A fluorescent immunochromatography reagent card based on carbon quantum dot microspheres and its preparation method and application

Abstract

The present invention provides a fluorescent immunochromatography reagent card based on carbon quantum dot microspheres and its preparation method and application. The fluorescent immunochromatography reagent card includes a test strip, the test strip includes a sample pad, a conjugation pad and a chromatographic membrane, and a detection line and a quality control line are provided on the chromatographic membrane. The detection line is coated with an anti-D-dimer first monoclonal antibody, and the quality control line is coated with a negative control antibody; the conjugation pad is coated with a carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody and a carbon quantum dot microsphere-negative control antigen. The fluorescent immunochromatography reagent card uses carbon quantum dot microspheres as fluorescent markers, has the advantages of high photoluminescence efficiency, stable luminescence, strong anti-bleaching, no flickering phenomenon, good biocompatibility, etc., can improve detection sensitivity and detection stability, and because the carbon quantum dot microspheres are an environmentally friendly material, do not contain heavy metals, after the reagent card detection is completed, the biological sample can be inactivated and directly treated as domestic garbage.

Description

Technical Field

[0001] The present invention relates to the field of medical devices, and in particular to a fluorescent immunochromatography reagent card based on carbon quantum dot microspheres, and a preparation method and application thereof. Background Art

[0002] Fluorescent immunochromatography reagent cards are the development and supplement of colloidal gold immunochromatography reagent cards, and their sensitivity is higher than that of colloidal gold immunochromatography reagent cards. The fluorescent substances used in current fluorescent immunochromatography reagent cards mainly include fluorescent dye microspheres, time-resolved fluorescent microspheres, quantum dot microspheres, etc. Fluorescent dye microspheres are mostly prepared by dyeing polystyrene microspheres with fluorescent dyes, but fluorescent dyes generally have high carcinogenicity, poor resistance to photobleaching, small Stoke's shift, resulting in large background signals, and fading may occur after dyeing the microspheres. It can be seen that there are many shortcomings in the fluorescent dye microspheres prepared using fluorescent dyes. Time-resolved fluorescent microspheres are mostly prepared by doping polystyrene microspheres with lanthanide metal chelates, which have the advantages of long fluorescence lifetime, high photoluminescence efficiency, large Stoke's shift, and stable luminescence. However, due to the use of lanthanide metal elements, their cost is high, the preparation process is complicated, and the damage to the natural environment after abandonment is large. Quantum dot microspheres are widely used in the field of point-of-care testing (POCT) due to their high photoluminescence efficiency and stable luminescence. Their detection performance is comparable to that of large-scale chemiluminescence methods. However, quantum dots are mostly prepared using heavy metals, which also have disadvantages such as high cost, complicated preparation process and great damage to the environment. Summary of the invention

[0003] The purpose of the present invention is to provide a fluorescent immunochromatography reagent card based on carbon quantum dot microspheres and a preparation method and application thereof. The reagent card uses carbon quantum dots as fluorescent substances. The carbon quantum dots do not contain heavy metal elements and will not pollute the environment. They have the characteristics of high photoluminescence efficiency, strong resistance to photobleaching, good biocompatibility, low toxicity, etc. They are applied to fluorescent immunochromatography reagent cards, so that the detection sensitivity of the reagent card is improved and the detection stability is good.

[0004] According to a first aspect of the present invention, a fluorescent immunochromatography reagent card based on carbon quantum dot microspheres is provided, the reagent card comprising a test strip, the test strip comprising a sample pad, a conjugation pad and a chromatographic membrane, a detection line and a quality control line are provided on the chromatographic membrane, the detection line is coated with an anti-D-dimer first monoclonal antibody, and the quality control line is coated with a negative control antibody; the conjugation pad is coated with a carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody and a carbon quantum dot microsphere-negative control antigen.

[0005] The fluorescent immunochromatography reagent card provided by the present invention uses carbon quantum dots as fluorescent substances, and the reagent card includes a test strip, the test strip includes a sample pad, a conjugate pad and a chromatographic membrane, and a detection line and a quality control line are respectively arranged in the chromatographic membrane. By detecting and calculating the fluorescence intensity of the detection line and the quality control line, the concentration of D-dimer can be accurately quantified. The first anti-D-dimer monoclonal antibody and the second anti-D-dimer monoclonal antibody involved in the present invention are both monoclonal antibodies against D-dimer, and both can bind to D-dimer. The sample to be tested is dripped onto the sample pad, and the sample to be tested can be chromatographed from the sample pad to the binding pad; the sample to be tested is chromatographed forward to the binding pad, and when the sample to be tested contains D-dimer (i.e., antigen), the D-dimer can combine with the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody on the binding pad to form carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody-D-dimer; the chromatography from the binding pad to the chromatography membrane is continued, and when the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody-D-dimer passes through the detection line, it can be fixed on the detection line coated with the anti-D-dimer first monoclonal antibody, forming a double sandwich antibody structure of carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody-D-dimer-anti-D-dimer first monoclonal antibody. The carbon quantum dot microsphere-negative control antigen coated on the binding pad moves forward during the chromatography process of the sample. When it reaches the quality control line, it can bind to the negative control antibody coated on the quality control line and be fixed. Its function is to monitor the correctness of the detection process and whether the fluorescent immunochromatography reagent card is invalid.

