CN114354924A - 2019-nCoV novel coronavirus antigen detection reagent of AIE nano-microspheres and preparation method and application thereof - Google Patents
2019-nCoV novel coronavirus antigen detection reagent of AIE nano-microspheres and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of in-vitro diagnosis virus detection, in particular to a 2019-nCoV novel coronavirus antigen detection reagent of AIE nano microspheres and a preparation method and application thereof. The 2019-nCoV novel coronavirus antigen detection reagent is characterized in that AIE nano microspheres with different particle sizes are used for marking a detection antibody, and the detection antibody adopts a 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 and a goat anti-rabbit IgG antibody. The method has the advantages that the sampling is simple, convenient and quick, and more processing on the sample is not needed; the detection process is simple to operate, and professional technicians are not needed; the detection is rapid, and the result is rapidly obtained within 10-15 min; the preparation process of the reagent is relatively simple, and the requirement on production environment is not high; the detection accuracy is high, and the detection sensitivity is high (10 pg/mL); the aggregation-induced emission nano-microsphere can effectively prevent photobleaching, so that the detection result is more accurate and stable.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis virus detection, in particular to a 2019-nCoV novel coronavirus antigen detection reagent of AIE nano microspheres and a preparation method and application thereof.
Background
The novel coronavirus (2019-nCoV) is a coronavirus of the genus Beta, a single-stranded plus-strand RNA virus with an envelope, the particles being circular or elliptical and having a diameter of 60-140 nm; is a human respiratory infectious disease, the incubation period is usually 3-14 days, and the human respiratory infectious disease is mainly transmitted through respiratory droplets or close contact; clinically, fever and hypodynamia are mostly manifested as dry cough, dyspnea gradually appears, and acute respiratory distress syndrome, septic shock, metabolic acidosis difficult to correct and blood coagulation dysfunction appear in severe cases.
Since the outbreak of new coronavirus in 2019, no effective method for completely destroying the virus has been found yet. With the large regional outbreak of the new 2019-nCoV coronavirus stage, more and more people need to be screened and corresponding measures need to be taken in time. In the process of preventing and controlling the novel coronavirus, the rapid diagnostic reagent plays a key role in higher sensitivity, stability and specificity. Rapid diagnosis is mainly divided into two categories: antibody detection and antigen detection. Antibody detection is commonly used to determine whether previous infection has occurred. The antigen detection mainly comprises the detection of virus specific nucleic acid fragments and virus surface specific proteins so as to determine whether pathogens exist in vivo, and is particularly suitable for early screening of new coronavirus latent stage, early onset stage and the like.
The immunochromatographic test strip technology is a detection technology for quickly and accurately detecting a specific substance to be detected by applying the specific immunology of an antigen and an antibody and the reaction principle of chromatography. Techniques such as colloidal gold and fluorescence immunochromatography are commonly used in vitro diagnosis.
The colloidal gold immunochromatography technology is widely applied in the fields of in-vitro diagnosis (early pregnancy detection and the like), food safety, drug detection and the like. However, the sensitivity and accuracy of the detection are relatively low, and the method is generally only used in the field of qualitative analysis or semi-quantitative analysis detection.
Compared with the colloidal gold immunochromatography technology, the fluorescence immunochromatography technology has the advantages of higher sensitivity, better stability and strong natural fluorescence interference resistance. The labels used for fluorescence immunochromatography mainly include fluorescein, quantum dots and the like. The fluorescein marker is easy to decompose under illumination, easy to generate a photobleaching phenomenon, has the defect of aggregation-induced quenching, and seriously influences the accuracy and reliability of an analysis result.
The appearance of Aggregation-Induced Emission (AIE) nano microspheres can effectively avoid the defect of Aggregation-Induced quenching, enrich the types of markers of fluorescence immunochromatography and enable people to have new knowledge on fluorescent materials. Compared with the traditional organic fluorescent dye, the AIE nano-microsphere has the advantages of strong modifiability, good light stability, stable fluorescent signal, strong background fluorescence interference resistance and the like in the aspects of biological imaging, fluorescence detection and the like.
