Immunochromatography test strip for quantitatively detecting monkey pox virus based on fluorescent latex microspheres

Abstract

The invention provides an immunochromatographic test strip for quantitatively detecting monkey pox virus based on fluorescent latex microspheres, which takes europium chelate Eu (DBM) 3Phen-loaded monodisperse carboxylated polystyrene copolymer microspheres as a signal substance, and the microspheres are subjected to acid-base treatment before loading to loosen the surface structure of the microspheres so as to improve the loading capacity; the surface compound of the test strip is prepared by coupling carboxyl on the surface of the microsphere with an antibody. The invention also discloses a preparation method of the test strip. The kit is convenient for rapidly and accurately detecting the monkey pox virus, and has the characteristics of simplicity, rapidness, no toxicity and low cost.

Description

Technical Field

[0001] The invention belongs to the technical field of immunochromatographic analysis, and in particular relates to an immunochromatographic test strip for quantitatively detecting monkeypox virus based on fluorescent latex microspheres and a preparation method thereof. Background Art

[0002] Monkeypox virus (MPXV) is a species of orthopoxvirus in the family of monkeypoxviridae, which was first discovered in monkeys in 1958 and used for scientific research. However, since May 2022, the number of monkeypox cases worldwide has increased dramatically, ultimately prompting the World Health Organization (WHO) to declare the monkeypox outbreak a global emergency. MPXV is a double-stranded DNA virus with two infectious forms, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV). MPXV binds to host cells through the A29L (heparin-binding IMV surface membrane fusion protein) and E8L (chondroitin sulfate-binding IMV surface membrane adsorption protein) proteins. Monkeypox virus infection produces the zoonotic disease monkeypox (MPX). The main transmission routes of MPXV are animal-to-human transmission and human-to-human transmission. The transmission route from animals to humans mainly occurs through direct contact with diseased blood, body fluids and infected sites of infected animals. Human-to-human transmission is mainly through contact with infected sites and respiratory droplets of infected patients. Human infection with MPX usually begins with fever, fatigue, and headache, followed by a rash that can spread to other parts of the body, with an incubation period of 5 to 21 days. The early symptoms of MPXV infection are similar to those of respiratory viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A (FluA), influenza B (FluB), and human adenovirus (HAV), and the long incubation period increases the risk of outbreaks. At the same time, the clinical manifestations of MPXV infection are not significantly different from those of other diseases.

[0003] Therefore, it is necessary to develop targeted methods for quantitative detection of monkeypox virus to facilitate rapid and accurate detection of monkeypox virus. Summary of the invention

[0004] Purpose of the invention: In view of the shortcomings and defects of the prior art, the present invention provides an immunochromatographic test strip for quantitative detection of monkeypox virus based on fluorescent latex microspheres and a preparation method thereof, which is convenient for rapid and accurate detection of monkeypox virus and has the characteristics of being simple, fast, non-toxic and low-cost.

[0005] Technical solution: The present invention discloses an immunochromatographic test strip for quantitatively detecting monkeypox virus based on fluorescent latex microspheres, characterized in that: the test strip uses monodisperse carboxylated polystyrene copolymer microspheres loaded with europium chelate Eu(DBM)3Phen as signal substances, and the microspheres are treated with acid and alkali before loading to make their surface structure loose so as to improve the loading capacity; the surface complex of the test strip is prepared by coupling the carboxyl groups on the surface of the microspheres with antibodies.

[0006] Among them, the test strip is a sandwich immunosensor that uses fluorescent latex microspheres as tracer markers, forming a quantitative detection of monkeypox virus.

[0007] The method for preparing an immunochromatographic test strip for quantitatively detecting monkeypox virus based on fluorescent latex microspheres of the present invention is characterized in that: a fluorescent dye is mixed with a latex microsphere solution, a certain amount of dichloromethane solution is added to dissolve the fluorescent substance, and then the solution is added to a latex solution with a mass fraction of 1%, reacted at room temperature for 12 hours, dichloromethane is removed by reduced pressure distillation using a rotary evaporator, and then excess dye is removed by centrifugation to obtain the fluorescent latex microspheres.

[0008] The fluorescent dye is europium chelate Eu(DBM)3Phen, and the latex microspheres are polystyrene-methacrylic acid, polystyrene-acrylic acid, and polystyrene maleic anhydride porous latex microspheres.