[0006] The fluorescent immunochromatographic reagent card provided by the present invention uses carbon quantum dot microspheres as fluorescent materials. Since the carbon quantum dot microspheres have the advantages of high photoluminescence efficiency, stable luminescence, strong bleaching resistance, no flickering phenomenon, good biocompatibility, etc., the signal-to-noise ratio can be improved in immunochromatographic detection, and has excellent signal amplification effect, thereby improving the sensitivity and stability of detection. In addition, the carbon quantum dot microspheres used in the fluorescent immunochromatographic reagent card do not contain heavy metals, are pollution-free, non-carcinogenic, non-toxic, and have low production costs. After the biological sample is inactivated after use, it can be directly treated as domestic waste and will not affect the environment.

[0007] Preferably, the negative control antibody is rabbit anti-chicken IgY, and the negative control antigen is chicken IgY.

[0008] Preferably, the particle size of the carbon quantum dot microspheres is 50 to 300 nm.

[0009] The particle size of the carbon quantum dot microspheres selected in this scheme is moderate, which can reduce steric hindrance, making it easier to bind to the corresponding specific antibodies, and at the same time can improve the fluorescence efficiency of the carbon quantum dot microspheres to a certain extent, thereby improving the detection sensitivity and stability. Specifically, the minimum detection limit of the fluorescent immunochromatography reagent card of the present invention is 0.050 mg / L, indicating that the detection sensitivity of the reagent card is high; and the intra-batch repeatability of the detection using the fluorescent immunochromatography reagent card is good, the coefficient of variation CV value is less than 15%, and the relative range between batches is also less than 15%, indicating that the detection stability of the reagent card is high.

[0010] Preferably, the carbon quantum microspheres include carbon quantum dots, the surface of which is sequentially coated with a first shell layer and a second shell layer, the carbon quantum dots are prepared from pyrene and toluene, the material of the first shell layer is bovine serum albumin, and the material of the second shell layer is silicon dioxide.

[0011] The carbon quantum dot microspheres used in this scheme have a core-shell structure, including a core and a first shell layer and a second shell layer coated on the core at one time, wherein the core is a carbon quantum dot prepared from pyrene and toluene, and the first shell layer coated on the carbon quantum dot is prepared using bovine serum albumin, and then the second shell layer is coated on the first shell layer. First, a layer of bovine serum albumin is coated on the surface of the carbon quantum dot to modify it into a water-soluble carbon quantum dot, and then a layer of silicon dioxide is further coated on the surface for curing. The carbon quantum dot microspheres obtained by this coating method are convenient for subsequent functionalization, and can avoid the phenomenon that the carbon quantum dot microspheres are quenched or partially quenched by fluorescence due to direct coating of silicon dioxide on the carbon quantum dots.

[0012] Preferably, the carbon quantum dot microspheres are prepared by the following method:

[0013] (1) Using pyrene as a carbon source and toluene as a solvent, a crude carbon quantum dot product is prepared by a solvothermal reaction;

[0014] (2) filtering the carbon quantum dot crude product using a 220 nm filter membrane and drying to obtain a carbon quantum dot powder;

[0015] (3) dissolving the carbon quantum dot powder in chloroform to obtain a carbon quantum dot chloroform solution;

[0016] (4) preparing a bovine serum albumin solution using deionized water and bovine serum albumin;

[0017] (5) adding the carbon quantum dot chloroform solution dropwise to the bovine serum albumin solution under ultrasonic conditions, centrifuging, discarding the supernatant, and washing to obtain the bovine serum albumin-coated carbon quantum dots;

[0018] (6) Add anhydrous ethanol to the bovine serum albumin-coated carbon quantum dots and mix well, then add concentrated ammonia water, slowly add tetraethyl orthosilicate dropwise while stirring, continue to stir evenly until the tetraethyl orthosilicate is completely hydrolyzed, centrifuge, discard the supernatant, wash, and obtain carbon quantum dot microspheres.