The AIE nano microsphere immunochromatography technology is adopted to mark specific protein, so that not only can the antigen be marked for antibody detection, but also the antigen detection antibody can be marked. In the FDA guidelines for the detection of SARS-COV-2, it is indicated that antibody detection alone cannot be used for the diagnosis of new coronaviruses, and that it is not possible to state the infection or to prove whether it is an infection. Therefore, a detection method capable of rapidly and directly detecting whether a human body carries a virus is more desirable.
At present, methods for detecting the new coronavirus mainly comprise nucleic acid detection (PCR or real-time PCR) and virus antigen detection. The PCR method has higher requirements on technical personnel, needs to be provided with professional detection equipment and a laboratory with the biological safety level of 3, and has the disadvantages of complex operation, long time consumption and high risk of infection on the detection personnel. The current method for detecting virus antigen adopts colloidal gold method, but has lower sensitivity and poorer repeatability. Chinese patent discloses a test strip, a kit and a detection method for detecting a novel coronavirus (2019-nCoV) antigen by using colloidal gold, wherein the minimum detection limit is 0.5 ng/mL. The detection limit of the patent is high, and the condition of missed detection may occur to positive samples with relatively low viral load.
Based on the method, the development of the novel 2019-nCoV coronavirus antigen detection reagent based on the AIE nano microspheres, which is simple, convenient and quick to operate, lower in detection limit, high in accuracy and short in detection period, has an important value.
Disclosure of Invention
The invention aims to provide a 2019-nCoV novel coronavirus antigen detection reagent based on AIE nano microspheres and a preparation method thereof, which are applicable to detection of novel coronavirus antigens in nasopharyngeal swabs, oropharyngeal swabs or sputum of suspected infected persons. The reagent has high sensitivity, good specificity and simple and quick operation, and is suitable for early screening in primary medical institutions or epidemic situation outbreak areas.
The invention provides an AIE nano-microsphere-based 2019-nCoV novel coronavirus antigen detection reagent, which comprises an AIE nano-microsphere-based 2019-nCoV novel coronavirus antigen detection card, wherein a quality control protein goat anti-rabbit IgG antibody and a 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 on a binding pad are marked by aggregation-induced emission nano-microspheres, capture is completed by the 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb2 and rabbit IgG fixed on an NC membrane, and a sample pad, the binding pad, the NC membrane and absorbent paper are sequentially overlapped and pasted on a bottom plate in an overlapping manner and form a detection reagent together with a virus extracting solution and the like.
The purpose of the invention is realized by the following technical scheme:
A2019-nCoV novel coronavirus antigen detection reagent of AIE nano microspheres comprises a detection antibody marked by the AIE nano microspheres, wherein the detection reagent comprises a 2019-nCoV novel coronavirus antigen detection card of the AIE nano microspheres and a virus extracting solution, and the detection antibody adopts a 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 and a goat anti-rabbit IgG antibody.
Further, the 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 is a monoclonal antibody.
Furthermore, the AIE nano-microspheres are AIE molecules wrapped by polystyrene.
Furthermore, the AIE nano-microsphere is prepared by wrapping aggregation-induced emission materials with polystyrene, the surface of the AIE nano-microsphere is modified by high-density carboxyl, the particle size is 100nm-400nm, the excitation wavelength is 350nm-400nm, and the emission wavelength is 400nm-600 nm.
Furthermore, the detection reagent can effectively detect the 2019-nCoV novel coronavirus with the concentration of 10pg/mL or more.
Further, the virus extracting solution is used for extracting the virus from the sample, so that the detection is convenient.
Further, the detection card comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane (NC membrane) and absorbent paper, and the bottom plate, the sample pad, the combination pad, the NC membrane and the absorbent paper are overlapped and overlapped in sequence.
