration in sample pad treatment solution on lateral flow immunochromatography

[0241] The sample pad was fully infiltrated with sample pad treatment solution containing 0.5%, 0.75%, 1%, 1.25%, 1.5% and 1.75% Tween (v / v) respectively. The preparation method of other immunochromatographic test strips was the same as that of Example 2.

[0242] Negative group and positive group were set up. Negative group: 100 μL PB solution (0.025 mol / L) was taken into the microwells of the ELISA plate, and the MQDs-BSA-Ab prepared in Example 1 was added. Sildenafil 2.5 μL of probe, i.e. each well contains MQDs-BSA-Ab Sildenafil Probe 0.6μg, mix well.

[0243] Positive group: 100 μL of PB solution (0.025 mol / L) containing 100 ng / mL sildenafil was taken into the microwells of the ELISA plate, and the MQDs-BSA-Ab prepared in Example 1 was added. Sildenafil 2.5 μL of probe, i.e. each well contains MQDs-BSA-Ab Sildenafil Probe 0.6μg, mix well.

[0244] The other detection processes are the same as those in Example 3.

[0245] 6. Effect of phosphate concentration in the chromatography fluid on lateral flow immunochromatography

[0246] The negative group chromatographic fluid and the positive group chromatographic fluid were prepared with PB solutions with phosphate concentrations of 0.0125, 0.025, 0.05, 0.1, 0.2, 0.3 and 0.4 mol / L, respectively.

[0247] Negative group: 100 μL of PB solution was taken into the microwells of the ELISA plate, and the MQDs-BSA-Ab prepared in Example 1 was added. Sildenafil 2.5 μL of probe, i.e. each well contains MQDs-BSA-Ab Sildenafil Probe 0.6μg, mix well.

[0248] Positive group: 100 μL of PB solution containing 100 ng / mL sildenafil was taken into the microwells of the ELISA plate, and the MQDs-BSA-Ab prepared in Example 1 was added. Sildenafil 2.5 μL of probe, i.e. each well contains MQDs-BSA-AbSildenafil Probe 0.6μg, mix well.

[0249] The other detection processes are the same as those in Example 3.

[0250] 2. Experimental Results

[0251] 1. Ag Tadalafil and Ag Sildenafil Effect of stripping concentration on lateral flow immunochromatography

[0252] The concentration of the antigen fixed on the T line of the paper strip will affect its binding ability with the antibody probe, and ultimately affect the sensitivity of the method. Fig.15 As shown in A and B, when the concentration of the fixed antigen is too low, the ability to capture the antibody is relatively weak, resulting in a low fluorescence intensity of the T line of the test strip; as the concentration of the fixed antigen increases, more and more probes are captured on the T line, and the fluorescence becomes stronger and stronger; but when the concentration of the fixed antigen is too low, the fluorescence intensity of the T line of the test strip is relatively low. Sildenafil Concentration higher than 0.38 mg / mL, Ag Tadalafil When the concentration is higher than 0.19 mg / mL, the T line of the positive test strip also begins to show a fluorescent response, indicating that the concentration of the fixed antigen is too high, which reduces the sensitivity of the method. Sildenafil The concentration is 0.38mg / mL, the best Ag Tadalafil The concentration is 0.19 mg / mL.

[0253] 2. Effect of probe dosage on lateral flow immunochromatography

[0254] When the amount of immune probe is too small, the fixed antigen can capture less signal probe, resulting in weaker color of the T line of the test strip, which is not convenient for subsequent observation ( Fig.15 C) MQDs-BSA-Ab prepared in Example 1 Sildenafil and MQDs-BSA-Ab Tadalafil As the amount of probe increases, the fluorescence response on the two T lines of the test strip also increases. When the amount of both probes reaches 2.5μL, the fluorescence of the double T lines of the negative test strip is relatively strong, and the double T lines of the positive test strip have no fluorescence; and when the amount continues to increase, the inhibition effect of the positive test strip begins to deteriorate, and at the same time, the probe consumption is high, which increases the detection cost. Therefore, the optimal amount of both probes is 2.5μL, that is, each well contains MQDs-BSA-Ab Sildenafil and MQDs-BSA-Ab Tadalafil The probes were 0.6 μg each.

[0255] 4. Effect of running time on lateral flow immunochromatography

[0256] The chromatography time also has a great influence on the sensitivity of the method. If the chromatography time is too short, only a small part of the probe will run onto the paper strip and be captured by the antigen on the T line, resulting in a weak fluorescence signal; extending the chromatography time can increase the chance of binding between the fixed antigen and the antibody probe, thereby enhancing the signal response of the T line of the test strip ( Fig.15 D) When the chromatography time reaches 6 minutes, the fluorescence signal basically reaches the strongest point, so the most suitable running time is 6 minutes.

[0257] 5. Effect of Tween-20 concentration in sample pad treatment solution on lateral flow immunochromatography

[0258] The NC membrane is hydrophobic, which is not conducive to the capillary flow of the probe. Therefore, treating the sample pad with a solution containing an appropriate amount of surfactant Tween-20 can enhance the hydrophilicity of the test strip, improve the chromatography effect, and thus shorten the detection time. After treating the sample pad with different concentrations of Tween-20 (0.5%, 0.75%, 1.0%, 1.25%, 1.5% and 1.75%) and assembling it into a test strip, the optimal Tween-20 concentration was determined by measuring the negative solution and a 100ng / mL mixed solution of sildenafil and tadalafil. Fig.15 As shown in Figure E, when the Tween-20 content gradually increases (0.5% to 1.0%), the fluorescence brightness of the two T lines also gradually increases; but when the Tween-20 content continues to increase, the T line of the positive test strip also begins to fluoresce, and the inhibition effect gradually becomes worse. Therefore, the optimal Tween-20 concentration of the sample pad treatment solution is 1.0%.