[0019] This scheme relates to a method for preparing carbon quantum dot microspheres, firstly, oil-soluble carbon quantum dots are prepared by a solvothermal method, then a layer of bovine serum albumin is coated on the surface of the oil-soluble carbon quantum dots to modify them into water-soluble carbon quantum dot microspheres, and finally a layer of silicon dioxide is coated on the surface. The coating of silicon dioxide can improve the stability of carbon quantum dot microspheres, but directly coating the surface of carbon quantum dots with silicon dioxide can easily lead to fluorescence quenching or partial quenching of carbon quantum dot microspheres. Before coating the surface of carbon quantum dots with silicon dioxide, the carbon quantum dots are first modified with bovine serum albumin to obtain water-soluble carbon quantum dots, and then coated with silicon dioxide, which can effectively improve the quantum efficiency of carbon quantum dot microspheres and make the carbon quantum dot microspheres have very good light stability and chemical stability.

[0020] Preferably, in step (1) of the above-mentioned method for preparing carbon quantum dot microspheres, the temperature of the solvothermal reaction is 160 to 200° C. and the time is 10 to 14 hours.

[0021] Preferably, in step (1) of the above-mentioned method for preparing carbon quantum dot microspheres, the temperature of the solvothermal reaction is 180° C. and the time is 12 h.

[0022] Preferably, in step (5) and step (6) of the above-mentioned method for preparing carbon quantum dot microspheres, the centrifugal conditions are: 8000 r / min, 15 min.

[0023] Preferably, the carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody is prepared by the following steps: dissolving the carbon quantum dot microspheres in 2-morpholineethanesulfonic acid (MES), adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and mixing, then adding N-hydroxysuccinimide (NHS), shaking and activating for 20 to 40 minutes, centrifuging, discarding the supernatant, adding the anti-D-dimer second monoclonal antibody thereto, and obtaining the carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody.

[0024] The present scheme relates to a method for preparing carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody, including two steps of activation and coupling. The carbon quantum dot microspheres are first activated using MES, EDC, and NHS, so that the carbon quantum dot microspheres are more easily combined with the anti-D-dimer second monoclonal antibody, thereby improving the coupling efficiency between the two.

[0025] Preferably, the carbon quantum dot microsphere-negative control antigen is prepared by the following steps: dissolving the carbon quantum dot microspheres in 2-morpholineethanesulfonic acid (MES), adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and mixing, then adding N-hydroxysuccinimide (NHS), shaking and activating for 20 to 40 minutes, centrifuging, discarding the supernatant, adding the negative control antibody thereto, and obtaining the carbon quantum dot microsphere-negative control antigen.

[0026] Preferably, the sample pad is prepared by the following steps: soaking a glass cellulose membrane in a PBS buffer solution containing 2.5 wt % sucrose, 1 wt % bovine serum albumin, and 1 wt % surfactant S9, and drying to obtain a sample pad.

[0027] Preferably, in the step of preparing the sample pad, the pH of the PBS buffer is 7.4 and the concentration is 10 mM.

[0028] Preferably, the conjugate pad is prepared by the following steps: soaking the glass cellulose membrane in a BB buffer containing 1wt% trehalose, 1vol% bovine serum albumin, and 0.5wt% polyethylene glycol 4000, drying, and evenly mixing carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody and carbon quantum dot microspheres-negative control antigen and spraying them on the glass cellulose membrane to obtain a conjugate pad.

[0029] Preferably, in the step of preparing the conjugate pad, the pH of the BB buffer is 8.0 and the concentration is 2 mM.

[0030] Preferably, in the fluorescent immunochromatographic reagent card based on carbon quantum dot microspheres, the test strip further comprises a water-absorbing pad and a bottom plate, and the sample pad, the binding pad, the chromatography membrane, and the water-absorbing pad are partially overlapped in sequence and then pasted on the surface of the bottom plate.

[0031] According to the second aspect of the present invention, a method for quantitatively detecting D-dimer concentration is provided, comprising the following steps: dropping a sample to be tested onto the above-mentioned fluorescent immunochromatography reagent card based on carbon quantum dot microspheres, irradiating the reagent card with a laser diode under the action of a hemispherical lens, and collecting the signal with a signal collector.

[0032] The beneficial effects of the present invention are:

[0033] 1. The present invention provides a fluorescent immunochromatographic reagent card based on carbon quantum dot microspheres, which uses carbon quantum dot microspheres prepared by a specific method as fluorescent markers, and has the advantages of high photoluminescence efficiency, stable luminescence, strong anti-bleaching, no flickering, good biocompatibility, etc., can improve the signal-to-noise ratio in immunochromatographic detection, and has excellent signal amplification effect, thereby improving the sensitivity and stability of detection

[0034] 2. The synthesis process of the carbon quantum dot microspheres used in the fluorescent immunochromatography reagent card provided by the present invention is simple, and they can be obtained in one step by a hydrothermal method, with low production cost. After the synthesis of the carbon quantum dot microspheres, the photoluminescence performance is enhanced, and the detection performance is comparable to that of the large-scale chemiluminescence method. At the same time, the carbon quantum dot microspheres are an environmentally friendly material, do not contain heavy metal elements, are pollution-free, non-carcinogenic, and non-toxic, and can be discarded after inactivating the biological sample after use, without affecting the environment.

