CN114544974A - Carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card and preparation method and application thereof - Google Patents

Carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card and preparation method and application thereof Download PDF

Info

Publication number
CN114544974A
CN114544974A CN202210181199.7A CN202210181199A CN114544974A CN 114544974 A CN114544974 A CN 114544974A CN 202210181199 A CN202210181199 A CN 202210181199A CN 114544974 A CN114544974 A CN 114544974A
Authority
CN
China
Prior art keywords
carbon quantum
quantum dot
reagent card
microsphere
dimer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210181199.7A
Other languages
Chinese (zh)
Inventor
刘召应
巫永胜
佘利民
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangdong Haolang Medical Technology Co ltd
Original Assignee
Guangdong Haolang Medical Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangdong Haolang Medical Technology Co ltd filed Critical Guangdong Haolang Medical Technology Co ltd
Priority to CN202210181199.7A priority Critical patent/CN114544974A/en
Publication of CN114544974A publication Critical patent/CN114544974A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/65—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/548—Carbohydrates, e.g. dextran
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/75—Fibrin; Fibrinogen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G01N2800/226—Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Pathology (AREA)
  • Materials Engineering (AREA)
  • Organic Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Nanotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

The invention provides a fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres and a preparation method and application thereof. The fluorescence immunochromatographic reagent card comprises a test strip, wherein the test strip comprises a sample pad, a combination 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 a first monoclonal antibody of anti-D-dimer, and the quality control line is coated with a negative control antibody; the bonding 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 fluorescence immunochromatography reagent card takes the carbon quantum dot microspheres as a fluorescence marker, has the advantages of high photoluminescence efficiency, stable luminescence, strong bleaching resistance, no flicker phenomenon, good biocompatibility and the like, can improve the detection sensitivity and the detection stability, and can be directly used as domestic garbage for treatment after the inactivation of biological samples after the detection of the reagent card because the carbon quantum dot microspheres are environment-friendly materials and do not contain heavy metals.

Description

Carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card and preparation method and application thereof
Technical Field
The invention relates to the field of medical instruments, in particular to a fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres and a preparation method and application thereof.
Background
The fluorescence immunochromatography reagent card is the development and supplement of the colloidal gold immunochromatography reagent card, and the sensitivity of the fluorescence immunochromatography reagent card is higher than that of the colloidal gold immunochromatography reagent card. The fluorescent materials adopted by the current fluorescence immunochromatography reagent card mainly comprise fluorescent dye microspheres, time-resolved fluorescent microspheres, quantum dot microspheres and the like. The fluorescent dye microspheres are mostly prepared by dyeing polystyrene microspheres with fluorescent dye, but the fluorescent dye generally has high carcinogenic performance, poor photobleaching resistance and large background signal caused by small Stokes shift, and can also fade after the microspheres are dyed, so that the fluorescent dye microspheres prepared by the fluorescent dye have many defects. The time-resolved fluorescent microspheres are mostly prepared by doping lanthanide series metal element chelate with polystyrene microspheres, and have the advantages of long fluorescence service life, high photoluminescence efficiency, large Stokes shift, stable luminescence and the like. The quantum dot microsphere has the characteristics of high photoluminescence efficiency and stable luminescence, so that the quantum dot microsphere has wide application in the field of point-of-care testing (POCT), the detection performance of the quantum dot microsphere can be higher than that of a large-scale chemiluminescence method, but the quantum dot is prepared by adopting heavy metals, and the quantum dot microsphere also has the defects of high cost, complex preparation process, great damage to the environment and the like.
Disclosure of Invention
The invention aims to provide a fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres and a preparation method and application thereof.
According to a first aspect of the invention, a fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres is provided, the reagent card comprises a test strip, the test strip comprises a sample pad, a combination 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 a first anti-D-dimer monoclonal antibody, and the quality control line is coated with a negative control antibody; the bonding 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 fluorescence immunochromatography reagent card provided by the invention takes carbon quantum dots as fluorescent substances, the reagent card comprises a test strip, the test strip comprises a sample pad, a combination pad and a chromatographic membrane, a detection line and a quality control line are respectively arranged in the chromatographic membrane, and the concentration of D-dimer can be accurately quantified by detecting and calculating the fluorescence intensity of the detection line and the quality control line. The anti-D-dimer first monoclonal antibody and the anti-D-dimer second monoclonal antibody are anti-D-dimer monoclonal antibodies, and both can be combined with the D-dimer. Dropwise adding a sample to be detected on the sample pad, wherein the sample to be detected can be chromatographed from the sample pad to the combination pad; the sample to be detected is chromatographed forwards to the combination pad, and when the sample to be detected contains D-dimer (namely antigen), the D-dimer can be combined with the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody on the combination pad to form the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody-D-dimer; and (4) continuing chromatography from the binding pad to the chromatographic membrane, and when the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody-D-dimer passes through the detection line, the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody-D-dimer can be immobilized on the detection line coated with the anti-D-dimer first monoclonal antibody to form a double sandwich antibody structure of the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody-D-dimer-anti-D-dimer first monoclonal antibody. And the carbon quantum dot microsphere-negative control antigen coated on the combination pad moves forwards in the process of carrying out chromatography on a sample, and can be combined with the negative control antibody coated on the quality control line to be fixed when reaching the quality control line, and the function of the combination pad is to monitor the correctness of the detection process and whether the fluorescence immunochromatographic reagent card fails.