[0009] The method for preparing the immunochromatographic test strip for quantitatively detecting monkeypox virus based on fluorescent latex microspheres of the present invention is characterized in that it comprises the following steps:

[0010] 1) Add styrene, methacrylic acid and ammonium persulfate to water under mechanical stirring, the mass ratio of styrene, methacrylic acid and ammonium persulfate is 7-10:1-3:1, preheat at 120° C. for 30 min and then heat to react for 3 h to obtain latex microspheres;

[0011] 2) diluting the latex microsphere solution to 100 mL, adding 5 ml of 2-butanone and 0.1 g of sodium dodecylbenzene sulfonate to the system; adjusting the pH value of the latex to 12.2 with a 2.5 mol / L sodium hydroxide aqueous solution; immersing the flask in a 90° C. water bath and stirring at a speed of 450 rpm / min for 3 h to obtain alkali-treated latex particles; cooling the alkali-treated latex to room temperature, adjusting the pH value to 2.2 with a 2.5 mol / L hydrochloric acid aqueous solution, and performing an acid treatment at 90° C. for 3 h; cooling the latex to room temperature, adjusting the pH to 8-9 with a 2.5 mol / L sodium hydroxide aqueous solution, and obtaining porous latex microspheres;

[0012] 3) The porous latex microspheres were diluted to a mass fraction of 1%, and after adding 0.25% of sodium dodecylbenzene sulfonate, 1 mL of Eu(DBM)3Phen dichloride solution was added. After ultrasonication for 20 min, stirring was performed at room temperature for 8 h. The fluorescent porous latex microspheres were obtained by vacuum distillation at 40°C for 15 min to remove the swelling agent.

[0013] Wherein, the particle size of the fluorescent porous latex microspheres in step 3) is 200nm-300nm.

[0014] Fluorescent porous latex microsphere-monkeypox antibody immune complex is formed on the surface of the test strip of the invention.

[0015] When preparing the complex, the carboxyl groups on the surface of the microspheres are first activated by using EDC and NHS, then monkeypox antibodies are added for coupling, and then bovine serum albumin is added for blocking to obtain fluorescent latex microspheres-monkeypox antibody immune complexes.

[0016] The test strip includes a sample pad, an NC membrane and a water absorbent pad arranged in sequence along the chromatography direction.

[0017] The method for preparing the immunochromatographic test strip for quantitatively detecting monkeypox virus based on fluorescent latex microspheres of the present invention is characterized in that it comprises the following steps:

[0018] 1) Preparation of NC membrane: 0.04 mg / cm 2 -0.07mg / cm 2 The concentration of monkeypox capture antibody is 0.04 mg / cm2 for line T and 0.04 mg / cm2 for line C. 2 -0.07mg / cm 2 ;

[0019] 2) Assembly of the test strip: The sample pad, NC membrane and absorbent pad are laid in sequence along the chromatography direction on the PVC bottom plate, and a part of the sample pad is overlapped on the NC membrane, and a part of the absorbent pad is overlapped on the other side of the NC membrane to prepare the test strip.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The preparation method of the present invention is simple to operate, and monodisperse polystyrene microspheres with large loading, strong fluorescence performance and good uniformity can be obtained without copolymerizing other monomers. The surface of the microspheres obtained by the present invention is rich in carboxyl groups, and no additional method is required to introduce carboxyl groups. It can be directly coupled to antibodies and used in biological detection, achieving the purpose of sensitive, rapid and simple clinical monitoring and quantitative analysis. It is convenient for rapid and accurate detection of monkeypox virus, and has the characteristics of simplicity, speed, non-toxicity and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a flow chart for the preparation of monodisperse carboxylated polystyrene microspheres;

[0022] Figure 2 Flow chart of the use of fluorescent latex microspheres for monkeypox virus detection;

[0023] Figure 3 TEM image of monodispersed carboxylated polystyrene microspheres;

[0024] Figure 4 is the TEM image of the microspheres after alkali treatment;

[0025] Figure 5It is the fluorescence spectrum of fluorescent latex microspheres; the abscissa is the wavelength, and the ordinate is the fluorescence intensity;

[0026] Figure 6 The infrared spectra of monodisperse carboxylated polystyrene microspheres and fluorescent latex microspheres; the abscissa is the wave number and the ordinate is the transmittance;

[0027] Figure 7 This is a graph showing the results of the flow-through immunochromatographic test strips of fluorescent latex microspheres testing different concentrations of monkeypox virus;

[0028] Figure 8 This is a linear response detection diagram of the fluorescent latex microsphere flow-through immunochromatographic test strip for different concentrations of monkeypox virus detection; the abscissa is the logarithm of the monkeypox virus concentration, and the ordinate is the fluorescence intensity of the T line. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods.