[0035] 3. When the fluorescent immunochromatography reagent card provided by the present invention is used to detect the D-dimer concentration, the minimum detection limit is 0.050 mg / L, and the detection sensitivity is high; and the intra-batch repeatability is good, the CV value is less than 15%, and the relative range between batches is also less than 15%, and the detection stability is high.

[0036] 4. The fluorescent immunochromatography reagent card based on carbon quantum dot microspheres provided by the present invention is simple to use and operate, has a short detection time, and greatly improves the detection speed.

[0037] 5. D-dimer mainly reflects the fibrinolytic function. When there is activated thrombosis and fibrinolytic activity in the body's blood vessels, the concentration of D-dimer will increase. As a marker for judging fibrinolysis, the accurate quantification of D-dimer concentration is extremely important in clinically judging and evaluating fibrinolysis and thrombus formation. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The present invention provides a fluorescent immunochromatography reagent card based on carbon quantum dot microspheres.

[0039] The reference numerals in the accompanying drawings are: 1 sample pad, 2 binding pad, 3 detection line, 4 quality control line, 5 chromatography membrane, 6 water absorbent pad, 7 bottom plate.

[0040] Figure 2 The present invention is a linear curve graph drawn with the concentration as the abscissa and the T / C value as the ordinate after detecting D-dimer antigen dilutions of different concentrations using the fluorescent immunochromatography reagent card based on carbon quantum dot microspheres provided by the present invention. DETAILED DESCRIPTION

[0041] The following is a further clear and complete description of the technical features in the technical solution provided by the present invention in conjunction with the specific implementation methods. 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 those skilled in the art without creative work are within the scope of protection of the present invention.

[0042] Example 1

[0043] A fluorescent immunochromatography reagent card based on carbon quantum dot microspheres is prepared by the following steps:

[0044] (1) Preparation of carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody

[0045] 25 μL of carbon quantum dot microspheres were dissolved in 200 μL of 2-morpholineethanesulfonic acid (MES) solution, 20 μL of 1 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was added and mixed, and then 10 μL of 1 mg / mL N-hydroxysuccinimide (NHS) was added, and the mixture was activated by shaking for 30 min, centrifuged at 8000 r / min for 15 min, and the supernatant was discarded. The anti-D-dimer second monoclonal antibody was added thereto for coupling for 2 h to obtain carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody;

[0046] Among them, the particle size of carbon quantum dot microspheres is 150nm;

[0047] Carbon quantum dot microspheres were prepared by the following method:

[0048] ① Dissolve 1 g of pyrene in toluene, quickly transfer into a 100 mL polytetrafluoroethylene hydrothermal reactor, and react at 180 ° C for 12 h to obtain a crude carbon quantum dot product;

[0049] ② The crude carbon quantum dot product was filtered through an oil-soluble 220 nm filter membrane and then dried on a rotary evaporator to obtain a black carbon quantum dot powder;

[0050] ③ Take 0.5g of carbon quantum dot powder and dissolve it in 5mL of chloroform to obtain a carbon quantum dot chloroform solution;

[0051] ④ Weigh 1g of bovine serum albumin (BSA) into a 50mL beaker, add 20mL of secondary deionized water, and sonicate until BSA is completely dissolved to obtain a BSA solution;

[0052] ⑤ Under ultrasonic conditions, the carbon quantum dot chloroform solution was added dropwise to the BSA solution. After dispersion, it was in an emulsified state. Centrifuged at 8000r / min for 15min, the supernatant was discarded, and the precipitate was washed twice with secondary deionized water to obtain BSA-coated carbon quantum dots.

[0053] ⑥ Take 2 mL of 200 mg / mL BSA-coated carbon quantum dots in a 100 mL beaker, add 30 mL of anhydrous ethanol to disperse evenly, then add 1 mL of concentrated ammonia, slowly add 0.5 mL of tetraethyl orthosilicate under stirring, continue stirring for about 4 h until the tetraethyl orthosilicate is completely hydrolyzed, centrifuge at 8000 r / min for 15 min, discard the supernatant, wash the precipitate twice with secondary deionized water, and make up to 5 mL with anhydrous ethanol to obtain carbon quantum dot microspheres;

[0054] According to the above preparation method, carbon quantum dot microspheres-chicken IgY were prepared;

[0055] (2) Preparation of sample pad

[0056] The glass cellulose membrane was immersed in a PBS buffer containing 2.5 wt % sucrose, 1 wt % bovine serum albumin, and 1 wt % surfactant S9, and then dried at 37° C. for 24 h to obtain a sample pad 1;

[0057] Among them, the pH value of PBS buffer was 7.4 and the concentration was 10 mM;