The fluorescence immunochromatographic reagent card provided by the invention adopts the carbon quantum dot microspheres as the fluorescent material, and the carbon quantum dot microspheres have the advantages of high photoluminescence efficiency, stable luminescence, strong bleaching resistance, no flicker phenomenon, good biocompatibility and the like, so that the signal-to-noise ratio can be improved in immunochromatographic detection, and the fluorescence immunochromatographic reagent card has an excellent signal amplification effect, thereby improving the sensitivity and stability of detection. In addition, the carbon quantum dot microspheres adopted in the fluorescence immunochromatography reagent card do not contain heavy metals, are pollution-free, carcinogenic and nontoxic, have low manufacturing cost, can be directly treated as household garbage after the inactivation of biological samples after use, and do not influence the environment.
Preferably, the negative control antibody is rabbit anti-chicken IgY and the negative control antigen is chicken IgY.
Preferably, the particle size of the carbon quantum dot microspheres is 50-300 nm.
The carbon quantum dot microspheres selected by the scheme have moderate particle size, can reduce steric hindrance, are easier to combine with corresponding specific antibodies, and can improve the fluorescence efficiency of the carbon quantum dot microspheres to a certain extent, so that the detection sensitivity and stability are improved. The concrete embodiment is as follows: the lowest detection limit of the fluorescence immunochromatographic reagent card is 0.050mg/L, which shows that the detection sensitivity of the reagent card is high; the repeatability in batches of detection by using the fluorescence immunochromatography reagent card is good, the coefficient of variation CV values are less than 15%, the relative range between batches is also less than 15%, and the reagent card is proved to have high detection stability.
Preferably, the carbon quantum microsphere comprises carbon quantum dots, the surface of the carbon quantum dots is sequentially coated with a first shell layer and a second shell layer, the carbon quantum dots are prepared from pyrene and toluene, the first shell layer is made of bovine serum albumin, and the second shell layer is made of silicon dioxide.
The carbon quantum dot microsphere adopted by the scheme has a core-shell structure and comprises a core, a first shell layer and a second shell layer, wherein the first shell layer and the second shell layer are coated on the core at one time, the core is the carbon quantum dot prepared from pyrene and methylbenzene, the first shell layer coated on the carbon quantum dot is prepared by utilizing bovine serum albumin, and then the second shell layer is coated on the first shell layer. The carbon quantum dot microspheres obtained by the coating method are convenient for subsequent functionalization, and the phenomenon that the carbon quantum dot microspheres are quenched by fluorescence or partially quenched due to the fact that the carbon quantum dots are directly coated with the silicon dioxide can be avoided.
Preferably, the carbon quantum dot microsphere is prepared by the following method:
(1) taking pyrene as a carbon source and toluene as a solvent, and preparing a carbon quantum dot crude product through solvothermal reaction;
(2) filtering the carbon quantum dot crude product by using a 220nm filter membrane, and drying to obtain carbon quantum dot powder;
(3) dissolving carbon quantum dot powder in chloroform to obtain a carbon quantum dot chloroform solution;
(4) preparing a bovine serum albumin solution by using deionized water and bovine serum albumin;
(5) dropwise adding a carbon quantum dot chloroform solution into a bovine serum albumin solution under an ultrasonic condition, centrifuging, discarding the supernatant, and washing to obtain a bovine serum albumin-coated carbon quantum dot;
(6) adding absolute ethyl alcohol into the carbon quantum dots coated with the bovine serum albumin, uniformly mixing, then adding concentrated ammonia water, slowly dropwise adding tetraethoxysilane under stirring, continuously uniformly stirring until the tetraethoxysilane is completely hydrolyzed, centrifuging, discarding supernatant, and washing to obtain the carbon quantum dot microspheres.
The scheme relates to a preparation method of carbon quantum dot microspheres, which comprises the steps of firstly preparing oil-soluble carbon quantum dots by a solvothermal method, then coating a layer of bovine serum albumin on the surfaces of the oil-soluble carbon quantum dots to modify the oil-soluble carbon quantum dots into water-soluble carbon quantum dot microspheres, and finally coating a layer of silicon dioxide on the surfaces of the water-soluble carbon quantum dot microspheres. The coating of the silicon dioxide can improve the stability of the carbon quantum dot microsphere, but the direct coating of the silicon dioxide on the surface of the carbon quantum dot can easily cause the carbon quantum dot microsphere to be quenched or partially quenched by fluorescence, before the coating of the silicon dioxide on the surface of the carbon quantum dot, the carbon quantum dot is modified by using bovine serum albumin to obtain water-soluble carbon quantum dot, and then the silicon dioxide coating is carried out, so that the quantum efficiency of the carbon quantum dot microsphere can be effectively improved, and the carbon quantum dot microsphere has very good light stability and chemical stability.
Preferably, in the step (1) of the preparation method of the carbon quantum dot microsphere, the temperature of the solvothermal reaction is 160-200 ℃ and the time is 10-14 h.