[0030] The present invention provides an immunochromatographic test strip for quantitatively detecting monkeypox virus based on fluorescent latex microspheres and a preparation method thereof, which specifically comprises the following parts:

[0031] Part I: The preparation method provided by the present invention comprises the following steps:

[0032] 1) using a boiling system soap-free emulsion polymerization method to carry out emulsion polymerization of styrene and carboxyl functional monomers to obtain a carboxyl polystyrene microsphere latex solution;

[0033] 2) treating the obtained latex solution with alkali to obtain carboxyl polystyrene latex microspheres with rough surfaces;

[0034] 3) The europium complex is embedded into the polystyrene microspheres by a swelling method to obtain the fluorescent dye latex microspheres.

[0035] The invention prepares carboxyl polystyrene microsphere seed emulsion by soap-free emulsion polymerization of copolymers such as styrene and methacrylic acid, embeds europium rare earth complex fluorescent dye in polymer nanoparticles by swelling method, and obtains polystyrene fluorescent microspheres with spherical structure.

[0036] Among them, carboxyl polystyrene microspheres are prepared by soap-free emulsion polymerization in a boiling system. When hydrophobic monomers such as styrene are polymerized, the initiation rate of the monomer is faster than the growth rate of the polymer chain, resulting in the generation of a large number of oligomer free radicals. The free radicals continue to grow. When their degree of polymerization reaches a certain critical concentration, they will condense to form primary particles. The primary particles absorb the oligomers and monomers and gradually form a polystyrene latex microsphere seed solution.

[0037] The purpose of the boiling system is that under boiling conditions, there are almost no oxygen free radicals in the reaction water phase, so no inert gas protection is required during the entire reaction process. In addition, compared with the reaction temperature of 60-80°C for traditional soap-free emulsion polymerization, the higher temperature of the boiling system helps to increase the reaction rate. Traditional soap-free emulsion polymerization usually requires a reaction time of 7-12 hours, while the boiling reaction system soap-free emulsion polymerization only takes 2-3 hours.

[0038] When the microspheres are treated with alkali, the shell of the microspheres is mainly composed of -(MAA)m-(St)n chains, and m is much larger than n. During the alkali treatment, as the pH value increases, most of the carboxyl groups are converted to -COO-. The ionization of the carboxyl groups destroys the hydrogen bonds between the macromolecular chains of the microspheres, which causes the carboxyl-rich polymer chains to extend into the water phase, the nanospheres swell, and the surface becomes rough. This is conducive to more fluorescent molecules migrating into the nanospheres during the subsequent swelling and coating process of the fluorescent dye.

[0039] The swelling method is mainly based on the hydrophobic interaction between the europium complex as a fluorescent dye and the carbon-oxygen structure inside the microspheres. The europium complex fluorescent dye is embedded in the microspheres. When the swelling agent is removed, the volume of the microspheres shrinks rapidly, and the pores on the surface of the microspheres will also decrease accordingly, thereby embedding the fluorescent dye inside the microspheres.

[0040] Furthermore, in step 1), styrene, methacrylic acid monomer and water are first mixed, heated until the system boils for a period of time, and then the initiator is added to the reaction system to dissolve, and an emulsion polymerization reaction is carried out to obtain the seed emulsion of the carboxyl polystyrene microspheres; step 1) needs to be carried out under boiling conditions of the solution system.

[0041] The boiling time is 15 min-30 min; the boiling temperature is 120° C.; the emulsion polymerization reaction time is 2 h-4 h, such as 2 h, 2.5 h, 3 h, 3.5 h, 4 h, etc.; the total mass of styrene is 100%, and the mass percentage of the initiator is 0.6%-1%, such as 0.6%, 0.8%, 1.0%; the volume ratio of styrene to the carboxyl functional monomer is 7:(0.1-0.6), wherein 0.1-0.6 can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, etc.; the carboxyl functional monomer includes acrylic acid, methacrylic acid, maleic anhydride, etc.; the initiator includes an aqueous phase initiator; the initiator includes any one of potassium sulfate, potassium persulfate or sodium thiosulfate or a combination of at least two thereof, wherein typical but non-limiting combinations include: a combination of potassium sulfate and potassium persulfate, a combination of potassium persulfate and sodium thiosulfate, a combination of potassium sulfate, potassium persulfate and sodium thiosulfate, etc.

[0042] As a preferred embodiment, step 1) specifically includes the following steps: adding styrene and methacrylic acid into water at once for dispersion, mixing evenly, raising the temperature until the solution boils, maintaining the temperature for 15 minutes, adding the initiator into the reaction system, and continuing heating for 2 hours to 4 hours to obtain the seed solution of the monodisperse carboxylated polystyrene microspheres.

[0043] Furthermore, in step 2), the obtained seed solution is subjected to alkali treatment to obtain a rough surface carboxyl polystyrene latex microsphere solution. The alkali treatment reaction should be carried out under uniform stirring; the alkali treatment reaction time is 2h-6h, such as 2h, 3h, 4h, 5h, 6h, etc.; the alkali treatment reaction pH is 12-13; and the alkali treatment reaction also includes washing.

[0044] As a preferred embodiment, in step 2), the seed solution in step 1) is redispersed in water to obtain a latex solution with a mass fraction of 1%, the pH value of the solution is adjusted to 13, the temperature is increased to 90°C, and maintained for 2 hours to obtain a polystyrene latex microsphere solution with a rough surface.

[0045] Further, in step 3), the obtained rough surface carboxyl polystyrene latex microsphere solution and the europium complex solution are mixed to perform a swelling reaction to obtain the fluorescent latex microsphere. Wherein, the swelling reaction is carried out under light protection and uniform stirring; the stirring time is 12h-24h, such as 12h, 14h, 16h, 18h, 20h, 22h, 24h, etc.; the reaction temperature is at room temperature, i.e., 18°C-25°C; the stirring rate of the swelling reaction is 300rpm-450rpm, such as 300rpm, 350rpm, 400rpm, 450rpm, etc.; the swelling reaction includes subsequent solvent removal and excess dye removal; the swelling agent includes any one of tetrahydrofuran, dichloromethane, chloroform, acetone, and toluene, or a combination of at least two; wherein typical but non-limiting groups include: a combination of tetrahydrofuran, dichloromethane and chloroform, and a combination of chloroform, acetone, and toluene.

[0046] As a preferred embodiment, step 3) specifically includes the following steps: mixing the europium complex solution and the seed solution of rough-surfaced carboxyl polystyrene microspheres, performing a swelling reaction for 12h-24h at a stirring rate of 300rpm-500rpm and a temperature of 20℃-40℃ under light-proof conditions, and then washing by rotary evaporation and centrifugation to obtain the fluorescent latex particles.

[0047] Part II: The present invention provides the use of monodisperse carboxylated fluorescent microspheres in monkeypox detection, comprising the following steps:

[0048] 1) coupling the carboxylated fluorescent microspheres to the monkeypox conjugated antibody;

[0049] 2) Construct a monkeypox detection immunoassay system and obtain test strips.

[0050] Furthermore, in step 1), the obtained fluorescent microsphere solution is redispersed in a PBS solution, and then EDC, NHS and monkeypox conjugated antibody are added. After incubation at 37°C, the obtained conjugate is centrifuged and redispersed in a PBS solution containing bovine serum albumin, sucrose, Tween 20 and sodium chloride, and stored at 4°C.

[0051] The mass fraction of the fluorescent microspheres is 0.5%-1.5%, and the added amount is 100μL-200μL; the concentration of the PBS solution is 0.1mM / L, and the pH is 7.4; the concentration of EDC and NHS is 2mg / mL-6mg / mL, and the added amount is 20μL-50μL; the mass of the monkeypox conjugated antibody is 150μg and 300μg; the mass fraction of bovine serum albumin is 0.5%-1.5%; the mass fraction of sucrose is 0.5%-1.5%; the mass fraction of Tween 20 is 0.15%-0.35%; the mass fraction of sodium chloride is 0.5%-1.5%; and the storage time is 0.5h-2.5h.