[0058] (3) Preparation of conjugate pad

[0059] The glass cellulose membrane is immersed in a BB buffer solution containing 1 wt% trehalose, 1 wt% bovine serum albumin, and 0.5 wt% polyethylene glycol 4000 for a period of time, and then dried at 37° C. for 18 h. The carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody and carbon quantum dot microspheres-chicken IgY prepared in step (1) are evenly mixed and then sprayed on the glass cellulose membrane to obtain a conjugate pad 2;

[0060] Among them, the pH value of BB buffer was 8.0 and the concentration was 2 mM;

[0061] (4) Preparation of chromatographic membrane

[0062] Cut the nitrocellulose membrane (NC membrane) into strips of 2.5 cm*30 cm, and paste the obtained strips in the middle of the bottom plate. Use a membrane marker to draw anti-D-dimer first monoclonal antibody and rabbit anti-chicken IgY as detection line 3 and quality control line 4 on different positions of the NC membrane, respectively, and dry at 37°C to obtain a chromatographic membrane 5;

[0063] (5) Assembling immunochromatographic test strips

[0064] The sample pad 1, the conjugate pad 2 and the absorbent pad 7 are respectively pasted on both sides of the chromatographic membrane 5, ensuring that the pads are stacked alternately with a spacing of 1.5 mm, and then pasted on the PVC bottom plate and cut into 3.5 mm wide test strips to obtain the following: Figure 1 The fluorescent immunochromatography reagent card based on carbon quantum dot microspheres is shown.

[0065] Example 2

[0066] A fluorescent immunochromatography reagent card based on carbon quantum dot microspheres is prepared by the following steps:

[0067] (1) Preparation of carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody

[0068] 25 μL of carbon quantum dot microspheres were dissolved in 200 μL of 2-morpholineethanesulfonic acid (MES) solution, 20 μL of 1 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was added and mixed, and then 10 μL of 1 mg / mL N-hydroxysuccinimide (NHS) was added, and the mixture was activated by shaking for 20 min, centrifuged at 8000 r / min for 15 min, and the supernatant was discarded. The anti-D-dimer second monoclonal antibody was added thereto for coupling for 2 h to obtain carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody;

[0069] Among them, the particle size of carbon quantum dot microspheres is 50nm;

[0070] Carbon quantum dot microspheres were prepared by the following method:

[0071] ① Dissolve 1 g of pyrene in toluene, quickly transfer into a 100 mL polytetrafluoroethylene hydrothermal reactor, and react at 200 ° C for 10 h to obtain a crude carbon quantum dot product;

[0072] ② The crude carbon quantum dot product was filtered through an oil-soluble 220 nm filter membrane and then dried on a rotary evaporator to obtain a black carbon quantum dot powder;

[0073] ③ Take 0.5g of carbon quantum dot powder and dissolve it in 5mL of chloroform to obtain a carbon quantum dot chloroform solution;

[0074] ④ Weigh 1g of bovine serum albumin (BSA) into a 50mL beaker, add 20mL of secondary deionized water, and sonicate until BSA is completely dissolved to obtain a BSA solution;

[0075] ⑤ Under ultrasonic conditions, the carbon quantum dot chloroform solution was added dropwise to the BSA solution. After dispersion, it was in an emulsified state. Centrifuged at 8000r / min for 15min, the supernatant was discarded, and the precipitate was washed twice with secondary deionized water to obtain BSA-coated carbon quantum dots.

[0076] ⑥ Take 2 mL of 200 mg / mL BSA-coated carbon quantum dots in a 100 mL beaker, add 30 mL of anhydrous ethanol to disperse evenly, then add 1 mL of concentrated ammonia, slowly add 0.5 mL of tetraethyl orthosilicate under stirring, continue stirring for about 4 h until the tetraethyl orthosilicate is completely hydrolyzed, centrifuge at 8000 r / min for 15 min, discard the supernatant, wash the precipitate twice with secondary deionized water, and make up to 5 mL with anhydrous ethanol to obtain carbon quantum dot microspheres;

[0077] According to the above preparation method, carbon quantum dot microspheres-chicken IgY were prepared;

[0078] (2) Preparation of sample pad

[0079] The glass cellulose membrane was immersed in a PBS buffer containing 2.5 wt % sucrose, 1 wt % bovine serum albumin, and 1 wt % surfactant S9, and then dried at 37° C. for 24 h to obtain a sample pad 1;

[0080] Among them, the pH value of PBS buffer was 7.4 and the concentration was 10 mM;

[0081] (3) Preparation of conjugate pad

[0082] The glass cellulose membrane is immersed in a BB buffer solution containing 1 wt% trehalose, 1 wt% bovine serum albumin, and 0.5 wt% polyethylene glycol 4000 for a period of time, and then dried at 37° C. for 18 h. The carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody and carbon quantum dot microspheres-chicken IgY prepared in step (1) are evenly mixed and then sprayed on the glass cellulose membrane to obtain a conjugate pad 2;