Preferably, in the step (1) of the preparation method of the carbon quantum dot microsphere, the temperature of the solvothermal reaction is 180 ℃ and the time is 12 hours.
Preferably, in step (5) and step (6) of the method for preparing carbon quantum dot microspheres, the centrifugation conditions are as follows: 8000r/min, 15 min.
Preferably, the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody is prepared by the following steps: dissolving carbon quantum dot microspheres in 2-morpholine ethanesulfonic acid (MES), adding 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), uniformly mixing, then adding N-hydroxysuccinimide (NHS), vibrating and activating for 20-40 min, centrifuging, discarding supernatant, and adding a second anti-D-dimer monoclonal antibody to obtain the carbon quantum dot microspheres-the second anti-D-dimer monoclonal antibody.
The scheme relates to a preparation method of a carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody, which comprises two steps of activation and coupling, wherein MES, EDC and NHS are firstly used for activating the carbon quantum dot microsphere, so that the carbon quantum dot microsphere is more easily combined with the anti-D-dimer second monoclonal antibody, and the coupling efficiency between the two is improved.
Preferably, the carbon quantum dot microsphere-negative control antigen is prepared by the following steps: dissolving carbon quantum dot microspheres in 2-morpholine ethanesulfonic acid (MES), adding 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), uniformly mixing, then adding N-hydroxysuccinimide (NHS), vibrating and activating for 20-40 min, centrifuging, discarding supernatant, and adding a negative control antibody to obtain the carbon quantum dot microspheres-negative control antigen.
Preferably, the sample pad is prepared by the following steps: the glass cellulose membrane was immersed in a PBS buffer solution containing 2.5 wt% sucrose, 1 wt% bovine serum albumin, 1 wt% surfactant S9, and dried to obtain a sample pad.
Preferably, in the sample pad preparation step, the PBS buffer has a pH of 7.4 and a concentration of 10 mM.
Preferably, the conjugate pad is prepared by the steps of: soaking the glass cellulose membrane in BB buffer solution containing 1 wt% of trehalose, 1 vol% of bovine serum albumin and 0.5 wt% of polyethylene glycol 4000, drying, uniformly mixing the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody and the carbon quantum dot microsphere-negative control antigen, and spraying the mixture on the glass cellulose membrane to obtain the binding pad.
Preferably, in the preparation step of the conjugate pad, the BB buffer has a pH of 8.0 and a concentration of 2 mM.
Preferably, in the above fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres, the test strip further comprises a water absorption pad and a bottom plate, and the sample pad, the binding pad, the chromatographic membrane and the water absorption pad are partially overlapped in sequence and then adhered to the surface of the bottom plate.
According to a second aspect of the present invention, there is provided a method for quantitatively detecting the concentration of D-dimer, comprising the steps of: and dropwise adding a sample to be detected on the fluorescence immunochromatographic reagent card based on the carbon quantum dot microspheres, irradiating the reagent card by using a laser diode under the action of a hemispherical lens, and collecting signals by using a signal collector.
The invention has the beneficial effects that:
1. the invention provides a fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres, which takes the carbon quantum dot microspheres prepared by a specific method as a fluorescence marker, has the advantages of high photoluminescence efficiency, stable luminescence, strong bleaching resistance, no flicker phenomenon, good biocompatibility and the like, can improve the signal-to-noise ratio in immunochromatographic detection, and has excellent signal amplification effect, thereby improving the sensitivity and stability of the detection
2. The carbon quantum dot microspheres adopted in the fluorescence immunochromatography reagent card provided by the invention are simple in synthesis process, can be obtained in one step by adopting a hydrothermal method, are low in manufacturing cost, have enhanced photoluminescence performance after the carbon quantum dot microspheres are synthesized, have detection performance of a larger-scale chemiluminescence method than that of a conventional chemiluminescence method, are environment-friendly materials, do not contain heavy metal elements, are pollution-free, carcinogenic and nontoxic, can be discarded after being inactivated for biological samples after being used, and cannot influence the environment.
3. When the fluorescence immunochromatographic reagent card provided by the invention is used for detecting the concentration of the D-dimer, the lowest detection limit is 0.050mg/L, and the detection sensitivity is high; and the repeatability in batches is good, the CV values are all less than 15%, the relative range between batches is also less than 15%, and the detection stability is high.
4. The fluorescence immunochromatographic reagent card based on the carbon quantum dot microspheres is simple to use and operate, short in detection time and greatly improved in detection speed.
D-dimer mainly reflects the fibrinolytic function, and when activated thrombus formation and fibrinolytic activity occur in the blood vessels of a human body, the concentration of the D-dimer is increased, and the D-dimer is used as a marker for judging fibrinolysis, and accurate quantification of the concentration of the D-dimer is clinically very important for judging and evaluating the fibrinolysis and the formation of thrombus.
Drawings
FIG. 1 shows a fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres provided by the invention.
The reference signs are: 1 sample pad, 2 combination pads, 3 detection lines, 4 quality control lines, 5 chromatographic membranes, 6 water absorption pads and 7 bottom plates.
FIG. 2 is a linear curve diagram which is drawn by taking the concentration as abscissa and the T/C value as ordinate after the fluorescence immunochromatographic reagent card based on the carbon quantum dot microspheres provided by the invention is used for detecting D-dimer antigen diluents with different concentrations.