[0259] 6. Effect of phosphate concentration in the chromatography fluid on lateral flow immunochromatography

[0260] Adding an appropriate amount of salt ions to the chromatography fluid can stabilize the protein conformation and facilitate antigen-antibody binding. When the phosphate concentration of the chromatography fluid increases from 0.0125 mol / L to 0.025 mol / L, the fluorescence of the double T lines of the test strip increases; however, as the phosphate concentration continues to increase, the T line signal weakens instead ( Fig.15 F). The reason may be that high concentration of salt ions will neutralize the electronegativity of the probe, causing the probe to aggregate in the chromatography fluid prematurely and difficult to chromatograph onto the paper strip. Therefore, the optimal phosphate concentration of the chromatography fluid is 0.025 mol / L.

[0261] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing a fluorescent immunoprobe, characterized in that: The steps of the preparation method are as follows: S1: mixing fluorescent quantum dots and n-hexane to obtain a fluorescent quantum dot solution, wherein the concentration of the fluorescent quantum dots is 1 to 3 mg / mL; the fluorescent quantum dots are ZnCdSe / ZnS QDs; S2: mixing the fluorescent quantum dot solution of step S1 with a PBS solution, and stirring for 2 to 4 hours, wherein the volume of the PBS and n-hexane is 4 to 12:1, the PBS solution contains BSA and antibodies to nafil-like substances, and the concentration of the BSA is 0.25 to 2 mg / mL; the antibodies to nafil-like substances are sildenafil and / or tadalafil, and the concentration of the sildenafil antibodies is 0.15 to 0.45 mg / mL, and the concentration of the tadalafil antibodies is 0.2 to 0.4 mg / mL; S3: Centrifuge, remove the supernatant, and obtain the fluorescent immunoprobe.

2. The method for preparing the fluorescent immunoprobe according to claim 1, characterized in that: In step S1, the concentration of the fluorescent quantum dots is 1.5-2.5 mg / mL.

3. The method for preparing the fluorescent immunoprobe according to claim 1, characterized in that: In step S2, the stirring time is 2.5 to 3.5 h, the volume of the PBS and n-hexane is 6 to 10:1, and the BSA concentration is 0.5 to 1.5 mg / mL.

4. The method for preparing the fluorescent immunoprobe according to claim 1, characterized in that: The antibody to sildenafil is Ab Sildenafil , the antibody to tadalafil is Ab Tadalafil .

5. A fluorescent immunoprobe, characterized in that: The probe is prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the fluorescent immunoprobe according to claim 5 in the preparation of products for detecting narcolepsy substances and / or narcolepsy substance derivatives.

7. A kit for detecting phenotype substances and / or phenotype substance derivatives, characterized in that: The kit contains the fluorescent immunoprobe described in claim 5 and a lateral flow immunochromatography test strip, wherein the lateral flow immunochromatography test strip consists of a sample pad, a nitrocellulose membrane, absorbent paper and a PVC bottom plate, and the detection line of the lateral flow immunochromatography test strip is coated with nafil antigens, and the quality control line is coated with sheep anti-rabbit IgG.

8. The kit for detecting phenotype substances and / or phenotype substance derivatives according to claim 7, characterized in that: The processing solution of the sample pad contains 4.5-5.5 mg / mL PVP, 4.5-5.5 mg / mL sucrose, 4.5-5.5 mg / mL BSA, 0.5%-1.5% Tween-20 (v / v) and 0.05 mol / L PB solution.

9. The kit for detecting phenotype substances and / or phenotype substance derivatives according to claim 7, characterized in that: The nafil-like substance antigen is an antigen of sildenafil and / or tadalafil, the concentration of the sildenafil antigen is 0.095-1.5 mg / mL, and the concentration of the tadalafil antigen is 0.10-0.75 mg / mL.

10. A method for using the kit for detecting phenotype substances and / or phenotype substance derivatives according to any one of claims 7 to 8, characterized in that: The method of use is a non-disease diagnosis and treatment method, and the steps are as follows: S21: loading a sample extract containing narcolepsy substances and / or narcolepsy substance derivatives using a solid phase extraction microfluidic chip; S22: using a methanol solution (v / v) containing 0.05% to 0.15% acetic acid as an eluent to elute the sample extract containing the phenazone-like substance in step S21; S23: using 0.024-0.026 mol / L PB solution as a chromatography solution, collecting the nalidixic substances and / or nalidixic substance derivatives in step S22, and obtaining a PB solution containing nalidixic substances and / or nalidixic substance derivatives; S24: Dispense the PB solution containing pheniramine-like substances and / or pheniramine-like substance derivatives described in step S23 into a reaction microtiter plate, 99-101 μL per well, mix with the fluorescent immunoprobe described in claim 5, 0.48-0.72 μg of each probe per well, and then react with the lateral flow immunochromatographic test strip described in claims 8-9 for 4-8 min, take out the lateral flow immunochromatographic test strip after the reaction, and collect the fluorescent signal.

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