[0052] Furthermore, in step 2), the sample pad needs to undergo certain pre-treatment steps before use. Use a streaking pen to apply monkeypox capture antibody and goat anti-mouse IgG antibody to the T line and C line of the NC membrane, respectively, and dry at 37°C. The prepared fluorescent microsphere antibody conjugate and monkeypox antigen of different concentrations are mixed and dripped onto the sample pad. After waiting for a while, the fluorescent image of the test strip is taken under 365nm ultraviolet light with an Apple mobile phone, and the fluorescence intensity of the T line and C line is analyzed using ImageJ software.

[0053] The pretreatment steps are as follows: immersing the sample pad in a PBS solution with a mass fraction of 0.5%-1.5% bovine serum albumin, a mass fraction of 0.5%-1.5% sucrose, a mass fraction of 0.15%-0.35% Tween 20, and a mass fraction of 0.5%-1.5% sodium chloride for 15 minutes, then taking it out and drying it at room temperature; the concentrations of monkeypox capture antibody and goat anti-mouse IgG antibody are 1.5 mg / mL-2.5 mg / mL; the concentration of monkeypox antigen is 0-500 ng / mL; and the waiting time is 5 minutes to 25 minutes.

[0054] Example:

[0055] The main reagents and instruments of this embodiment are as follows: methacrylic acid, potassium persulfate, sodium dodecyl sulfate, sodium hydroxide, sodium dihydrogen phosphate dihydrate, sodium hydrogen phosphate dodecahydrate, Tween 20, and dichloromethane were purchased from Sinopharm Chemical Reagent Co., Ltd. Bovine serum albumin, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysulfosuccinimide, and 2-morpholineethanesulfonic acid (MES) were purchased from Shenggong Biotechnology Co., Ltd. Styrene and tri(dibenzoylmethane)mono(phenanthroline)europium, sucrose, and maleic anhydride were purchased from Shanghai Aladdin Co., Ltd. The particle size distribution and zeta potential of the microspheres were measured using a dynamic light scattering instrument (MarvernZetasizer). Fluorescence spectra were performed on a fluorescence spectrophotometer (FluoroMax-4). The chemical composition transformation of different components was observed using a Fourier transform infrared spectrometer (Nicolet 5700). The content of carboxyl groups on the surface of the microspheres was measured using a conductivity meter (DLS-11A). The changes in the microsphere morphology were observed using a high-resolution transmission electron microscope (Talos F200X).

[0056] 1. Preparation of carboxylated styrene microspheres by high temperature soap-free emulsion polymerization:

[0057] 1) High temperature soap-free emulsion polymerization was used to quickly prepare styrene maleic anhydride copolymer microspheres PSMA. Styrene needs to be cleaned with 10% sodium hydroxide before use to remove the stabilizer in the reagent. Purified styrene and 30 mL of deionized water were added to a three-necked flask, and a certain amount of maleic anhydride was weighed and added to the flask. The mixture was mechanically stirred at a speed of 300 rpm / min, and the temperature was raised to the boiling point of the solution and maintained for 15 minutes to remove the dissolved oxygen in the solution. After the dissolved oxygen in the solution was basically removed, potassium persulfate was added to initiate the polymerization reaction, and mechanical stirring was continued for 3 hours until the reaction was completed.

[0058] 2) PSMA cleaning: The obtained emulsion was centrifuged at 10000 rpm for 30 min, and the supernatant was removed and then washed three times with ethanol and deionized water to obtain pure PSMA microspheres.

[0059] 2. Alkali treatment and swelling of PSMA microspheres:

[0060] After adding 0.25% SDS to 10mL of the prepared microspheres, the microspheres were mixed with 1mL of dichloromethane solution of europium complexes of different concentrations. After ultrasonic treatment for 20 minutes, the solution was placed in a single-necked flask and reacted at 50°C for 24 hours. The dichloromethane was removed by heating at 40°C for 20 minutes using a rotary evaporation method, and the microspheres were washed three times with deionized water and redispersed to obtain the corresponding PSMA microspheres with fluorescent properties.

[0061] 3. Conjugation of PSMA microspheres:

[0062] 1) Using hydrochloric acid to adjust the pH value of the PSMA microsphere solution to 1, after 12 hours of magnetic stirring, washing with an equal volume of a mixed solution of deionized water and ethanol to obtain surface carboxylation polystyrene microspheres;

[0063] 2) 100 μl of 1% PSMA solution was redispersed in PBS solution (10 mM, pH = 7.4), and EDC (5 mg / mL), NHS (5 mg / mL) and 200 μg of monkeypox conjugated antibody were added. After incubation at 37°C for 2 h, the resulting conjugate was centrifuged and redispersed in PBS solution containing bovine serum albumin (1%), sucrose (0.5%), Tween 20 (0.5%) and sodium chloride (1%), and stored at 4°C.

[0064] 4. Preparation of Monkeypox Immunochromatographic Test Strips:

[0065] 1) Sample pad pretreatment: Cut the sample pad into 8 mm strips and soak it in a PBS solution of bovine serum albumin (1%), sucrose (0.5%), Tween 20 (0.5%) and sodium chloride (1%) for 15 min, then take it out and dry it at 37°C for later use;

[0066] 2) Preparation of NC membrane: C line is coated with goat anti-mouse antibody, and T line is coated with monkeypox capture antibody; 2 mg / mL goat anti-mouse antibody is evenly streaked on nitrocellulose membrane to obtain C line, and 2 mg / mL monkeypox capture antibody is evenly streaked on nitrocellulose membrane to obtain T line. Place in a 37°C oven to dry for 2 hours and set aside;

[0067] 3) Preparation of monkeypox detection immunochromatographic test strips: Lay the black PVC base plate, the cellulose membrane, the sample pad and the absorbent pad together. Paste the scribed nitrocellulose membrane in the middle of the black base plate, and then paste the sample pad and the absorbent pad at both ends of the black base plate, respectively. The nitrocellulose membrane and the sample pad and the nitrocellulose membrane and the absorbent pad overlap by 1mm-2mm. Use 4% bovine serum albumin blocking solution to block the nonspecific adsorption sites on the nitrocellulose membrane. After drying, use a cutting machine to cut the assembled black base plate into strips with a width of 3mm to obtain the fluorescent immunoassay strips for detecting monkeypox components. Store in a 4°C refrigerator for future use.

[0068] 5. Detection of monkeypox virus by monkeypox immunochromatographic test strips:

[0069] 1) Preparation of different concentrations of monkeypox antigen: Use PBS buffer solution to prepare monkeypox antigen solutions of 500 ng / mL; 100 ng / mL; 50 ng / mL; 10 ng / mL; 1 ng / mL; 0;

[0070] 2) Take 5 μL of the PSMA antibody complex obtained above and mix it with 30 μL of monkeypox antigen solution, incubate at room temperature for 20 minutes, drop the mixture onto the sample point, and chromatograph the solution on the nitrocellulose membrane under the capillary action of the absorbent pad for 15 minutes, and observe the experimental results under a 365nm ultraviolet lamp. The specific experimental results are as follows Figure 6 As shown in the figure, when the monkeypox antigen is 500ng / mL, the T line has a brighter red color because of its high concentration. However, when the concentration drops to 1ng / mL or below, the brightness of the T line is very weak and it is basically impossible to observe its brightness. Through the above experimental phenomena, it can be found that this method is feasible for monkeypox detection.

[0071] The present invention has the following significant advantages: the preparation method of the present invention is simple to operate, and monodisperse polystyrene microspheres with large loading, strong fluorescence performance and good uniformity can be obtained without copolymerizing other monomers. The surface of the microspheres obtained by the present invention is rich in carboxyl groups, and no additional method is required to introduce carboxyl groups. The microspheres can be directly coupled to antibodies and used in biological detection, achieving the purpose of sensitive, rapid and simple clinical monitoring and quantitative analysis. It is convenient for rapid and accurate detection of monkeypox virus, and has the characteristics of being simple, fast, non-toxic and low-cost.

Claims

1. An immunochromatographic test strip for quantitative detection of monkeypox virus based on fluorescent latex microspheres, characterized in that: The test strip uses monodisperse carboxylated polystyrene copolymer microspheres loaded with europium chelate Eu(DBM)3Phen as signal substances. The microspheres are treated with acid and alkali before loading to make their surface structure loose so as to improve the loading capacity. The surface complex of the test strip is prepared by coupling the carboxyl groups on the surface of the microspheres with antibodies.

2. The immunochromatographic test strip for quantitative detection of monkeypox virus based on fluorescent latex microspheres according to claim 1, characterized in that: The test strip is a sandwich immunosensor that uses fluorescent latex microspheres as tracer markers, forming a quantitative detection of monkeypox virus.