Detailed Description
Technical features in the technical solutions provided by the present invention are further clearly and completely described below with reference to specific embodiments, and it is obvious that the described embodiments are only a part of embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres is prepared by the following steps:
(1) preparation of carbon Quantum dot microsphere-anti-D-dimer second monoclonal antibody
Dissolving 25 mu L of carbon quantum dot microspheres in 200 mu L of 2-morpholine ethanesulfonic acid (MES) solution, adding 20 mu L of 1mg/mL 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and uniformly mixing, then adding 10 mu L of 1mg/mL N-hydroxysuccinimide (NHS), shaking and activating for 30min, centrifuging for 15min at 8000r/min, discarding supernatant, adding a second anti-D-dimer monoclonal antibody into the mixture and coupling for 2h to obtain the carbon quantum dot microspheres-the second anti-D-dimer monoclonal antibody;
wherein the particle size of the carbon quantum dot microspheres is 150 nm;
the carbon quantum dot microsphere is prepared by the following method:
firstly, dissolving 1g of pyrene in toluene, quickly transferring the solution into a 100mL polytetrafluoroethylene hydrothermal reaction kettle, and reacting the kettle at 180 ℃ for 12 hours to obtain a carbon quantum dot crude product;
filtering the carbon quantum dot crude product by an oil-soluble 220nm filter membrane, and then performing rotary evaporation drying on the filtered product by a rotary evaporator to obtain black carbon quantum dot powder;
dissolving 0.5g of carbon quantum dot powder in 5mL of chloroform to obtain a carbon quantum dot chloroform solution;
weighing 1g of Bovine Serum Albumin (BSA) in a 50mL beaker, adding 20mL of secondary deionized water, and performing ultrasonic treatment until the BSA is completely dissolved to obtain a BSA solution;
dropwise adding a carbon quantum dot chloroform solution into a BSA solution under an ultrasonic condition, dispersing to form an emulsion state, centrifuging at 8000r/min for 15min, discarding supernatant, and washing and precipitating twice by using secondary deionized water to obtain a BSA-coated carbon quantum dot;
sixthly, putting 2mL of the carbon quantum dots coated by 200mg/mL BSA in a 100mL beaker, adding 30mL of absolute ethyl alcohol, uniformly dispersing, then adding 1mL of concentrated ammonia water, slowly dropwise adding 0.5mL of ethyl orthosilicate under stirring, continuously stirring for about 4 hours until the ethyl orthosilicate is completely hydrolyzed, centrifuging at 8000r/min for 15min, discarding the supernatant, washing and precipitating twice by using secondary deionized water, and adding absolute ethyl alcohol to 5mL to obtain the carbon quantum dot microspheres;
preparing carbon quantum dot microspheres-chicken IgY according to the preparation method;
(2) preparation of sample pad
Soaking a glass cellulose membrane in PBS (phosphate buffer solution) containing 2.5 wt% of sucrose, 1 wt% of bovine serum albumin and 1 wt% of surfactant S9, and drying at 37 ℃ for 24 hours to obtain a sample pad 1;
wherein the pH value of the PBS buffer solution is 7.4, and the concentration is 10 mM;
(3) preparation of the bonding pad
Soaking a glass cellulose membrane in BB buffer solution containing 1 wt% of trehalose, 1 wt% of bovine serum albumin and 0.5 wt% of polyethylene glycol 4000 for a period of time, drying at 37 ℃ for 18 hours, uniformly mixing the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody prepared in the step (1) and the carbon quantum dot microsphere-chicken IgY, and spraying the mixture on the glass cellulose membrane to obtain a binding pad 2;
wherein the BB buffer solution has a pH value of 8.0 and a concentration of 2 mM;
(4) preparation of chromatographic membranes
Cutting a nitrocellulose membrane (NC membrane) into strips with the specification of 2.5cm x 30cm, pasting the obtained strips in the middle of a bottom plate, respectively scribing a first anti-D-dimer monoclonal antibody and rabbit anti-chicken IgY on different positions of the NC membrane by using a scribing instrument as a detection line 3 and a quality control line 4, and drying at 37 ℃ to obtain a chromatographic membrane 5;
(5) assembled immunochromatography test strip
Respectively sticking the sample pad 1, the combination pad 2 and the water absorption pad 7 on two sides of the chromatographic membrane 5, ensuring that the pad intervals are 1.5mm, and then, alternately laminating, sticking the sample pad, the combination pad and the water absorption pad on a PVC (polyvinyl chloride) bottom plate, and cutting the sample pad, the combination pad and the water absorption pad into test strips with the width of 3.5mm to obtain the fluorescent immunochromatographic reagent card based on the carbon quantum dot microspheres shown in figure 1.