[0083] Among them, the pH value of BB buffer was 8.0 and the concentration was 2 mM;

[0084] (4) Preparation of chromatographic membrane

[0085] Cut the nitrocellulose membrane (NC membrane) into strips of 2.5 cm*30 cm, and paste the obtained strips in the middle of the bottom plate. Use a membrane marker to draw anti-D-dimer first monoclonal antibody and rabbit anti-chicken IgY as detection line 3 and quality control line 4 on different positions of the NC membrane, respectively, and dry at 37°C to obtain a chromatographic membrane 5;

[0086] (5) Assembling immunochromatographic test strips

[0087] The sample pad 1, the conjugate pad 2 and the absorbent pad 7 are respectively pasted on both sides of the chromatographic membrane 5, ensuring that the pads are stacked alternately with a spacing of 1.5 mm, and then pasted on the PVC bottom plate and cut into 3.5 mm wide test strips to obtain the following: Figure 1 The fluorescent immunochromatography reagent card based on carbon quantum dot microspheres is shown.

[0088] Example 3

[0089] A fluorescent immunochromatography reagent card based on carbon quantum dot microspheres is prepared by the following steps:

[0090] (1) Preparation of carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody

[0091] 25 μL of carbon quantum dot microspheres were dissolved in 200 μL of 2-morpholineethanesulfonic acid (MES) solution, 20 μL of 1 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) was added and mixed, and then 10 μL of 1 mg / mL N-hydroxysuccinimide (NHS) was added, and the mixture was activated by shaking for 40 min, centrifuged at 8000 r / min for 15 min, and the supernatant was discarded. The anti-D-dimer second monoclonal antibody was added thereto for coupling for 2 h to obtain carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody;

[0092] Among them, the particle size of carbon quantum dot microspheres is 300nm;

[0093] Carbon quantum dot microspheres were prepared by the following method:

[0094] ① Dissolve 1 g of pyrene in toluene, quickly transfer into a 100 mL polytetrafluoroethylene hydrothermal reactor, and react at 160 ° C for 14 h to obtain a crude carbon quantum dot product;

[0095] ② The crude carbon quantum dot product was filtered through an oil-soluble 220 nm filter membrane and then dried on a rotary evaporator to obtain a black carbon quantum dot powder;

[0096] ③ Take 0.5g of carbon quantum dot powder and dissolve it in 5mL of chloroform to obtain a carbon quantum dot chloroform solution;

[0097] ④ Weigh 1g of bovine serum albumin (BSA) into a 50mL beaker, add 20mL of secondary deionized water, and sonicate until BSA is completely dissolved to obtain a BSA solution;

[0098] ⑤ Under ultrasonic conditions, the carbon quantum dot chloroform solution was added dropwise to the BSA solution. After dispersion, it was in an emulsified state. Centrifuged at 8000r / min for 15min, the supernatant was discarded, and the precipitate was washed twice with secondary deionized water to obtain BSA-coated carbon quantum dots.

[0099] ⑥ Take 2 mL of 200 mg / mL BSA-coated carbon quantum dots in a 100 mL beaker, add 30 mL of anhydrous ethanol to disperse evenly, then add 1 mL of concentrated ammonia, slowly add 0.5 mL of tetraethyl orthosilicate under stirring, continue stirring for about 4 h until the tetraethyl orthosilicate is completely hydrolyzed, centrifuge at 8000 r / min for 15 min, discard the supernatant, wash the precipitate twice with secondary deionized water, and make up to 5 mL with anhydrous ethanol to obtain carbon quantum dot microspheres;

[0100] According to the above preparation method, carbon quantum dot microspheres-chicken IgY were prepared;

[0101] (2) Preparation of sample pad

[0102] The glass cellulose membrane was immersed in a PBS buffer containing 2.5 wt % sucrose, 1 wt % bovine serum albumin, and 1 wt % surfactant S9, and then dried at 37° C. for 24 h to obtain a sample pad 1;

[0103] Among them, the pH value of PBS buffer was 7.4 and the concentration was 10 mM;

[0104] (3) Preparation of conjugate pad

[0105] The glass cellulose membrane is immersed in a BB buffer solution containing 1 wt% trehalose, 1 wt% bovine serum albumin, and 0.5 wt% polyethylene glycol 4000 for a period of time, and then dried at 37° C. for 18 h. The carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody and carbon quantum dot microspheres-chicken IgY prepared in step (1) are evenly mixed and then sprayed on the glass cellulose membrane to obtain a conjugate pad 2;

[0106] Among them, the pH value of BB buffer was 8.0 and the concentration was 2 mM;

[0107] (4) Preparation of chromatographic membrane

[0108] Cut the nitrocellulose membrane (NC membrane) into strips of 2.5 cm*30 cm, and paste the obtained strips in the middle of the bottom plate. Use a membrane marker to draw anti-D-dimer first monoclonal antibody and rabbit anti-chicken IgY as detection line 3 and quality control line 4 on different positions of the NC membrane, respectively, and dry at 37°C to obtain a chromatographic membrane 5;

[0109] (5) Assembling immunochromatographic test strips

[0110] The sample pad 1, the conjugate pad 2 and the absorbent pad 7 are respectively pasted on both sides of the chromatographic membrane 5, ensuring that the pads are stacked alternately with a spacing of 1.5 mm, and then pasted on the PVC bottom plate and cut into 3.5 mm wide test strips to obtain the following: Figure 1 The fluorescent immunochromatography reagent card based on carbon quantum dot microspheres is shown.