3. The method for preparing an immunochromatographic test strip for quantitative detection of monkeypox virus based on fluorescent latex microspheres according to claim 1 or 2, characterized in that: The fluorescent dye was mixed with the latex microsphere solution, and a certain amount of dichloromethane solution was added to dissolve the fluorescent substance, and then added to a latex solution with a mass fraction of 1%, and reacted at room temperature for 12 hours. The dichloromethane was removed by reduced pressure distillation using a rotary evaporator, and then the excess dye was removed by centrifugation to obtain fluorescent latex microspheres.

4. The method for preparing an immunochromatographic test strip for quantitative detection of monkeypox virus based on fluorescent latex microspheres according to claim 3, characterized in that: The fluorescent dye is europium chelate Eu(DBM)3Phen, and the latex microspheres are polystyrene-methacrylic acid, polystyrene-acrylic acid, and polystyrene maleic anhydride porous latex microspheres.

5. The method for preparing the immunochromatographic test strip for quantitative detection of monkeypox virus based on fluorescent latex microspheres according to claim 3, characterized in that: The steps include: 1) Add styrene, methacrylic acid and ammonium persulfate to water under mechanical stirring, the mass ratio of styrene, methacrylic acid and ammonium persulfate is 7-10:1-3:1, preheat at 120° C. for 30 min and then heat to react for 3 h to obtain latex microspheres; 2) diluting the latex microsphere solution to 100 mL, adding 5 ml of 2-butanone and 0.1 g of sodium dodecylbenzene sulfonate to the system; adjusting the pH value of the latex to 12.2 with a 2.5 mol / L sodium hydroxide aqueous solution; immersing the flask in a 90° C. water bath and stirring at a speed of 450 rpm / min for 3 h to obtain alkali-treated latex particles; cooling the alkali-treated latex to room temperature, adjusting the pH value to 2.2 with a 2.5 mol / L hydrochloric acid aqueous solution, and performing an acid treatment at 90° C. for 3 h; cooling the latex to room temperature, adjusting the pH to 8-9 with a 2.5 mol / L sodium hydroxide aqueous solution, and obtaining porous latex microspheres; 3) The porous latex microspheres were diluted to a mass fraction of 1%, and after adding 0.25% of sodium dodecylbenzene sulfonate, 1 mL of Eu(DBM)3Phen dichloride solution was added. After ultrasonication for 20 min, stirring was performed at room temperature for 8 h. The fluorescent porous latex microspheres were obtained by vacuum distillation at 40°C for 15 min to remove the swelling agent.

6. The method for preparing an immunochromatographic test strip for quantitative detection of monkeypox virus based on fluorescent latex microspheres according to claim 3, characterized in that: The particle size of the fluorescent porous latex microspheres in step 3) is 200nm-300nm.

7. The immunochromatographic test strip for quantitative detection of monkeypox virus based on fluorescent latex microspheres according to claim 1 or 2, characterized in that: Fluorescent porous latex microspheres-monkeypox antibody immune complexes are formed on the surface of the test strip.

8. The immunochromatographic test strip for quantitative detection of monkeypox virus based on fluorescent latex microspheres according to claim 7, characterized in that: When the complex is prepared, EDC and NHS are first used to activate the carboxyl groups on the surface of the microspheres, then monkeypox antibodies are added for coupling, and then bovine serum albumin is added for blocking to obtain fluorescent latex microspheres-monkeypox antibody immune complexes.

9. The immunochromatographic test strip for quantitative detection of monkeypox virus based on fluorescent latex microspheres according to claim 7, characterized in that: The test strip is provided with a sample pad, an NC membrane and a water-absorbing pad in sequence along the chromatography direction.

10. The method for preparing the immunochromatographic test strip for quantitative detection of monkeypox virus based on fluorescent latex microspheres according to claim 9, characterized in that: The steps include: 1) Preparation of NC membrane: 0.04 mg / cm 2 -0.07mg / cm 2 The concentration of monkeypox capture antibody is 0.04 mg / cm2 for line T and 0.04 mg / cm2 for line C. 2 -0.07mg / cm 2 ; 2) Assembly of the test strip: The sample pad, NC membrane and absorbent pad are laid in sequence along the chromatography direction on the PVC bottom plate, and a part of the sample pad is overlapped on the NC membrane, and a part of the absorbent pad is overlapped on the other side of the NC membrane to prepare the test strip.

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