Example 2
A fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres is prepared by the following steps:
(1) preparation of carbon Quantum dot microsphere-anti-D-dimer second monoclonal antibody
Dissolving 25 mu L of carbon quantum dot microspheres in 200 mu L of 2-morpholine ethanesulfonic acid (MES) solution, adding 20 mu L of 1mg/mL 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and uniformly mixing, then adding 10 mu L of 1mg/mL N-hydroxysuccinimide (NHS), shaking and activating for 20min, centrifuging for 15min at 8000r/min, discarding supernatant, adding a second anti-D-dimer monoclonal antibody into the mixture and coupling for 2h to obtain the carbon quantum dot microspheres-the second anti-D-dimer monoclonal antibody;
wherein the particle size of the carbon quantum dot microspheres is 50 nm;
the carbon quantum dot microsphere is prepared by the following method:
firstly, dissolving 1g of pyrene in toluene, quickly transferring the solution into a 100mL polytetrafluoroethylene hydrothermal reaction kettle, and reacting the kettle at 200 ℃ for 10 hours to obtain a carbon quantum dot crude product;
filtering the carbon quantum dot crude product by an oil-soluble 220nm filter membrane, and then performing rotary evaporation drying on the filtered product by a rotary evaporator to obtain black carbon quantum dot powder;
dissolving 0.5g of carbon quantum dot powder in 5mL of chloroform to obtain a carbon quantum dot chloroform solution;
weighing 1g of Bovine Serum Albumin (BSA) in a 50mL beaker, adding 20mL of secondary deionized water, and performing ultrasonic treatment until the BSA is completely dissolved to obtain a BSA solution;
dropwise adding a carbon quantum dot chloroform solution into a BSA solution under an ultrasonic condition, dispersing to form an emulsion state, centrifuging at 8000r/min for 15min, discarding supernatant, and washing and precipitating twice by using secondary deionized water to obtain a BSA-coated carbon quantum dot;
sixthly, putting 2mL of the carbon quantum dots coated by 200mg/mL BSA in a 100mL beaker, adding 30mL of absolute ethyl alcohol, uniformly dispersing, then adding 1mL of concentrated ammonia water, slowly dropwise adding 0.5mL of ethyl orthosilicate under stirring, continuously stirring for about 4 hours until the ethyl orthosilicate is completely hydrolyzed, centrifuging at 8000r/min for 15min, discarding the supernatant, washing and precipitating twice by using secondary deionized water, and adding absolute ethyl alcohol to 5mL to obtain the carbon quantum dot microspheres;
preparing carbon quantum dot microspheres-chicken IgY according to the preparation method;
(2) preparation of sample pad
Soaking a glass cellulose membrane in PBS (phosphate buffer solution) containing 2.5 wt% of sucrose, 1 wt% of bovine serum albumin and 1 wt% of surfactant S9, and drying at 37 ℃ for 24 hours to obtain a sample pad 1;
wherein the pH value of the PBS buffer solution is 7.4, and the concentration is 10 mM;
(3) preparation of the bonding pad
Soaking a glass cellulose membrane in BB buffer solution containing 1 wt% of trehalose, 1 wt% of bovine serum albumin and 0.5 wt% of polyethylene glycol 4000 for a period of time, drying at 37 ℃ for 18 hours, uniformly mixing the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody prepared in the step (1) and the carbon quantum dot microsphere-chicken IgY, and spraying the mixture on the glass cellulose membrane to obtain a binding pad 2;
wherein the BB buffer solution has a pH value of 8.0 and a concentration of 2 mM;
(4) preparation of chromatographic membranes
Cutting a nitrocellulose membrane (NC membrane) into strips with the specification of 2.5cm x 30cm, pasting the obtained strips in the middle of a bottom plate, respectively scribing a first anti-D-dimer monoclonal antibody and rabbit anti-chicken IgY on different positions of the NC membrane by using a scribing instrument as a detection line 3 and a quality control line 4, and drying at 37 ℃ to obtain a chromatographic membrane 5;
(5) assembled immunochromatography test strip
Respectively sticking the sample pad 1, the combination pad 2 and the water absorption pad 7 on two sides of the chromatographic membrane 5, ensuring that the pad intervals are 1.5mm, and then, alternately laminating, sticking the sample pad, the combination pad and the water absorption pad on a PVC (polyvinyl chloride) bottom plate, and cutting the sample pad, the combination pad and the water absorption pad into test strips with the width of 3.5mm to obtain the fluorescent immunochromatographic reagent card based on the carbon quantum dot microspheres shown in figure 1.