[0111] Test Case

[0112] 1. Experimental Construction Method

[0113] The test object of this test example is the fluorescent immunochromatography reagent card based on carbon quantum dot microspheres prepared in Examples 1 to 3, which is used to detect D-dimer antigen (purchased from Guangdong Feipeng Biotechnology Co., Ltd.), wherein the antigen diluent is a PBS buffer (pH 7.4) containing 1wt% bovine serum albumin. After the D-dimer antigen is diluted with the antigen diluent, the chemiluminescence analyzer of Guangzhou Wondfo Biotechnology and its supporting D-dimer detection kit (chemiluminescence method) are used to assign values.

[0114] (1) Linear analysis

[0115] The D-dimer antigen was diluted to the following concentrations: 0.2, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0 mg / L, and the D-dimer antigens of different concentrations were dripped onto the sample pads of the fluorescent immunochromatographic reagent cards prepared in Examples 1 to 3, respectively. The reagent cards were irradiated with a laser diode under the action of a hemispherical lens, and the signals were collected by a signal collector to detect the D-dimer antigen of each concentration, and the fluorescence intensity of the test line (T) and the quality control line (C) of the test reagent card was recorded each time, and the T / C ratio and the coefficient of variation CV value were calculated. At the same time, the concentration of the D-dimer antigen was used as the horizontal coordinate and the T / C value was used as the vertical coordinate. A linear curve was drawn using data analysis software and the correlation coefficient R was calculated. 2 , where the calculation formula for the coefficient of variation CV value is as follows: SD stands for standard deviation, Indicates the average value.

[0116] (2) Minimum detection limit detection

[0117] The fluorescent immunochromatographic reagent cards prepared in Examples 1 to 3 were used to detect the antigen dilutions 20 times respectively. The minimum detection limit is calculated by the formula: represents mean, and SD represents standard deviation.

[0118] (3) Repeatability test

[0119] The fluorescent immunochromatographic reagent cards prepared in Examples 1 to 3 were used to detect low-concentration and high-concentration antigen dilutions 10 times respectively, and the coefficient of variation CV value and the relative range between batches were calculated. The correlation coefficient R was calculated using data analysis software. 2 , where the calculation formula for the coefficient of variation CV value is as follows: SD stands for standard deviation, Represents the average value; the calculation formula for the relative range between batches is as follows: x max Indicates the maximum concentration detected within the batch, x min Indicates the minimum concentration detected within the batch.

[0120] 2. Experimental results

[0121] (1) Linear analysis

[0122] Table 1 Linear analysis results

[0123]

[0124] The results of the linear analysis experiment are shown in Table 1. The linear curve and correlation coefficient R2 The result of the value is Figure 2 As shown in Table 1, each concentration gradient was tested using the fluorescent immunochromatography reagent card of Examples 1 to 3, and the values ​​of the three groups of experimental results were very close and there was no significant difference, indicating that when the fluorescent immunochromatography reagent card based on carbon quantum dot microspheres provided by the present invention was used to detect the antigen to be tested, the test results had good reproducibility. Figure 2 It can be seen that when the concentration of the antigen to be tested is in the range of 0.2 to 12 mg / L, the correlation coefficient R 2 It reached 0.9999, indicating that the linear relationship between the antigen concentration and T / C value was good within this range.

[0125] (2) Minimum detection limit

[0126] Table 2 Minimum detection limit results

[0127]

[0128] The test results of the minimum detection limit are shown in Table 2. As can be seen from Table 2, the fluorescent immunochromatographic reagent card prepared in Examples 1 to 3 was used to detect the antigen dilution solution 20 times, and the calculated minimum detection limit was 0.050 mg / L, which is much lower than other detection reagents with the same methodology, indicating that the fluorescent immunochromatographic reagent card based on carbon quantum dot microspheres provided by the present invention has high detection sensitivity.

[0129] (3) Repeatability

[0130] Table 3 Repeatability test results

[0131]

[0132] The repeatability test results are shown in Table 3. As can be seen from Table 3, the fluorescent immunochromatographic reagent cards prepared in Examples 1 to 3 were used to detect low-concentration and high-concentration antigen dilutions 10 times, respectively. The calculation results show that the CV values ​​are all less than 15%, and the relative range between batches is also less than 15%, indicating that the fluorescent immunochromatographic reagent card based on carbon quantum dot microspheres provided by the present invention has high detection stability.