Further, the detection card comprises a detection line and a quality control line.
Further, the detection card comprises a detection line coated with a 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb2 and a quality control line coated with rabbit IgG.
Further, the bottom plate of the detection card is selected from a PVC material or a PS plate.
Further, the binding pad of the detection card is selected from a cellophane film or a polyester film.
Furthermore, a binding pad of the detection card is sprayed with a 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 marked by AIE nano microspheres and a quality control protein goat anti-rabbit IgG antibody marked by AIE nano microspheres.
Furthermore, a nitrocellulose membrane (NC membrane) of the detection card is provided with a detection line and a quality control line which are respectively coated with a 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb2 and rabbit IgG.
Further, the absorbent paper is used for absorbing unreacted labeled antibody and detection solution.
Furthermore, the sample pad, the combination pad, the NC membrane and the absorbent paper are sequentially laminated and fixed on the bottom plate according to the transverse chromatography direction taking the sample pad as the starting position.
A preparation method of 2019-nCoV novel coronavirus antigen detection reagent of AIE nano microspheres comprises the following steps:
s1, preparation of an AIE nano microsphere labeled antibody solution: activating the AIE nano-microspheres by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), wherein the mass ratio of the AIE nano-microspheres to EDC and NHS is respectively 1 (0.1-5) and 1 (0.2-20), the activation time is 0.5h-1h, after the activation is finished, adding an antibody for coupling, wherein the mass ratio of the AIE nano-microspheres to the antibody is 1 (0.03-0.24), the coupling time is 1h-4h, and after the coupling is finished, adding a BSA solution for blocking, wherein the concentration of the BSA solution is 5g/L-20g/L, and the blocking reaction time is 0.5h-2 h;
s2, preparing a bonding pad: spraying the prepared AIE nano microsphere marked antibody on a bonding pad, wherein the gold spraying concentration of the AIE nano microsphere marked 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 solution is 20g/L-100g/L, the gold spraying concentration of the AIE nano microsphere marked goat anti-rabbit IgG antibody solution is 10g/L-50g/L, uniformly mixing, spraying the pad parameters which are 1 muL/cm-5 muL/cm for film spraying, and after the film spraying is finished, putting the pad in an oven at 30-50 ℃ for drying for 16-24 h;
s3, preparation of a coating film: coating 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb2 at a detection line T line position of a nitrocellulose membrane, wherein the scribing concentration is 0.5-3 mg/mL, the scribing dosage is 0.5-2 muL/cm, coating rabbit IgG at a quality control line C line position, the scribing concentration is 0.5-2.4 mg/mL, the scribing dosage is 0.5-2 muL/cm, and after completion, placing the coated rabbit IgG in an oven at the temperature of 30-50 ℃ for baking for 16-24 hours;
s4, assembling a detection card: the combined pad, the sample pad and the absorbent paper are sequentially lapped on the bottom plate stuck with the nitrocellulose membrane to be assembled into a large plate, and the large plate is cut into detection cards with the width of 4mm by a slitter.
Further, the virus extract mainly comprises: citric acid at pH 6.0 or Phosphate Buffered Saline (PBS) at pH 7.4, casein, surfactants, sodium chloride, BSA, EDTA and Proclin 300.
Further, the surfactant is a nonionic surfactant Tritonx-100.
Further, the concentration of the Tritonx-100 is 0.1g/L-2 g/L.
Further, the concentration of the citric acid or phosphate buffer solution is 0.02-0.1M.
Furthermore, the concentration of the casein is 0.1g/L-2g/L, the concentration of EDTA is 1g/L-5g/L, and the concentration of BSA is 1g/L-5 g/L.
Further, the pH of the virus extract is 6-8.
The novel 2019-nCoV coronavirus antigen detection reagent based on the AIE nano microspheres can be used for detecting whether samples to be detected such as nasopharyngeal swabs, sputum, blood, saliva, urine, anal swabs and alveolar lavage fluid and samples collected in the environment contain the novel 2019-nCoV coronavirus.
A2019-nCoV novel coronavirus antigen detection reagent based on AIE nano-microspheres can be used for detecting nasopharyngeal swabs, sputum, blood, saliva, urine, anal swabs, alveolar lavage fluid and samples collected in the environment.
Furthermore, the detection reagent directly detects the specific antigen of the 2019-nCoV novel coronavirus, can detect asymptomatic infectors of the 2019-nCoV novel coronavirus, and has important significance for early screening of the 2019-nCoV novel coronavirus.
Furthermore, the detection reagent can detect whether the new 2019-nCoV coronavirus exists in the environment (water and air) and on the surface of an object, and prevent the new 2019-nCoV coronavirus from being transmitted through the environment and articles such as food and articles for daily use.
Further, when the detection reagent is used for qualitative detection by using the 2019-nCoV novel coronavirus antigen detection card based on the AIE nano microspheres, a sample to be detected is mixed with a certain amount of virus extracting solution, the mixture is dripped on the detection card, and after the detection card is irradiated by an ultraviolet lamp, if the test line and the quality control line show fluorescence, the existence of the 2019-nCoV novel coronavirus is proved to be detected.
The invention has the advantages that:
1. the sampling is simple, convenient and quick, and more processing on the sample is not needed.
2. The detection process is simple to operate, and professional technicians are not needed; the detection is rapid, and the result is rapidly obtained within 10-15 min.
3. The preparation process of the reagent is relatively simple, and the requirement on production environment is not high.
4. High accuracy and high sensitivity (10 pg/mL).
5. The Aggregation Induced Emission (AIE) nano-microspheres can effectively prevent photobleaching, so that the detection result is more accurate and stable.
Drawings
Fig. 1 is a schematic structural diagram of the detection card of the present invention.
FIG. 2 is a schematic structural diagram of the detection card housing of the present invention.
FIG. 3 is a schematic diagram illustrating the determination of the test result of the test card according to the present invention.
Detailed Description
The following embodiments of the present invention are further illustrated with reference to the drawings and examples, and the examples described herein are only for clearly and clearly explaining the present invention and are not intended to limit the scope of the present invention.
Example 1
FIG. 1 is a schematic view of the structure of the test card of the present invention (1 is a base plate, 2 is a sample pad, 3 is a conjugate pad, 4 is an NC membrane, 5 is absorbent paper, 6 is a test line T, and 7 is a quality control line C).
FIG. 2 is a schematic view of the structure of the detection card case of the present invention (8 is the detection window, 9 is the sample application hole).
FIG. 3 is a schematic diagram illustrating the determination of the test result of the test card according to the present invention.
Preparation of 2019-nCoV novel coronavirus antigen detection card based on AIE nano-microspheres
(1) Preparation of AIE nano microsphere labeled antibody solution
Taking 100 μ L (10 g/L concentration) of AIE nano microspheres with 100nm particle size and carboxyl, adding appropriate amount of MES buffer solution, cleaning, centrifuging at 15000rpm for 10min in a centrifuge, and redissolving in MES buffer solution. Adding activators EDC and NHS, wherein the mass ratio of the added AIE nano microspheres to the added activators EDC is 1:0.1, the mass ratio of the added AIE nano microspheres to the added activators NHS is 1:0.2, rotationally mixing in the dark for 0.5h, centrifuging in a centrifuge at 15000rpm for 10min, redissolving in 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution, adding 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 for coupling, adding AIE nano microspheres and adding 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1, and the mass ratio of the added AIE nano microspheres to the added 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 is 1: 0.03, spin mix in the dark for 1h, centrifuge at 15000rpm for 10min, after which the supernatant is removed, 1mL blocking buffer (containing BSA at 5g/L) is added, spin mix for 0.5h, wash 3 times with 0.02M PBS pH 7.4 (containing 2g/L BSA and 1g/L trehalose), re-dissolve the labeled product in 100. mu.l phosphate buffer PBS 0.02MpH 7.4.4 (containing 2g/L BSA and 1g/L trehalose), put at 2 ℃ -8 ℃ for use;
the modification scheme of the AIE nano microsphere labeled goat anti-rabbit IgG antibody is as above, wherein the goat anti-rabbit IgG antibody is added, and the mass ratio of the added AIE nano microsphere to the added goat anti-rabbit IgG antibody is 1: 0.02;
(2) bond pad preparation
The conjugate pad was soaked with 0.02M Tris-HCl pH 7.4 (1 g/L trehalose, 2g/L bovine serum albumin, 0.2g/L LTween-20) for 2h and baked in an oven at 50 ℃ for 16 h. Diluting the 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 marked by the AIE nano microspheres and the goat anti-rabbit IgG antibody marked by the AIE nano microspheres according to the dilution concentration, wherein the gold spraying concentration of a solution of the 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 marked by the AIE nano microspheres is 20g/L, and the gold spraying concentration of a solution of the goat anti-rabbit IgG antibody marked by the AIE nano microspheres is 10 g/L. After mixing, spraying a pad by using a film-scratching gold spraying instrument with the parameter of 1 mu L/cm, putting the pad in a drying oven at 30 ℃ for drying for 16h, and sealing the pad for later use after drying;
(3) preparation of coating film
Diluting the N protein antibody COV19-PS-MAb2 of the 2019-nCoV novel coronavirus and the rabbit IgG respectively by using PBS (containing 2g/L BSA and 5g/L trehalose) with the pH of 7.4 and 0.02M, and then carrying out scribing, wherein the detection line is the N protein antibody COV19-PS-MAb2 of the 2019-nCoV novel coronavirus, the scribing concentration is 0.5mg/mL, and the scribing dosage is 0.5 muL/cm; the control line is rabbit IgG, the streaking concentration is 0.5mg/mL, and the streaking dosage is 0.5 muL/cm. After finishing, placing the coating film in a drying oven at 30 ℃ for drying for 16h, and sealing for later use after drying;
(4) test card assembly
As shown in fig. 1, the dried coating film, the bonding pad, the sample pad and the absorbent paper were attached to the base plate. After the assembly is finished, the test strip is cut into test strips with the width of 4mm by a slitter, the test strips are arranged in the detection card shown in figure 2 according to the direction that the sample pads are aligned with the sample adding holes, 8 sample adding holes correspond to the sample pads, and 9 detection windows correspond to the detection line T and the quality control line C area on the NC film. The detection card is put into an aluminum foil bag containing a drying agent, sealed and stored at room temperature (10-30 ℃).
Example 2
Preparation of 2019-nCoV novel coronavirus antigen detection card based on AIE nano-microspheres
(1) Preparation of AIE nano microsphere labeled antibody solution
Taking 100 μ L (10 g/L concentration) of AIE nano microspheres with 100nm particle size and carboxyl, adding appropriate amount of MES buffer solution, cleaning, centrifuging at 15000rpm for 10min in a centrifuge, and redissolving in MES buffer solution. Adding activators EDC and NHS, wherein the mass ratio of the added AIE nano microspheres to the added activators EDC is 1:2, the mass ratio of the added AIE nano microspheres to the added activators NHS is 1:5, rotationally mixing in the dark for 0.7h, centrifuging in a centrifuge at 15000rpm for 10min, redissolving in 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution, adding a 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 for coupling, adding the AIE nano microspheres and adding a 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1, and the mass ratio of the added AIE nano microspheres to the added 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 is 1: 0.15, spin-mix in the dark for 2h, centrifuge at 15000rpm for 10min, then remove the supernatant, add 1mL blocking buffer (containing BSA concentration of 12g/L), spin-mix for 1h, wash 3 times with 0.02M PBS pH 7.4 (containing 2g/L BSA and 1g/L trehalose), re-dissolve the labeled product in 100. mu.l phosphate buffer PBS 0.02M pH 7.4 (containing 2g/L BSA and 1g/L trehalose), put at 2 ℃ -8 ℃ for use;
the modification scheme of the AIE nano microsphere labeled goat anti-rabbit IgG antibody is as above, wherein the goat anti-rabbit IgG antibody is added, and the mass ratio of the added AIE nano microsphere to the added goat anti-rabbit IgG antibody is 1: 0.1;
(2) bond pad preparation
The conjugate pad was soaked with 0.02M Tris-HCl pH 7.4 (1 g/L trehalose, 2g/L bovine serum albumin, 0.2g/L LTween-20) for 2h and baked in an oven at 50 ℃ for 16 h. Diluting the 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 marked by the AIE nano microspheres and the goat anti-rabbit IgG antibody marked by the AIE nano microspheres according to the dilution concentration, wherein the gold spraying concentration of a solution of the 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 marked by the AIE nano microspheres is 50g/L, and the gold spraying concentration of a solution of the goat anti-rabbit IgG antibody marked by the AIE nano microspheres is 30 g/L. After mixing, spraying a pad by using a film-scratching gold spraying instrument with the parameter of 3 mu L/cm, putting the pad in a drying oven at 40 ℃ for drying for 20 hours, and sealing the pad for later use after drying;
(3) preparation of coating film
Diluting the N protein antibody COV19-PS-MAb2 of the 2019-nCoV novel coronavirus and the rabbit IgG respectively by using PBS (containing 2g/L BSA and 5g/L trehalose) with the pH of 7.4 and 0.02M, and then carrying out scribing, wherein the detection line is the N protein antibody COV19-PS-MAb2 of the 2019-nCoV novel coronavirus, the scribing concentration is 2mg/mL, and the scribing dosage is 1 muL/cm; the control line is rabbit IgG, the streaking concentration is 1.5mg/mL, and the streaking dosage is 1 muL/cm. After finishing, placing the coating film in a drying oven at 40 ℃ for drying for 20h, and sealing for later use after drying;
(4) test card assembly
As shown in fig. 1, the dried coating film, the bonding pad, the sample pad and the absorbent paper were attached to the base plate. After the assembly is finished, the test strip is cut into test strips with the width of 4mm by a slitter, the test strips are arranged in the detection card shown in figure 2 according to the direction that the sample pads are aligned with the sample adding holes, 8 sample adding holes correspond to the sample pads, and 9 detection windows correspond to the detection line T and the quality control line C area on the NC film. The detection card is put into an aluminum foil bag containing a drying agent, sealed and stored at room temperature (10-30 ℃).
The same procedure as in step (4) of example 1.
Example 3
Preparation of 2019-nCoV novel coronavirus antigen detection card based on AIE nano-microspheres
(1) Preparation of AIE nano microsphere labeled antibody solution
Taking 100 μ L (10 g/L concentration) of AIE nano microspheres with 100nm particle size and carboxyl, adding appropriate amount of MES buffer solution, cleaning, centrifuging at 15000rpm for 10min in a centrifuge, and redissolving in MES buffer solution. Adding activators EDC and NHS, wherein the mass ratio of the added AIE nano microspheres to the added activators EDC is 1:5, the mass ratio of the added AIE nano microspheres to the added activators NHS is 1:20, rotationally mixing the mixture for 1h in the dark, centrifuging the mixture for 10min at 15000rpm in a centrifuge, redissolving the mixture in 2- (N-morpholine) ethanesulfonic acid (MES) buffer solution, adding a 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 for coupling, adding the AIE nano microspheres and adding a 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1, wherein the mass ratio of the added AIE nano microspheres to the added 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 is 1: 0.24, spin mix in the dark for 4h, centrifuge at 15000rpm for 10min, then remove the supernatant, add 1mL blocking buffer (containing 20g/L BSA concentration), spin mix for 2h, wash 3 times with 0.02M phosphate buffer PBS pH 7.4 (containing 2g/L BSA and 1g/L trehalose), re-dissolve the labeled product in 100. mu.L 0.02M PBS pH 7.4 (containing 2g/L BSA and 1g/L trehalose), put at 2 ℃ -8 ℃ for use;
the modification scheme of the AIE nano microsphere labeled goat anti-rabbit IgG antibody is as above, wherein a goat anti-IgG rabbit antibody is added, and the mass ratio of the added AIE nano microsphere to the added goat anti-rabbit IgG antibody is 1: 0.2;
(2) bond pad preparation
The conjugate pad was soaked with 0.02M Tris-HCl pH 7.4 (1 g/L trehalose, 2g/L bovine serum albumin, 0.2g/L LTween-20) for 2h and baked in an oven at 50 ℃ for 16 h. Diluting the 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 marked by the AIE nano microspheres and the goat anti-rabbit IgG antibody marked by the AIE nano microspheres according to the dilution concentration, wherein the gold spraying concentration of a solution of the 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 marked by the AIE nano microspheres is 100g/L, and the gold spraying concentration of a solution of the goat anti-rabbit IgG antibody marked by the AIE nano microspheres is 50 g/L. After mixing, spraying a pad by using a film-scratching metal spraying instrument with the parameter of 5 mu L/cm, putting the pad in a 50 ℃ oven for drying for 24 hours, and sealing the pad for later use after drying;
(3) preparation of coating film
Diluting the N protein antibody COV19-PS-MAb2 of the 2019-nCoV novel coronavirus and the rabbit IgG respectively by using PBS (containing 2g/L BSA and 5g/L trehalose) with the pH of 7.4 and the pH of 0.02M, and then carrying out scribing, wherein the detection line is the N protein antibody COV19-PS-MAb2 of the 2019-nCoV novel coronavirus, the scribing concentration is 3mg/mL, and the scribing dosage is 2 mu L/cm; the control line is rabbit IgG, the streaking concentration is 2.4mg/mL, and the streaking dosage is 2 muL/cm. After finishing, placing the coating film in a drying oven at 50 ℃ for drying for 24 hours, and sealing for later use after drying;
(4) test card assembly
As shown in fig. 1, the dried coating film, the bonding pad, the sample pad and the absorbent paper were attached to the base plate. After the assembly is finished, the test strip is cut into test strips with the width of 4mm by a slitter, the test strips are arranged in the detection card shown in figure 2 according to the direction that the sample pads are aligned with the sample adding holes, 8 sample adding holes correspond to the sample pads, and 9 detection windows correspond to the detection line T and the quality control line C area on the NC film. The detection card is put into an aluminum foil bag containing a drying agent, sealed and stored at room temperature (10-30 ℃).
The same procedure as in step (4) of example 1.
Example 4
Detection limit test of 2019-nCoV novel coronavirus antigen detection reagent of AIE nano-microspheres
The detection cards prepared in example 1 were tested with 2019-nCoV novel coronavirus antigens to be tested at different concentrations, respectively, and the data are shown in Table 1.
TABLE 1 detection Limit test
According to tests, the detection is rapid, the result is obtained quickly within 10-15min, the detection card prepared in the embodiment 1 of the invention still shows a positive judgment result when the addition concentration of the 2019-nCoV novel coronavirus antigen to be detected is 10pg/mL, and the detection sensitivity is high.
Example 5
24 negative throat swab samples were tested using the 2019-nCoV novel coronavirus antigen detection card based on AIE nanospheres prepared in example 3, and the results are shown in Table 2.
TABLE 2
As can be seen from Table 2, the 24 negative pharyngeal swab samples all showed negative test results and high detection accuracy.
Finally, it should be noted that: the above embodiments are only used for illustrating the technical solution of the present invention, and not for limiting the same; while the invention has been described in detail with reference to the foregoing examples, those skilled in the art will appreciate that: the technical solutions described in the foregoing embodiments may be modified, or some or all of the technical features may be equally replaced; and the modifications and the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The reagent for detecting the 2019-nCoV novel coronavirus antigen of the AIE nano microspheres is characterized by comprising a detection antibody marked by the AIE nano microspheres, wherein the detection reagent comprises a 2019-nCoV novel coronavirus antigen detection card of the AIE nano microspheres and a virus extracting solution, and the detection antibody adopts a 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 and a goat anti-rabbit IgG antibody.
2. The reagent of claim 1, wherein the 2019-nCoV novel coronavirus antigen detection reagent of AIE nanospheres is a monoclonal antibody, and the 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 is a monoclonal antibody.
3. The reagent of claim 1, wherein the AIE nanospheres are AIE molecules coated with polystyrene.
4. The reagent of claim 1, wherein the reagent is capable of detecting 2019-nCoV coronavirus antigens with a concentration of at least 10 pg/mL.
5. The reagent of claim 1, wherein the virus extract is used to extract virus from a sample for detection.
6. The reagent of claim 1, wherein the detection card comprises a base plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorbent paper, which are overlapped.
7. The reagent of claim 1, wherein the detection card comprises a detection line and a quality control line.
8. The reagent of claim 1, wherein the detection card comprises a detection line coated with 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb2 and a quality control line coated with rabbit IgG.
9. A preparation method of 2019-nCoV novel coronavirus antigen detection reagent of AIE nano microspheres is characterized in that the preparation of a detection card in the detection reagent comprises the following steps:
s1, preparation of an AIE nano microsphere labeled antibody solution: activating AIE nano microspheres by adopting EDC and NHS, wherein the mass ratio of the AIE nano microspheres to EDC and NHS is 1 (0.1-5) and 1 (0.2-20), the activation time is 0.5h-1h, after the activation is finished, adding an antibody for coupling, wherein the mass ratio of the AIE nano microspheres to the antibody is 1 (0.02-0.24), the coupling time is 1h-4h, after the coupling is finished, adding a BSA solution for blocking, wherein the concentration of the BSA solution is 5g/L-20g/L, and the blocking reaction time is 0.5h-2 h;
s2, preparing a bonding pad: spraying the prepared AIE nano microsphere marked antibody on a bonding pad, wherein the gold spraying concentration of the AIE nano microsphere marked 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb1 solution is 20g/L-100g/L, the gold spraying concentration of the AIE nano microsphere marked goat anti-rabbit IgG antibody solution is 10g/L-50g/L, uniformly mixing, spraying the pad parameters which are 1 muL/cm-5 muL/cm for film spraying, and after the film spraying is finished, putting the pad in an oven at 30-50 ℃ for drying for 16-24 h;
s3, preparation of a coating film: coating 2019-nCoV novel coronavirus N protein antibody COV19-PS-MAb2 at a detection line T line position of a nitrocellulose membrane, wherein the scribing concentration is 0.5-3 mg/mL, the scribing dosage is 0.5-2 muL/cm, coating rabbit IgG at a quality control line C line position, the scribing concentration is 0.5-2.4 mg/mL, the scribing dosage is 0.5-2 muL/cm, and after completion, placing the coated rabbit IgG in an oven at the temperature of 30-50 ℃ for baking for 16-24 hours;
s4, assembling a detection card: the combined pad, the sample pad and the absorbent paper are sequentially lapped on the bottom plate stuck with the nitrocellulose membrane to be assembled into a large plate, and the large plate is cut into detection cards with the width of 4mm by a slitter.
10. A2019-nCoV novel coronavirus antigen detection reagent based on AIE nano-microspheres can be used for detecting nasopharyngeal swabs, sputum, blood, saliva, urine, anal swabs, alveolar lavage fluid and samples collected in the environment.
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