Example 3
A fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres is prepared by the following steps:
(1) preparation of carbon Quantum dot microsphere-anti-D-dimer second monoclonal antibody
Dissolving 25 mu L of carbon quantum dot microspheres in 200 mu L of 2-morpholine ethanesulfonic acid (MES) solution, adding 20 mu L of 1mg/mL 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and uniformly mixing, then adding 10 mu L of 1mg/mL N-hydroxysuccinimide (NHS), shaking and activating for 40min, centrifuging for 15min at 8000r/min, discarding supernatant, adding a second anti-D-dimer monoclonal antibody into the mixture and coupling for 2h to obtain the carbon quantum dot microspheres-the second anti-D-dimer monoclonal antibody;
wherein the particle size of the carbon quantum dot microspheres is 300 nm;
the carbon quantum dot microsphere is prepared by the following method:
firstly, dissolving 1g of pyrene in toluene, quickly transferring the solution into a 100mL polytetrafluoroethylene hydrothermal reaction kettle, and reacting for 14h at 160 ℃ to obtain a carbon quantum dot crude product;
filtering the carbon quantum dot crude product by an oil-soluble 220nm filter membrane, and then performing rotary evaporation drying on the filtered product by a rotary evaporator to obtain black carbon quantum dot powder;
dissolving 0.5g of carbon quantum dot powder in 5mL of chloroform to obtain a carbon quantum dot chloroform solution;
weighing 1g of Bovine Serum Albumin (BSA) in a 50mL beaker, adding 20mL of secondary deionized water, and performing ultrasonic treatment until the BSA is completely dissolved to obtain a BSA solution;
dropwise adding a carbon quantum dot chloroform solution into a BSA solution under an ultrasonic condition, dispersing to form an emulsion state, centrifuging at 8000r/min for 15min, discarding supernatant, and washing and precipitating twice by using secondary deionized water to obtain a BSA-coated carbon quantum dot;
sixthly, putting 2mL of the carbon quantum dots coated by 200mg/mL BSA in a 100mL beaker, adding 30mL of absolute ethyl alcohol, uniformly dispersing, then adding 1mL of concentrated ammonia water, slowly dropwise adding 0.5mL of ethyl orthosilicate under stirring, continuously stirring for about 4 hours until the ethyl orthosilicate is completely hydrolyzed, centrifuging at 8000r/min for 15min, discarding the supernatant, washing and precipitating twice by using secondary deionized water, and adding absolute ethyl alcohol to 5mL to obtain the carbon quantum dot microspheres;
preparing carbon quantum dot microspheres-chicken IgY according to the preparation method;
(2) preparation of sample pad
Soaking a glass cellulose membrane in PBS (phosphate buffer solution) containing 2.5 wt% of sucrose, 1 wt% of bovine serum albumin and 1 wt% of surfactant S9, and drying at 37 ℃ for 24 hours to obtain a sample pad 1;
wherein the pH value of the PBS buffer solution is 7.4, and the concentration is 10 mM;
(3) preparation of the bonding pad
Soaking a glass cellulose membrane in BB buffer solution containing 1 wt% of trehalose, 1 wt% of bovine serum albumin and 0.5 wt% of polyethylene glycol 4000 for a period of time, drying at 37 ℃ for 18 hours, uniformly mixing the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody prepared in the step (1) and the carbon quantum dot microsphere-chicken IgY, and spraying the mixture on the glass cellulose membrane to obtain a binding pad 2;
wherein the BB buffer solution has a pH value of 8.0 and a concentration of 2 mM;
(4) preparation of chromatographic membranes
Cutting a nitrocellulose membrane (NC membrane) into strips with the specification of 2.5cm x 30cm, pasting the obtained strips in the middle of a bottom plate, respectively scribing a first anti-D-dimer monoclonal antibody and rabbit anti-chicken IgY on different positions of the NC membrane by using a scribing instrument as a detection line 3 and a quality control line 4, and drying at 37 ℃ to obtain a chromatographic membrane 5;
(5) assembled immunochromatography test strip
Respectively sticking the sample pad 1, the combination pad 2 and the water absorption pad 7 on two sides of the chromatographic membrane 5, ensuring that the pad intervals are 1.5mm, and then, alternately laminating, sticking the sample pad, the combination pad and the water absorption pad on a PVC (polyvinyl chloride) bottom plate, and cutting the sample pad, the combination pad and the water absorption pad into test strips with the width of 3.5mm to obtain the fluorescent immunochromatographic reagent card based on the carbon quantum dot microspheres shown in figure 1.
Test example
1. Construction mode of experiment
The reference object of this test example is the fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres prepared in examples 1 to 3, and the fluorescence immunochromatographic reagent card is used for detecting a D-dimer antigen (purchased from guangdong feng peng biotechnology, ltd), wherein the antigen diluent is PBS buffer (ph7.4) containing 1 wt% bovine serum albumin, and the chemiluminescence analyzer of guangzhou shifu organisms and a matched D-dimer detection kit (chemiluminescence method) are used for assigning values after the D-dimer antigen is diluted by the antigen diluent.
(1) Linear analysis
D-dimer antigen was diluted to several concentrations: 0.2, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0mg/L, respectively dripping the D-dimer antigens with different concentrations on the sample pad of the fluorescence immunochromatography reagent card prepared in the embodiment 1-3, irradiating the reagent card by using a laser diode under the action of a hemispherical lens, collecting signals by using a signal collector to detect the D-dimer antigens with each concentration, recording the fluorescence intensity of a detection line (T) and a quality control line (C) of each test of the reagent card, calculating a T/C ratio and a Coefficient of Variation (CV), simultaneously drawing a linear curve chart by using the concentration of the D-dimer antigens as a horizontal coordinate and the T/C value as a vertical coordinate, and using data analysis software to calculate a correlation coefficient R2Wherein, the calculation formula of the coefficient of variation CV value is as follows:
Figure BDA0003521025700000111
SD represents the standard deviation of the measured signal,
Figure BDA0003521025700000112
the average value is shown.
(2) Minimum detection limit detection
The antigen dilutions were detected 20 times using the fluorescence immunochromatographic reagent cards prepared in examples 1 to 3, respectively, and the detection was carried out
Figure BDA0003521025700000119
The formula calculates the lowest detection limit, wherein
Figure BDA0003521025700000114
Mean values are indicated and SD standard deviation.