[0133] 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. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the technical solutions of the present invention can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present invention.

Claims

1. A fluorescent immunochromatography reagent card based on carbon quantum dot microspheres, characterized in that: The reagent card comprises a test strip, the test strip comprises a sample pad, a conjugate pad and a chromatographic membrane, the chromatographic membrane is provided with a detection line and a quality control line, the detection line is coated with an anti-D-dimer first monoclonal antibody, and the quality control line is coated with rabbit anti-chicken IgY; The conjugate pad is coated with carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody and carbon quantum dot microspheres-chicken IgY; The carbon quantum dot microspheres include carbon quantum dots, the surface of which is sequentially coated with a first shell layer and a second shell layer, the carbon quantum dots are prepared from pyrene and toluene, the material of the first shell layer is bovine serum albumin, and the material of the second shell layer is silicon dioxide; The carbon quantum dot microspheres are prepared by the following method: (1) using the pyrene as a carbon source and the toluene as a solvent, preparing a crude carbon quantum dot product through a solvothermal reaction; (2) filtering the carbon quantum dot crude product using a 220 nm filter membrane and drying to obtain a carbon quantum dot powder; (3) dissolving the carbon quantum dot powder in chloroform to obtain a carbon quantum dot chloroform solution; (4) preparing a bovine serum albumin solution using deionized water and bovine serum albumin; (5) adding the carbon quantum dot chloroform solution dropwise to the bovine serum albumin solution under ultrasonic conditions, centrifuging, discarding the supernatant, and washing to obtain bovine serum albumin-coated carbon quantum dots; (6) Add anhydrous ethanol to the bovine serum albumin-coated carbon quantum dots and mix well, then add concentrated ammonia water, slowly drop ethyl orthosilicate under stirring, continue to stir evenly until ethyl orthosilicate is completely hydrolyzed, centrifuge, discard the supernatant, wash, and obtain the carbon quantum dot microspheres.

2. The fluorescent immunochromatography reagent card based on carbon quantum dot microspheres as claimed in claim 1, characterized in that: The particle size of the carbon quantum dot microspheres is 50-300 nm.

3. The fluorescent immunochromatography reagent card based on carbon quantum dot microspheres as claimed in claim 1, characterized in that: In the step (1), the temperature of the solvent thermal reaction is 160-200° C. and the time is 10-14 hours.

4. The fluorescent immunochromatography reagent card based on carbon quantum dot microspheres according to claim 1, characterized in that: In step (5) and step (6), the centrifugal conditions are: 8000 r / min, 15 min.

5. The fluorescent immunochromatography reagent card based on carbon quantum dot microspheres according to claim 1, characterized in that: The carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody is prepared by the following steps: dissolving the carbon quantum dot microsphere in 2-morpholineethanesulfonic acid, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and mixing, then adding N-hydroxysuccinimide, shaking and activating for 20 to 40 minutes, centrifuging, discarding the supernatant, adding the anti-D-dimer second monoclonal antibody thereto, and obtaining the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody.

6. The fluorescent immunochromatography reagent card based on carbon quantum dot microspheres according to claim 1, characterized in that: The sample pad is prepared by the following steps: soaking a glass cellulose membrane in a PBS buffer containing 2.5 wt % sucrose, 1 wt % bovine serum albumin, and 1 wt % surfactant S9, and drying to obtain the sample pad; The conjugate pad is prepared by the following steps: soaking a glass cellulose membrane in a BB buffer containing 1wt% trehalose, 1wt% bovine serum albumin, and 0.5wt% polyethylene glycol 4000, drying, and uniformly mixing the carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody and the carbon quantum dot microspheres-chicken IgY and spraying them on the glass cellulose membrane to obtain the conjugate pad; In the sample pad preparation step, the pH of the PBS buffer is 7.4 and the concentration is 10 mM; In the step of preparing the conjugate pad, the pH of the BB buffer is 8.0 and the concentration is 2 mM.

7. The fluorescent immunochromatography reagent card based on carbon quantum dot microspheres according to claim 1, characterized in that: The test strip also includes a water-absorbing pad and a bottom plate. The sample pad, the binding pad, the chromatography membrane, and the water-absorbing pad are partially overlapped in sequence and then pasted on the surface of the bottom plate.

8. A method for quantitatively detecting D-dimer concentration, characterized in that: The following steps are involved: The sample to be tested is dropped onto the fluorescent immunochromatography reagent card based on carbon quantum dot microspheres as described in any one of claims 1 to 7, the reagent card is irradiated by a laser diode under the action of a hemispherical lens, and the signal is collected by a signal collector.

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