(3) Repeatability test
The fluorescence immunochromatography reagent cards prepared in examples 1 to 3 were used to detect low and high concentrations, respectivelyDiluting the antigen for 10 times, calculating the coefficient of variation CV value and the relative range between batches, and calculating the correlation coefficient R by using data analysis software2Wherein, the calculation formula of the coefficient of variation CV value is as follows:
Figure BDA0003521025700000115
SD represents the standard deviation of the measured signal,
Figure BDA0003521025700000116
represents the average value; the calculation formula of relative range between batches is as follows:
Figure BDA0003521025700000117
xmaxrepresents the maximum value of the detected concentration, x, in a batchminRepresents the minimum of the detected concentration within the batch.
2. Results of the experiment
(1) Linear analysis
TABLE 1 results of Linear analysis
Figure BDA0003521025700000118
The results of the linear analysis are shown in Table 1, the linear curve and the correlation coefficient R2The results of the values are shown in FIG. 2. As can be seen from the data in Table 1, each concentration gradient is detected by using the fluorescence immunochromatographic reagent card of examples 1 to 3, the numerical values of the three groups of experimental results are very close, and no significant difference exists, which indicates that the reproducibility of the test results is good when the fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres provided by the invention is used for detecting the antigen to be detected. As shown in FIG. 2, the antigen concentration to be measured is in the range of 0.2-12 mg/L, and the correlation coefficient R thereof2Reaching 0.9999, which shows that the linear relation between the concentration of the antigen to be detected and the T/C value in the range is good.
(2) Minimum detection limit
TABLE 2 lowest detection Limit results
Figure BDA0003521025700000121
The test results for the lowest detection limit are shown in table 2. As can be seen from Table 2, the antigen diluent is detected 20 times respectively by using the fluorescence immunochromatographic reagent card prepared in the embodiments 1 to 3, and the minimum detection limit obtained by calculation is 0.050mg/L, which is far lower than other detection reagents in the same methodology, which indicates that the fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres provided by the present invention has high detection sensitivity.
(3) Repeatability of
TABLE 3 repeatability test results
Figure BDA0003521025700000131
The results of the repeatability tests are shown in table 3. As can be seen from table 3, the low-concentration and high-concentration antigen dilutions were detected 10 times using the fluorescence immunochromatographic reagent cards prepared in examples 1 to 3, and the calculated results showed that the CV values were all less than 15%, and the batch-to-batch relative range was also less than 15%, indicating that the fluorescence immunochromatographic reagent card based on carbon quantum dot microspheres provided by the present invention has high detection stability.
Although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. A fluorescence immunochromatography reagent card based on carbon quantum dot microspheres is characterized in that: the reagent card comprises a test strip, the test strip comprises a sample pad, a binding pad and a chromatographic membrane, a detection line and a quality control line are arranged on the chromatographic membrane, the detection line is coated with a first monoclonal antibody of anti-D-dimer, and the quality control line is coated with a negative control antibody;
the combination pad is coated with a carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody and a carbon quantum dot microsphere-negative control antigen.
2. The carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card of claim 1, wherein: the particle size of the carbon quantum dot microspheres is 50-300 nm.
3. The carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card of claim 1, wherein: the carbon quantum microsphere comprises carbon quantum dots, the surface of the carbon quantum dots is sequentially coated with a first shell layer and a second shell layer, the carbon quantum dots are prepared from pyrene and methylbenzene, the first shell layer is made of bovine serum albumin, and the second shell layer is made of silicon dioxide.
4. The carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card of claim 3, wherein the carbon quantum dot microsphere is prepared by the following method:
(1) taking the pyrene as a carbon source and the toluene as a solvent, and preparing a carbon quantum dot crude product through solvothermal reaction;
(2) filtering the crude product of the carbon quantum dots by using a 220nm filter membrane, and drying to obtain 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 by using deionized water and bovine serum albumin;
(5) dropwise adding the carbon quantum dot chloroform solution into the bovine serum albumin solution under an ultrasonic condition, centrifuging, discarding the supernatant, and washing to obtain bovine serum albumin-coated carbon quantum dots;
(6) and adding absolute ethyl alcohol into the bovine serum albumin coated carbon quantum dots, uniformly mixing, then adding concentrated ammonia water, slowly dropwise adding tetraethoxysilane under stirring, continuously and uniformly stirring until the tetraethoxysilane is completely hydrolyzed, centrifuging, discarding supernatant, and washing to obtain the carbon quantum dot microspheres.
5. The carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card of claim 4, wherein: in the step (1), the temperature of the solvothermal reaction is 160-200 ℃ and the time is 10-14 h.
6. The carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card of claim 4, wherein in the step (5) and the step (6), the centrifugation conditions are as follows: 8000r/min, 15 min.
7. The carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card of claim 1, wherein the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody is prepared by the following steps: dissolving the carbon quantum dot microspheres in 2-morpholine ethanesulfonic acid, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, mixing uniformly, then adding N-hydroxysuccinimide, oscillating and activating for 20-40 min, centrifuging, discarding supernatant, and adding the anti-D-dimer second monoclonal antibody to obtain the carbon quantum dot microspheres-anti-D-dimer second monoclonal antibody.
8. The carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card of claim 1, wherein: the sample pad is prepared by the following steps: soaking a glass cellulose membrane in PBS (phosphate buffer solution) containing 2.5 wt% of sucrose, 1 wt% of bovine serum albumin and 1 wt% of surfactant S9, and drying to obtain the sample pad;
the conjugate pad is prepared by the following steps: soaking a glass cellulose membrane in BB buffer solution containing 1 wt% of trehalose, 1 wt% of bovine serum albumin and 0.5 wt% of polyethylene glycol 4000, drying, uniformly mixing the carbon quantum dot microsphere-anti-D-dimer second monoclonal antibody and the carbon quantum dot microsphere-negative control antigen, and spraying the mixture on the glass cellulose membrane to obtain the binding pad;
in the sample pad preparation step, the PBS buffer was at pH7.4 and at a concentration of 10 mM;
in the preparation step of the conjugate pad, the BB buffer had a pH of 8.0 and a concentration of 2 mM.
9. The carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card of claim 1, wherein: the test strip also comprises a water absorption pad and a bottom plate, wherein the sample pad, the combination pad, the chromatographic membrane and the water absorption pad are partially overlapped in sequence and then are adhered to the surface of the bottom plate.
10. A method for quantitatively detecting the concentration of D-dimer, comprising the steps of: a sample to be detected is dripped on the carbon quantum dot microsphere-based fluorescence immunochromatography reagent card as claimed in any one of claims 1 to 9, the reagent card is irradiated by a laser diode under the action of a hemispherical lens, and a signal is collected by a signal collector.
CN202210181199.7A 2022-02-25 2022-02-25 Carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card and preparation method and application thereof Pending CN114544974A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210181199.7A CN114544974A (en) 2022-02-25 2022-02-25 Carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210181199.7A CN114544974A (en) 2022-02-25 2022-02-25 Carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card and preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN114544974A true CN114544974A (en) 2022-05-27

Family

ID=81679728

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210181199.7A Pending CN114544974A (en) 2022-02-25 2022-02-25 Carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN114544974A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115818621A (en) * 2022-11-28 2023-03-21 山西医科大学 Biomass-derived carbon nanoparticles with near-infrared two-zone light emission characteristic and preparation method and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115818621A (en) * 2022-11-28 2023-03-21 山西医科大学 Biomass-derived carbon nanoparticles with near-infrared two-zone light emission characteristic and preparation method and application thereof
CN115818621B (en) * 2022-11-28 2024-04-05 山西医科大学 Biomass-derived carbon nano particle with near infrared two-region light emission characteristic and preparation method and application thereof

Similar Documents

Publication Publication Date Title
CN109633144B (en) Fluorescence immunochromatography test strip prepared by using aggregation-induced emission fluorescent microspheres as beacon carrier
US9977018B2 (en) Electrochemical lateral flow bioassay and biosensor
EP0051183A1 (en) Multilayer analysis element
CN110987882B (en) Fluorescence-quenched colloidal gold immunochromatographic test strip, preparation method and application thereof
CN109613244B (en) Preparation method and application of Ag @ Pt-CuS labeled immunosensor
CN111693571A (en) Method for detecting GPC3 based on optical addressing potential sensor
CN104374909A (en) Chloramphenicol quantitative detection method based on up-conversion phosphor technology and immunochromatography technology
CN110133252A (en) For detecting kit and detection method and its application of carcinomebryonic antigen
CN111781351A (en) Electrochemical immunochromatographic test strip for quantitative detection of novel coronavirus antigen and preparation method thereof
CN111504909B (en) Photoelectrochemical biosensor for label-free detection of alpha fetoprotein, preparation method and application thereof
CN110220889A (en) A kind of pair of quenching electrochemical luminescence strategy is applied to transducer production method and the application of the detection of procalcitonin
CN109142748A (en) Human prostate specific antigen detection method and its kit
CN114544974A (en) Carbon quantum dot microsphere-based fluorescence immunochromatographic reagent card and preparation method and application thereof
CN111830255A (en) Norfloxacin detection method
CN112014369B (en) System and method for ultrasensitive digital chromatography rapid detection of analytes
CN112526135A (en) Preparation method and application of photoelectrochemical biosensor for detecting prostate specific antigen
CN111504959B (en) C peptide rare earth fluorescent microsphere kit, detection card and preparation method thereof
CN112858416A (en) Preparation method of coreactant-free electrochemical luminescence immunosensor
JP3841559B2 (en) Immunological examination method and immunological examination kit
CN115060893A (en) Three-dimensional homogeneous phase filling type magnetic-precious metal composite nano enzyme, neocorona antigen immunochromatographic test paper and application thereof
CN114660029A (en) Colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere and application thereof
CN114720437A (en) Fe-PDAN-based efficient fluorescence quenching test strip and preparation method and detection method thereof
CN109682964B (en) Au@Fe3O4MNPs-Ab2Preparation method of nano enzyme detection probe and method for detecting multi-component antigen
CN210923728U (en) Time-resolved fluorescence S-100 β protein detection card
CN210323044U (en) IL-6/PCT combined detection time resolution detection card and kit

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination