A human fluid fungus detection test paper and preparation method thereof

Abstract

The present invention relates to the field of biological detection technology, and specifically to a human fluid fungus detection test paper and a preparation method thereof, wherein the test paper comprises a PVC base plate, a sample pad, a fluorescent pad, a reaction membrane and a water-absorbing pad. Among them, the fluorescent pad is coated with an antibody labeled with fluorescent microspheres; a detection line and a quality control line are provided on the reaction membrane, the detection line is coated with a (1-3)-β-D glucan-bovine serum albumin conjugate, and the quality control line is coated with a sheep anti-mouse IgG secondary antibody. The test strip of the present invention can quickly detect (1-3)-β-D glucan, and has high sensitivity, good accuracy, and strong repeatability and stability.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a human fluid fungus detection test paper and a preparation method thereof. Background Art

[0002] (1-3)-β-D glucan is widely present in fungal cell walls, accounting for more than 50% of the dry mass of fungal cells. Except for the bound bacteria (mainly Rhizopus and Mucor), all fungal cell wall components contain (1-3)-β-D glucan, with the highest content in yeast-like fungi. Human cells, viruses, prokaryotes, etc. do not contain this component, which makes it an ideal marker for detecting deep fungal infections. When fungi invade the human blood or deep tissues and cause deep infections, the fungal cells are phagocytosed and digested by phagocytes, and (1-3)-β-D glucan is released from the cell wall, resulting in a significant increase in this component in the blood and body fluids. (1-3)-β-D glucan exists in two forms: one is a low-order structure in the form of a single chain, and the other is a high-order structure in the form of a double helix or triple helix. (1-3)-β-D glucan is neutral and contains a small amount of β-1,6 glycosidic bond connection structure. Under the action of multiple hydroxyl groups in the molecule, it forms a dense triple helix structure with the characteristics of high immune activity and low solubility. Therefore, quantitative detection of (1-3)-β-D glucan is a powerful basis for diagnosing deep fungal infection and can be used for the identification of invasive fungal disease (IFD). Due to the high risk and high incidence of IFD, it has become an increasingly important factor endangering health, especially for immunocompromised patients or hospitalized patients with serious underlying diseases. Therefore, rapid and accurate early diagnosis of deep fungal infection is of great significance. At present, a variety of dextran colorimetric detection kits based on horseshoe crab reagent have been developed, which have the advantages of rapidity, sensitivity, strong specificity, and good repeatability, and have been widely used in clinical practice. The disadvantage is that the detection time is long, and the horseshoe crab reagent produced by the traditional method is easily interfered by endotoxin, resulting in false positives. Horseshoe crabs are national second-class protected animals, so the development of glucose detection kits based on immunology is urgent. Such as magnetic particle chemiluminescence technology, but its disadvantage is that it is easily interfered by biotin in the sample and has low detection accuracy. Fluorescence immunochromatography is applied to IFD detection, which has the advantages of simple operation, rapid detection, low equipment requirements, plug-in card reading, easy results to judge, and greatly shortened detection time. It provides an effective auxiliary means for the detection of susceptible populations. In summary, there is currently a lack of immunochromatographic test strips for the detection of fungal (1-3)-β-D glucan, and the existing detection methods and kits have low detection accuracy and insufficient sensitivity. How to provide an immunochromatographic test strip for detecting (1-3)-β-D glucan and its preparation method, which does not require heating treatment while improving the accuracy and sensitivity of detection, has become one of the problems that technicians in this field need to solve. Summary of the invention

[0003] In view of the deficiencies in the prior art, the present invention provides a human fluid fungus detection test paper and a preparation method thereof.

[0004] The present invention is achieved through the following technical solutions:

[0005] A human fluid fungus detection test paper comprises a PVC base plate, a sample pad, a fluorescent pad, a reaction membrane and a water-absorbing pad; wherein the fluorescent pad is coated with an antibody labeled with fluorescent microspheres; a detection line and a quality control line are arranged on the reaction membrane, the detection line is coated with a (1-3)-β-D-glucan-bovine serum albumin conjugate, and the quality control line is coated with a goat anti-mouse IgG secondary antibody.

[0006] Furthermore, the method for preparing the sample pad comprises the following steps:

[0007] (1) In a nitrogen environment, ethylene glycol methyl ether is heated to 160°C, and a mixed solution of N-vinyl pyrrolidone, N,N-dimethylacrylamide, and di-tert-butyl peroxide is added dropwise. After the addition is completed, the temperature is kept at 160°C for 1 hour, then the temperature is lowered to 70°C, and the mixture is stirred at 180 rpm for 1 hour to obtain a polyvinyl pyrrolidone copolymer;

[0008] (2) adding N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to the polyvinylpyrrolidone copolymer obtained in step (1), stirring at 60°C and 100 rpm for 1 h, adding inulin, continuing stirring for 24 h, placing in a dialysis bag with a molecular weight cutoff of 7000 Da, and dialyzing with deionized water for 48 h, collecting the product in the dialysis bag, centrifuging at 4000 rpm for 10 min, and taking the supernatant for freeze-drying to obtain a composite polymer;

[0009] (3) adding the composite polymer obtained in step (2), casein and Tween-20 to a 20 mM PBS buffer solution (pH 7.4) and mixing them evenly to obtain a treatment solution A;

[0010] (4) The glass fiber membrane was immersed in the treatment solution A obtained in step (3) for 2 h and dried at 37°C to obtain a sample pad.

[0011] Furthermore, in step (1), the mass ratio of ethylene glycol methyl ether, N-vinyl pyrrolidone, N,N-dimethylacrylamide and di-tert-butyl peroxide is 40:20:3:2.7.

[0012] Furthermore, the dropping speed of the mixed solution in step (1) is 1.5 g / min.

[0013] Furthermore, in step (2), the mass ratio of the polyvinyl pyrrolidone copolymer to N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and inulin is 50:15:1:20.

[0014] Furthermore, in step (3), the dosage ratio of the PBS buffer, the composite polymer, Tween-20, and the casein is 1000 mL:10 g:10 mL:1 g.

[0015] Furthermore, the method for preparing the fluorescent pad comprises the following steps:

[0016] (a) The fluorescent microspheres were mixed with 50 mmol / L MES buffer (pH 6.0), sonicated at 150 W for 1 min, centrifuged at 8-10°C and 12,000 g for 20 min, the supernatant was removed, 50 mmol / L MES buffer (pH 6.0) was added again, and sonicated again for 1 min to obtain a fluorescent microsphere mixture;

[0017] (b) using 50 mM MES buffer at pH 6.0 to prepare 10 mg / mL NHS solution and 5 mg / mL EDAC solution, respectively, and adding them dropwise to the fluorescent microsphere mixture in step (a) to a final concentration of 0.8 mg / mL NHS and 0.3 mg / mL EDAC, and then ultrasonicating at 150 W for 1 min and reacting at 37°C in the dark for 1 h to obtain a mixture A;

[0018] (c) The mixed solution A in step (b) was centrifuged at 8°C and 12000 g for 20 min, the supernatant was discarded, the precipitate was washed twice with 50 mM pH8.0 HEPES buffer, the fungal (1-3)-β-D glucan antibody solution was added, the mixture was ultrasonicated at 150 W for 1 min, the mixture was reacted at 37°C in the dark for 1 h, 20 mg / mL bovine serum albumin solution was added, the mixture was ultrasonicated for 1 min, the mixture was rotated at 4°C and 60 rpm for 8 h in the dark, and the mixture was centrifuged at 8°C and 12000 g for 20 min, and the supernatant was discarded to obtain a microsphere antibody conjugate precipitate;

[0019] (d) Dissolve trehalose and oxalic anhydride in acetonitrile, add NaOH, stir at 70°C and 120 rpm for 7 h, concentrate under reduced pressure at 0.01 MPa and 60°C for 5-6 h, and recrystallize in a mixed solution of ethanol and ether to obtain carboxylated trehalose; dissolve ascorbic acid in dichloromethane, add thionyl chloride in an ice-water bath at 0°C, stir at 25°C and 100 rpm for 0.5 h, concentrate under reduced pressure at 0.01 MPa and 60°C for 10 h, wash with deionized water, dry at 80°C, dissolve the dried product in 1wt% acetic acid solution, add 3,4-dihydroxybenzaldehyde, stir at 30°C and 180 rpm for 3-4 h, add sodium borohydride and continue stirring for 1 h, filter and obtain a filter cake; mix the filter cake with carboxylated trehalose and deionized water to obtain a mixed solution B, add 3 mol / L hydrochloric acid solution and distilled water, mix well and stir at 70-90°C for 6-10 h, after centrifugation at 8000 rpm for 30 min, the supernatant was filtered through a 0.22 μm filter and freeze-dried to obtain the trehalose-ascorbic acid modified complex;

[0020] (e) adding the trehalose-ascorbic acid modified complex obtained in step (d), glycine and dithiothreitol to a 10 mmol / L HEPES buffer solution and mixing evenly to obtain a conjugate reconstitution storage solution; washing the microsphere-antibody conjugate precipitate obtained in step (c) twice with a pH 7.4 50 mM PBS buffer solution, adding the conjugate reconstitution storage solution to resuspend and mix evenly to obtain a microsphere-antibody solution;

[0021] (f) adding glucose and borax to a 4% w / v bovine serum albumin solution to obtain a treatment solution B, soaking the polyester film in the treatment solution B for 2 h, and then drying at 37° C. to obtain a pretreated polyester film;

[0022] (g) The microsphere-antibody solution prepared in step (e) was diluted to 1 mg / mL with 50 mM PBS buffer, and sprayed evenly on the pretreated polyester film obtained in step (f) at a flow rate of 3 μL / cm using a quantitative film sprayer, and dried at 37°C to obtain a fluorescent pad.

[0023] Furthermore, the fluorescent microspheres in step (a) include time-resolved fluorescent microspheres; the surface modification functional groups of the fluorescent microspheres include hydroxyl groups or carboxyl groups; and the fluorescent microspheres are made of polystyrene, polymethyl methacrylate or silica.

[0024] Furthermore, the concentration of the fluorescent microspheres in the fluorescent microsphere mixture in step (a) is 12.5 mg / mL.

[0025] Furthermore, in step (c), the mass ratio of the washed precipitate to the fungus (1-3)-β-D glucan antibody is 10:1.

[0026] Furthermore, in step (c), the ratio of the bovine serum albumin solution to the washed precipitate is 1 mL:0.1 g.

[0027] Furthermore, in step (d), the dosage ratio of trehalose, oxalic anhydride, acetonitrile and sodium hydroxide is 0.2 mol:0.1 mol:200 mL:0.3 mol.

[0028] Furthermore, the volume ratio of ethanol to ether in the ethanol and ether mixture in step (d) is 1:3.

[0029] Furthermore, in step (d), the ratio of ascorbic acid, dichloromethane and thionyl chloride is 0.12 mol:200 mL:0.1 mL.

[0030] Furthermore, in step (d), the usage ratio of the dried product, acetic acid solution, 3,4-dihydroxybenzaldehyde and sodium borohydride is 15 g:1000 mL:1 g:0.5 g.

[0031] Furthermore, in step (d), the ratio of carboxylated trehalose, filter cake and deionized water is 1 g:10 g:50 mL.

[0032] Furthermore, in step (d), the volume ratio of the mixed solution B, the hydrochloric acid solution and the distilled water is 25:1:1.

[0033] Furthermore, in step (e), the amount ratio of the HEPES buffer solution, the trehalose-ascorbic acid modified complex, glycine, and dithiothreitol is 100 mL: 2 g: 1 g: 0.5 g.

[0034] Furthermore, in step (e), the ratio of the washed microsphere-antibody conjugate precipitate to the conjugate reconstitution storage solution is 10 mg:1 mL.

[0035] Furthermore, in step (f), the dosage ratio of the bovine serum albumin solution, glucose and borax is 100 mL:3 g:2 mmol.

[0036] Furthermore, the method for preparing the reaction membrane comprises the following steps:

[0037] (i) mixing (1-3)-β-D-glucan and deionized water to obtain a 20 mg / L glucan solution, adding the glucan solution to a 10 wt % hydrogen peroxide solution, stirring at 120 rpm for 18-20 h, adding diethylene glycol, continuing to stir for 2 h, filtering through a 10 kDa ultrafiltration membrane, and freeze-drying to obtain oxidized glucan;

[0038] (ii) mixing equal volumes of 0.1 mol / L pH 8.0 phosphate buffer and 0.1 mol / L pH 5.0 acetate buffer to obtain a mixed buffer, adding bovine serum albumin (BSA) and the oxidized dextran obtained in step (i) to the mixed buffer, reacting at 4°C for 14-16 h, centrifuging at 4000 rpm for 10 min, taking the supernatant and placing it in a dialysis bag and dialyzing it with pH 7.4 50 mM PBS buffer at 4°C for 96 h to obtain a (1-3)-β-D-glucan-BSA conjugate;

[0039] (iii) adding the composite polymer obtained in step (2) and sodium chloride to deionized water to obtain a treatment solution C, soaking the nitrocellulose membrane in the treatment solution C for 15 minutes and then drying at 37° C. to obtain a pre-treated reaction membrane;

[0040] (iv) Dilute the (1-3)-β-D-glucan-BSA conjugate to 0.33 mg / mL and the goat anti-mouse IgG to 0.5 mg / mL with pH 7.4 15 mM PBS, and spray them onto the pretreated reaction membrane in step (iii) at a flow rate of 3 μL / cm using a film spotter to draw lines as the test line (T line) and the quality control line (C line), respectively, and dry at 37°C to obtain a reaction membrane.

[0041] Furthermore, in step (i), the volume ratio of the dextran solution, the hydrogen peroxide solution and the diethylene glycol is 10:1:1.

[0042] Furthermore, in step (ii), the dosage ratio of BSA, oxidized dextran and mixed buffer is 1 g:2 g:100 mL.

[0043] Furthermore, in step (iii), the usage ratio of deionized water, composite polymer and sodium chloride is 100 mL:0.3 g:1 g.

[0044] Furthermore, the absorbent pad is absorbent paper.

[0045] The present invention also provides a method for preparing the human fluid fungus detection test paper, comprising the following steps: using a PVC base plate as a backing, pasting a sample pad, a fluorescent pad, a reaction membrane and a water-absorbing pad on the base plate in order, and cutting the detection line on the reaction membrane close to one end of the fluorescent pad into strips with a width of 3.00 mm using a cutting machine to obtain a human fluid fungus detection test paper.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The materials used in the fluorescent immunochromatography of the present invention are nitrocellulose membrane and glass fiber, which contain (1-3)-β-D glucan. The present invention uses different treatment solutions to perform secondary treatment on these materials, thereby eliminating the interference of (1-3)-β-D glucan in the materials and enhancing the sensitivity of fluorescent immunochromatography detection. The sample treatment solution and treatment method in the present invention are also optimized. Compared with the sample alkali treatment and heating incubation after the sample of the dextran colorimetric detection kit based on the horseshoe crab reagent (the sample treatment in the enzyme-linked immunosorbent assay and magnetic microparticle chemiluminescence kit often requires incubation), the present invention does not require heating treatment, is simple and convenient to operate, and improves the sensitivity and specificity of fluorescent immunochromatography detection. The present invention prepares a composite polymer, which can effectively reduce the non-specific adsorption of antibodies, thereby improving the sensitivity of test strip detection, increasing the hydrophilicity of the sample pad, and its aqueous solution has a suspension and dispersion effect, which can be used to protect the antigen in the sample, which is conducive to the rapid reaction of the antigen in the sample and the antibody in the test strip; casein has good hydrophobicity, can be used well in sol, will not dissolve in water with the flow of the solution, and reduces the background increase caused by the use environment; the components of the treatment liquid A prepared by the present invention are reasonably compatible, synergistic, and the sample pad is treated, which can effectively improve the detection sensitivity and the detection speed. The treatment liquid of the reaction membrane is added with a composite polymer, which can reduce background interference, reduce the occurrence of non-specific binding, improve detection accuracy and sensitivity, improve hydrophilicity, help to improve the absorption efficiency of the sample on the membrane, enhance the sensitivity of the reaction, improve the antibody binding ability, and enhance the binding force between the antibody and the membrane, thereby improving the specificity and sensitivity of the detection, improving the reaction speed, and shortening the reaction time. The present invention prepares a trehalose-ascorbic acid modified complex. By preparing the trehalose-ascorbic acid modified complex, the biological activities of trehalose and ascorbic acid are retained, the stability and antibacterial and antioxidant effects of ascorbic acid are improved, and the introduction of trehalose further improves the water solubility and reduces the toxicity. The obtained trehalose-ascorbic acid modified complex is added to a conjugate re-dissolving storage solution, which can effectively kill bacteria and resist oxidation, improve the stability of a microsphere antibody conjugate, extend the shelf life, prevent antibody aggregation and precipitation, maintain the activity and function of the antibody, and effectively improve the detection sensitivity and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1 This is a schematic diagram of the structure of the human body fluid fungus detection test paper of the present invention;

[0050] Figure 2 The repeatability test diagram of the test paper of Examples 1-3 and Comparative Examples 1-3 of the present invention;

[0051] Figure 3 These are photos of the microsphere-antibody solution after storage described in Example 3 of the present invention and Comparative Example 2. DETAILED DESCRIPTION

[0052] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0053] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0054] Embodiment 1: This embodiment provides a human body fluid fungus detection test strip, comprising a PVC base plate, a sample pad, a fluorescent pad, a reaction membrane and an absorbent pad, wherein the absorbent pad is absorbent paper.

[0055] The method for preparing the sample pad comprises the following steps:

[0056] (1) In a nitrogen environment, ethylene glycol methyl ether was heated to 160°C, and a mixed solution of N-vinyl pyrrolidone, N,N-dimethylacrylamide, and di-tert-butyl peroxide was added dropwise at a rate of 1.5 g / min, wherein the mass ratio of ethylene glycol methyl ether, N-vinyl pyrrolidone, N,N-dimethylacrylamide, and di-tert-butyl peroxide was 40:20:3:2.7. After the addition was completed, the mixture was kept at 160°C for 1 h, cooled to 70°C, and stirred at 180 rpm for 1 h to obtain a polyvinyl pyrrolidone copolymer;

[0057] (2) adding N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to the polyvinyl pyrrolidone copolymer obtained in step (1), stirring at 60°C and 100 rpm for 1 h, and then adding inulin, wherein the mass ratio of the polyvinyl pyrrolidone copolymer to N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and inulin is 50:15:1:20, and stirring is continued for 24 h. The mixture is placed in a dialysis bag with a molecular weight cutoff of 7000 Da, and dialyzed with deionized water for 48 h. The product in the dialysis bag is collected, centrifuged at 4000 rpm for 10 min, and the supernatant is freeze-dried to obtain a composite polymer;

[0058] (3) Add the composite polymer obtained in step (2), casein and Tween-20 to a 20 mM PBS buffer solution at pH 7.4 and mix them evenly. The amount ratio of PBS buffer solution, composite polymer, Tween-20 and casein is 1000 mL:10 g:10 mL:1 g to obtain a treatment solution A.

[0059] (4) The glass fiber membrane was immersed in the treatment solution A obtained in step (3) for 2 h and dried at 37°C to obtain a sample pad.

[0060] The preparation method of the fluorescent pad comprises the following steps:

[0061] (a) mixing hydroxypolystyrene time-resolved fluorescent microspheres with 50 mmol / L MES buffer at pH 6.0, ultrasonicating at 150 W for 1 min, centrifuging at 10°C and 12000 g for 20 min, removing the supernatant, re-adding 50 mmol / L MES buffer at pH 6.0, and ultrasonicating again for 1 min to obtain a fluorescent microsphere mixed solution, wherein the concentration of the fluorescent microspheres in the fluorescent microsphere mixed solution is 12.5 mg / mL;

[0062] (b) using 50 mM MES buffer at pH 6.0 to prepare 10 mg / mL NHS solution and 5 mg / mL EDAC solution, respectively, and adding them dropwise to the fluorescent microsphere mixture in step (a) to a final concentration of 0.8 mg / mL NHS and 0.3 mg / mL EDAC, and then ultrasonicating at 150 W for 1 min and reacting at 37°C in the dark for 1 h to obtain a mixture A;

[0063] (c) The mixed solution A in step (b) was centrifuged at 8°C and 12000 g for 20 min, the supernatant was discarded, the precipitate was washed twice with 50 mM pH8.0 HEPES buffer, the fungal (1-3)-β-D glucan antibody solution was added, the mass ratio of the washed precipitate to the fungal (1-3)-β-D glucan antibody was 10:1, ultrasonicated at 150 W for 1 min, reacted at 37°C in the dark for 1 h, 20 mg / mL bovine serum albumin solution was added, the amount ratio of the bovine serum albumin solution to the washed precipitate was 1 mL:0.1 g, ultrasonicated for 1 min, rotated at 4°C and 60 rpm for 8 h in the dark, centrifuged at 8°C and 12000 g for 20 min, and the supernatant was discarded to obtain a microsphere antibody conjugate precipitate;

[0064] (d) Dissolve trehalose and oxalic anhydride in acetonitrile, add NaOH, the amount ratio of trehalose, oxalic anhydride, acetonitrile and sodium hydroxide is 0.2 mol:0.1 mol:200 mL:0.3 mol, stir at 70°C and 120 rpm for 7 h, concentrate under reduced pressure at 0.01 MPa and 60°C for 6 h, and recrystallize in a mixed solution of ethanol and ether (volume ratio 1:3) to obtain carboxylated trehalose; dissolve ascorbic acid in dichloromethane, add thionyl chloride in an ice-water bath at 0°C, the amount ratio of ascorbic acid, dichloromethane and thionyl chloride is 0.12 mol:200 mL:0.1 mL, stir at 25°C and 100 rpm for 0.5 h, and concentrate under reduced pressure at 0.01 MPa and 60°C for 10 h, washed with deionized water, dried at 80 °C, dissolved the dried product in 1wt% acetic acid solution, added 3,4-dihydroxybenzaldehyde, stirred at 30 °C and 180 rpm for 4 h, added sodium borohydride and continued to stir for 1 h, the amount ratio of the dried product, acetic acid solution, 3,4-dihydroxybenzaldehyde and sodium borohydride was 15 g:1000 mL:1 g:0.5 g, filtered to obtain a filter cake; the filter cake was mixed evenly with carboxylated trehalose and deionized water, the amount ratio of carboxylated trehalose, filter cake and deionized water was 1 g:10 g:50 mL, and a mixed solution B was obtained, 3 mol / L hydrochloric acid solution and distilled water were added, the volume ratio of mixed solution B, hydrochloric acid solution and distilled water was 25:1:1, stirred evenly at 90 °C for 10 h, centrifuged at 8000 rpm for 30 min, and the supernatant was filtered through 0.22 μm filter membrane and freeze-dried to obtain the trehalose-ascorbic acid modified complex;

[0065] (e) adding the trehalose-ascorbic acid modified complex obtained in step (d), glycine and dithiothreitol to a 10 mmol / L HEPES buffer solution and mixing evenly, wherein the amount ratio of the HEPES buffer solution, the trehalose-ascorbic acid modified complex, glycine and dithiothreitol is 100 mL:2 g:1 g:0.5 g, to obtain a conjugate reconstitution storage solution; washing the microsphere-antibody conjugate precipitate obtained in step (c) twice with pH 7.4 50 mM PBS buffer, adding the conjugate reconstitution storage solution to resuspend and mix evenly, wherein the amount ratio of the washed microsphere-antibody conjugate precipitate to the conjugate reconstitution storage solution is 10 mg:1 mL, to obtain a microsphere-antibody solution;

[0066] (f) adding glucose and borax to a 4% w / v bovine serum albumin solution to obtain a treatment solution B, wherein the amount ratio of the bovine serum albumin solution, glucose and borax is 100 mL:3 g:2 mmol, and the polyester film is immersed in the treatment solution B for 2 h and then dried at 37° C. to obtain a pretreated polyester film;

[0067] (g) The microsphere-antibody solution prepared in step (e) was diluted to 1 mg / mL with 50 mM PBS buffer, and sprayed evenly on the pretreated polyester film obtained in step (f) at a flow rate of 3 μL / cm using a quantitative film sprayer, and dried at 37°C to obtain a fluorescent pad.

[0068] The method for preparing the reaction film comprises the following steps:

[0069] (i) (1-3)-β-D glucan was mixed with deionized water to obtain a 20 mg / L glucan solution, the glucan solution was added to a 10 wt % hydrogen peroxide solution, and the mixture was stirred at 120 rpm for 20 h. Diethylene glycol was added, and the volume ratio of the glucan solution, hydrogen peroxide solution and diethylene glycol was 10:1:1. The mixture was stirred for 2 h, filtered through a 10 kDa ultrafiltration membrane, and freeze-dried to obtain oxidized glucan.

[0070] (ii) Mix equal volumes of 0.1 mol / L pH 8.0 phosphate buffer and 0.1 mol / L pH 5.0 acetate buffer to obtain a mixed buffer, add BSA and the oxidized dextran obtained in step (i) to the mixed buffer, the amount ratio of BSA, oxidized dextran and mixed buffer being 1 g:2 g:100 mL, react at 4°C for 16 h, centrifuge at 4000 rpm for 10 min, take the supernatant and place it in a dialysis bag and dialyze it with pH 7.4 50 mM PBS buffer for 96 h at 4°C to obtain (1-3)-β-D-glucan-BSA conjugate;

[0071] (iii) adding the composite polymer obtained in step (2) and sodium chloride to deionized water in a ratio of 100 mL:0.3 g:1 g to obtain a treatment solution C, soaking the nitrocellulose membrane in the treatment solution C for 15 min and drying it at 37° C. to obtain a pre-treated reaction membrane;

[0072] (iv) Dilute the (1-3)-β-D-glucan-BSA conjugate to 0.33 mg / mL and the goat anti-mouse IgG to 0.5 mg / mL with pH 7.4 15 mM PBS, and spray them onto the pretreated reaction membrane in step (iii) at a flow rate of 3 μL / cm using a film spotter to draw lines as the test line (T line) and the quality control line (C line), respectively, and dry at 37°C to obtain a reaction membrane.

[0073] This embodiment also provides a method for preparing the human fluid fungus detection test paper, comprising the following steps: using a PVC base plate as a backing, pasting a sample pad, a fluorescent pad, a reaction membrane and a water-absorbing pad on the base plate in order, and cutting the detection line on the reaction membrane close to one end of the fluorescent pad into strips with a 3.00 mm width using a cutting machine to obtain a human fluid fungus detection test paper, the structural schematic diagram of which is shown in FIG. Figure 1 shown.

[0074] Embodiment 2: This embodiment provides a human body fluid fungus detection test strip, comprising a PVC base plate, a sample pad, a fluorescent pad, a reaction membrane and an absorbent pad, wherein the absorbent pad is absorbent paper.

[0075] The method for preparing the sample pad comprises the following steps:

[0076] (1) In a nitrogen environment, ethylene glycol methyl ether was heated to 160°C, and a mixed solution of N-vinyl pyrrolidone, N,N-dimethylacrylamide, and di-tert-butyl peroxide was added dropwise at a rate of 1.5 g / min, wherein the mass ratio of ethylene glycol methyl ether, N-vinyl pyrrolidone, N,N-dimethylacrylamide, and di-tert-butyl peroxide was 40:20:3:2.7. After the addition was completed, the mixture was kept at 160°C for 1 h, cooled to 70°C, and stirred at 180 rpm for 1 h to obtain a polyvinyl pyrrolidone copolymer;

[0077] (2) adding N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to the polyvinyl pyrrolidone copolymer obtained in step (1), stirring at 60°C and 100 rpm for 1 h, and then adding inulin, wherein the mass ratio of the polyvinyl pyrrolidone copolymer to N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and inulin is 50:15:1:20, and stirring is continued for 24 h. The mixture is placed in a dialysis bag with a molecular weight cutoff of 7000 Da, and dialyzed with deionized water for 48 h. The product in the dialysis bag is collected, centrifuged at 4000 rpm for 10 min, and the supernatant is freeze-dried to obtain a composite polymer;

[0078] (3) Add the composite polymer obtained in step (2), casein and Tween-20 to a 20 mM PBS buffer solution at pH 7.4 and mix them evenly. The amount ratio of PBS buffer solution, composite polymer, Tween-20 and casein is 1000 mL:10 g:10 mL:1 g to obtain a treatment solution A.

[0079] (4) The glass fiber membrane was immersed in the treatment solution A obtained in step (3) for 2 h and dried at 37°C to obtain a sample pad.

[0080] The preparation method of the fluorescent pad comprises the following steps:

[0081] (a) mixing hydroxypolystyrene time-resolved fluorescent microspheres with 50 mmol / L MES buffer at pH 6.0, ultrasonicating at 150 W for 1 min, centrifuging at 8°C and 12000 g for 20 min, removing the supernatant, re-adding 50 mmol / L MES buffer at pH 6.0, and ultrasonicating again for 1 min to obtain a fluorescent microsphere mixed solution, wherein the concentration of the fluorescent microspheres in the fluorescent microsphere mixed solution is 12.5 mg / mL;

[0082] (b) using 50 mM MES buffer at pH 6.0 to prepare 10 mg / mL NHS solution and 5 mg / mL EDAC solution, respectively, and adding them dropwise to the fluorescent microsphere mixture in step (a) to a final concentration of 0.8 mg / mL NHS and 0.3 mg / mL EDAC, and then ultrasonicating at 150 W for 1 min and reacting at 37°C in the dark for 1 h to obtain a mixture A;

[0083] (c) The mixed solution A in step (b) was centrifuged at 8°C and 12000 g for 20 min, the supernatant was discarded, the precipitate was washed twice with 50 mM pH8.0 HEPES buffer, the fungal (1-3)-β-D glucan antibody solution was added, the mass ratio of the washed precipitate to the fungal (1-3)-β-D glucan antibody was 10:1, ultrasonicated at 150 W for 1 min, reacted at 37°C in the dark for 1 h, 20 mg / mL bovine serum albumin solution was added, the amount ratio of the bovine serum albumin solution to the washed precipitate was 1 mL:0.1 g, ultrasonicated for 1 min, rotated at 4°C and 60 rpm for 8 h in the dark, centrifuged at 8°C and 12000 g for 20 min, and the supernatant was discarded to obtain a microsphere antibody conjugate precipitate;

[0084] (d) Dissolve trehalose and oxalic anhydride in acetonitrile, add NaOH, the amount ratio of trehalose, oxalic anhydride, acetonitrile and sodium hydroxide is 0.2 mol:0.1 mol:200 mL:0.3 mol, stir at 70°C and 120 rpm for 7 h, concentrate under reduced pressure at 0.01 MPa and 60°C for 5 h, and recrystallize in a mixed solution of ethanol and ether (volume ratio 1:3) to obtain carboxylated trehalose; dissolve ascorbic acid in dichloromethane, add thionyl chloride in an ice-water bath at 0°C, the amount ratio of ascorbic acid, dichloromethane and thionyl chloride is 0.12 mol:200 mL:0.1 mL, stir at 25°C and 100 rpm for 0.5 h, and concentrate under reduced pressure at 0.01 MPa and 60°C for 10 h, washed with deionized water, dried at 80 °C, dissolved the dried product in 1wt% acetic acid solution, added 3,4-dihydroxybenzaldehyde, stirred at 30 °C and 180 rpm for 3 h, added sodium borohydride and continued to stir for 1 h, the amount ratio of the dried product, acetic acid solution, 3,4-dihydroxybenzaldehyde and sodium borohydride was 15 g:1000 mL:1 g:0.5 g, filtered to obtain a filter cake; the filter cake was mixed evenly with carboxylated trehalose and deionized water, the amount ratio of carboxylated trehalose, filter cake and deionized water was 1 g:10 g:50 mL, and a mixed solution B was obtained, 3 mol / L hydrochloric acid solution and distilled water were added, the volume ratio of mixed solution B, hydrochloric acid solution and distilled water was 25:1:1, stirred evenly at 70 °C for 6 h, centrifuged at 8000 rpm for 30 min, and the supernatant was filtered through 0.22 μm filter membrane and freeze-dried to obtain the trehalose-ascorbic acid modified complex;

[0085] (e) adding the trehalose-ascorbic acid modified complex obtained in step (d), glycine and dithiothreitol to a 10 mmol / L HEPES buffer solution and mixing evenly, wherein the amount ratio of the HEPES buffer solution, the trehalose-ascorbic acid modified complex, glycine and dithiothreitol is 100 mL:2 g:1 g:0.5 g, to obtain a conjugate reconstitution storage solution; washing the microsphere-antibody conjugate precipitate obtained in step (c) twice with pH 7.4 50 mM PBS buffer, adding the conjugate reconstitution storage solution to resuspend and mix evenly, wherein the amount ratio of the washed microsphere-antibody conjugate precipitate to the conjugate reconstitution storage solution is 10 mg:1 mL, to obtain a microsphere-antibody solution;

[0086] (f) adding glucose and borax to a 4% w / v bovine serum albumin solution to obtain a treatment solution B, wherein the amount ratio of the bovine serum albumin solution, glucose and borax is 100 mL:3 g:2 mmol, and the polyester film is immersed in the treatment solution B for 2 h and then dried at 37° C. to obtain a pretreated polyester film;

[0087] (g) The microsphere-antibody solution prepared in step (e) was diluted to 1 mg / mL with 50 mM PBS buffer, and sprayed evenly on the pretreated polyester film obtained in step (f) at a flow rate of 3 μL / cm using a quantitative film sprayer, and dried at 37°C to obtain a fluorescent pad.

[0088] The method for preparing the reaction film comprises the following steps:

[0089] (i) (1-3)-β-D glucan was mixed with deionized water to obtain a 20 mg / L glucan solution, the glucan solution was added to a 10 wt % hydrogen peroxide solution, and the mixture was stirred at 120 rpm for 18 h. Diethylene glycol was added, and the volume ratio of the glucan solution, hydrogen peroxide solution and diethylene glycol was 10:1:1. The mixture was stirred for 2 h, filtered through a 10 kDa ultrafiltration membrane, and freeze-dried to obtain oxidized glucan.

[0090] (ii) Mix equal volumes of 0.1 mol / L pH 8.0 phosphate buffer and 0.1 mol / L pH 5.0 acetate buffer to obtain a mixed buffer, add BSA and the oxidized dextran obtained in step (i) to the mixed buffer, the amount ratio of BSA, oxidized dextran and mixed buffer being 1 g:2 g:100 mL, react at 4°C for 14 h, centrifuge at 4000 rpm for 10 min, take the supernatant and place it in a dialysis bag and dialyze it with pH 7.4 50 mM PBS buffer at 4°C for 96 h to obtain (1-3)-β-D-glucan-BSA conjugate;

[0091] (iii) adding the composite polymer obtained in step (2) and sodium chloride to deionized water in a ratio of 100 mL:0.3 g:1 g to obtain a treatment solution C, soaking the nitrocellulose membrane in the treatment solution C for 15 min and drying it at 37° C. to obtain a pre-treated reaction membrane;

[0092] (iv) Dilute the (1-3)-β-D-glucan-BSA conjugate to 0.33 mg / mL and the goat anti-mouse IgG to 0.5 mg / mL with pH 7.4 15 mM PBS, and spray them onto the pretreated reaction membrane in step (iii) at a flow rate of 3 μL / cm using a film spotter to draw lines as the test line and quality control line, respectively, and dry at 37°C to obtain a reaction membrane.

[0093] This embodiment also provides a method for preparing the human fluid fungus detection test paper, comprising the following steps: using a PVC base plate as a backing, pasting a sample pad, a fluorescent pad, a reaction membrane and a water-absorbing pad on the base plate in order, and cutting the detection line on the reaction membrane close to one end of the fluorescent pad into strips with a cutting machine to obtain a human fluid fungus detection test paper.

[0094] Embodiment 3: This embodiment provides a human body fluid fungus detection test strip, comprising a PVC base plate, a sample pad, a fluorescent pad, a reaction membrane and an absorbent pad, wherein the absorbent pad is absorbent paper.

[0095] The method for preparing the sample pad comprises the following steps:

[0096] (1) In a nitrogen environment, ethylene glycol methyl ether was heated to 160°C, and a mixed solution of N-vinyl pyrrolidone, N,N-dimethylacrylamide, and di-tert-butyl peroxide was added dropwise at a rate of 1.5 g / min, wherein the mass ratio of ethylene glycol methyl ether, N-vinyl pyrrolidone, N,N-dimethylacrylamide, and di-tert-butyl peroxide was 40:20:3:2.7. After the addition was completed, the mixture was kept at 160°C for 1 h, cooled to 70°C, and stirred at 180 rpm for 1 h to obtain a polyvinyl pyrrolidone copolymer;

[0097] (2) adding N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to the polyvinyl pyrrolidone copolymer obtained in step (1), stirring at 60°C and 100 rpm for 1 h, and then adding inulin, wherein the mass ratio of the polyvinyl pyrrolidone copolymer to N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and inulin is 50:15:1:20, and stirring is continued for 24 h. The mixture is placed in a dialysis bag with a molecular weight cutoff of 7000 Da, and dialyzed with deionized water for 48 h. The product in the dialysis bag is collected, centrifuged at 4000 rpm for 10 min, and the supernatant is freeze-dried to obtain a composite polymer;

[0098] (3) Add the composite polymer obtained in step (2), casein and Tween-20 to a 20 mM PBS buffer solution at pH 7.4 and mix them evenly. The amount ratio of PBS buffer solution, composite polymer, Tween-20 and casein is 1000 mL:10 g:10 mL:1 g to obtain a treatment solution A.

[0099] (4) The glass fiber membrane was immersed in the treatment solution A obtained in step (3) for 2 h and dried at 37°C to obtain a sample pad.

[0100] The preparation method of the fluorescent pad comprises the following steps:

[0101] (a) mixing hydroxypolystyrene time-resolved fluorescent microspheres with 50 mmol / L MES buffer at pH 6.0, ultrasonicating at 150 W for 1 min, centrifuging at 9°C and 12000 g for 20 min, removing the supernatant, re-adding 50 mmol / L MES buffer at pH 6.0, and ultrasonicating again for 1 min to obtain a fluorescent microsphere mixed solution, wherein the concentration of the fluorescent microspheres in the fluorescent microsphere mixed solution is 12.5 mg / mL;

[0102] (b) using 50 mM MES buffer at pH 6.0 to prepare 10 mg / mL NHS solution and 5 mg / mL EDAC solution, respectively, and adding them dropwise to the fluorescent microsphere mixture in step (a) to a final concentration of 0.8 mg / mL NHS and 0.3 mg / mL EDAC, and then ultrasonicating at 150 W for 1 min and reacting at 37°C in the dark for 1 h to obtain a mixture A;

[0103] (c) The mixed solution A in step (b) was centrifuged at 8°C and 12000 g for 20 min, the supernatant was discarded, the precipitate was washed twice with 50 mM pH8.0 HEPES buffer, the fungal (1-3)-β-D glucan antibody solution was added, the mass ratio of the washed precipitate to the fungal (1-3)-β-D glucan antibody was 10:1, ultrasonicated at 150 W for 1 min, reacted at 37°C in the dark for 1 h, 20 mg / mL bovine serum albumin solution was added, the amount ratio of the bovine serum albumin solution to the washed precipitate was 1 mL:0.1 g, ultrasonicated for 1 min, rotated at 4°C and 60 rpm for 8 h in the dark, centrifuged at 8°C and 12000 g for 20 min, and the supernatant was discarded to obtain a microsphere antibody conjugate precipitate;

[0104] (d) Dissolve trehalose and oxalic anhydride in acetonitrile, add NaOH, the amount ratio of trehalose, oxalic anhydride, acetonitrile and sodium hydroxide is 0.2 mol:0.1 mol:200 mL:0.3 mol, stir at 70°C and 120 rpm for 7 h, concentrate under reduced pressure at 0.01 MPa and 60°C for 5.5 h, and recrystallize in a mixed solution of ethanol and ether (volume ratio 1:3) to obtain carboxylated trehalose; dissolve ascorbic acid in dichloromethane, add thionyl chloride in an ice-water bath at 0°C, the amount ratio of ascorbic acid, dichloromethane and thionyl chloride is 0.12 mol:200 mL:0.1 mL, stir at 25°C and 100 rpm for 0.5 h, and concentrate under reduced pressure at 0.01 MPa and 60°C for 10 h, washed with deionized water, dried at 80 ° C, dissolved the dried product in 1wt% acetic acid solution, added 3,4-dihydroxybenzaldehyde, stirred at 30 ° C, 180 rpm for 3.5 h, added sodium borohydride and continued to stir for 1 h, the amount ratio of the dried product, acetic acid solution, 3,4-dihydroxybenzaldehyde and sodium borohydride was 15 g:1000 mL:1 g:0.5 g, filtered to obtain a filter cake; the filter cake was mixed with carboxylated trehalose and deionized water, the amount ratio of carboxylated trehalose, filter cake and deionized water was 1 g:10 g:50 mL, and a mixed solution B was obtained. 3 mol / L hydrochloric acid solution and distilled water were added, and the volume ratio of mixed solution B, hydrochloric acid solution and distilled water was 25:1:1. After mixing evenly, it was stirred at 80 ° C for 8 h, centrifuged at 8000 rpm for 30 min, and the supernatant was filtered through 0.22 μm filter membrane and freeze-dried to obtain the trehalose-ascorbic acid modified complex;

[0105] (e) adding the trehalose-ascorbic acid modified complex obtained in step (d), glycine and dithiothreitol to a 10 mmol / L HEPES buffer solution and mixing evenly, wherein the amount ratio of the HEPES buffer solution, the trehalose-ascorbic acid modified complex, glycine and dithiothreitol is 100 mL:2 g:1 g:0.5 g, to obtain a conjugate reconstitution storage solution; washing the microsphere-antibody conjugate precipitate obtained in step (c) twice with pH 7.4 50 mM PBS buffer, adding the conjugate reconstitution storage solution to resuspend and mix evenly, wherein the amount ratio of the washed microsphere-antibody conjugate precipitate to the conjugate reconstitution storage solution is 10 mg:1 mL, to obtain a microsphere-antibody solution;

[0106] (f) adding glucose and borax to a 4% w / v bovine serum albumin solution to obtain a treatment solution B, wherein the amount ratio of the bovine serum albumin solution, glucose and borax is 100 mL:3 g:2 mmol, and the polyester film is immersed in the treatment solution B for 2 h and then dried at 37° C. to obtain a pretreated polyester film;

[0107] (g) The microsphere-antibody solution prepared in step (e) was diluted to 1 mg / mL with 50 mM PBS buffer, and sprayed evenly on the pretreated polyester film obtained in step (f) at a flow rate of 3 μL / cm using a quantitative film sprayer, and dried at 37°C to obtain a fluorescent pad.

[0108] The method for preparing the reaction film comprises the following steps:

[0109] (i) (1-3)-β-D glucan was mixed with deionized water to obtain a 20 mg / L glucan solution, the glucan solution was added to a 10 wt % hydrogen peroxide solution, and the mixture was stirred at 120 rpm for 19 h. Diethylene glycol was added, and the volume ratio of the glucan solution, hydrogen peroxide solution and diethylene glycol was 10:1:1. The mixture was stirred for 2 h, filtered through a 10 kDa ultrafiltration membrane, and freeze-dried to obtain oxidized glucan.

[0110] (ii) Mix equal volumes of 0.1 mol / L pH 8.0 phosphate buffer and 0.1 mol / L pH 5.0 acetate buffer to obtain a mixed buffer, add BSA and the oxidized dextran obtained in step (i) to the mixed buffer, the amount ratio of BSA, oxidized dextran and mixed buffer being 1 g:2 g:100 mL, react at 4°C for 15 h, centrifuge at 4000 rpm for 10 min, take the supernatant and place it in a dialysis bag and dialyze it with pH 7.4 50 mM PBS buffer for 96 h at 4°C to obtain (1-3)-β-D-glucan-BSA conjugate;

[0111] (iii) adding the composite polymer obtained in step (2) and sodium chloride to deionized water in a ratio of 100 mL:0.3 g:1 g to obtain a treatment solution C, soaking the nitrocellulose membrane in the treatment solution C for 15 min and drying it at 37° C. to obtain a pre-treated reaction membrane;

[0112] (iv) Dilute the (1-3)-β-D-glucan-BSA conjugate to 0.33 mg / mL and the goat anti-mouse IgG to 0.5 mg / mL with pH 7.4 15 mM PBS, and spray them onto the pretreated reaction membrane in step (iii) at a flow rate of 3 μL / cm using a film spotter to draw lines as the test line and quality control line, respectively, and dry at 37°C to obtain a reaction membrane.

[0113] This embodiment also provides a method for preparing the human fluid fungus detection test paper, comprising the following steps: using a PVC base plate as a backing, pasting a sample pad, a fluorescent pad, a reaction membrane and a water-absorbing pad on the base plate in order, and cutting the detection line on the reaction membrane close to one end of the fluorescent pad into strips with a cutting machine to obtain a human fluid fungus detection test paper.

[0114] The only difference between Comparative Example 1 and Example 1 is that polyvinyl pyrrolidone is used in the treatment liquid A instead of the composite polymer.

[0115] The only difference between Comparative Example 2 and Example 1 is that ascorbic acid is used instead of the trehalose-ascorbic acid modified complex.

[0116] The only difference between Comparative Example 3 and Example 1 is that polyvinyl alcohol is used in the treatment liquid C instead of the composite polymer.

[0117] Experimental Example 1: Take 1 tube of (1-3)-β-D-glucan standard, disinfect it with 75% alcohol, open it, take out the standard, add it to heat-free water to dissolve, put it in a vortex mixer to mix for 5 min, prepare a 100 pg / mL (1-3)-β-D-glucan solution reference, add it dropwise to the sample pad of the test paper obtained in Example 1-3 and Comparative Example 1-3, let it stand for chromatography, and use the test paper prepared in Example 1-3 and Comparative Example 1-3 to test the accuracy and repeatability of the reference product. The results are as follows: Figure 2 shown.

[0118] Figure 2 The results show that the reference product was tested 10 times using the test paper of the present invention, and the repeatability CV of the test paper prepared in Examples 1-3 for the reference product was <10%, and the repeatability of the test kit prepared in Comparative Examples 1-3 was worse than that of Examples 1-3, indicating that the test paper of the present invention has high detection accuracy and good repeatability. In addition, the test paper of Example 1-3 was stored at 30°C for 18 months, and its CV still met <10%, which were 4.12%, 4.15% and 4.21% respectively, indicating that the test paper of the present invention has good stability.

[0119] Experimental Example 2: The fluorescence intensity of the T line is stronger than or equal to that of the C line. The fluorescence detector detects the fluorescence values ​​of the T line and the C line, and the T / C value is greater than or equal to 1, which is negative. The T line has no fluorescence or the fluorescence intensity is weaker than that of the C line. The fluorescence detector detects the fluorescence values ​​of the T line and the C line, and the T / C value is less than 1, which is positive. The C line has no fluorescence, which is invalid.

[0120] The test papers of Examples 1-3 of the present invention and Comparative Examples 1-3 were used to simultaneously detect 200 clinical negative samples (serum of healthy adults without liver, kidney and respiratory diseases and who had not used any antibiotics in the recent period), and 200 positive samples (including 100 serum samples, 50 urine samples, 30 pleural effusions and ascites samples, and 20 bronchial lavage fluid samples). According to the yin and yang of the test results, the sensitivity and specificity were calculated. Sensitivity (%) = (number of positive cases detected / number of positive samples) × 100%, specificity (%) = (number of negative cases detected / number of negative samples) × 100%. Clinical compliance rate = (number of cases where the detection results were consistent with the clinical results / total number of samples) × 100%. At the same time, when the readings were stable, the detection time was recorded, and the results were shown in Table 1.

[0121] Table 1:

[0122]

[0123] The results in Table 1 show that when the test strips of Examples 1-3 are used to detect clinical samples, the clinical compliance rate can reach 99%, with high sensitivity and good specificity, and the effect is better than that of Comparative Examples 1-3. The human body fluid fungus detection test strip of the present invention has strong sensitivity and specificity, high accuracy and fast detection speed.

[0124] Experimental Example 3: Take 1 (1-3)-β-D-glucan standard, disinfect with 75% alcohol, open, take out the standard, add pyrogen-free water, place in a vortex mixer and mix for 5 min, prepare a 100 pg / mL (1-3)-β-D-glucan solution, and dilute with pyrogen-free water to 80 pg / mL, 40 pg / mL, 20 pg / mL, 10 pg / mL to obtain a calibrator. The calibrator was added dropwise to the sample pads of the test papers obtained in Example 1 and Comparative Examples 1-3, respectively, and allowed to stand for chromatography. The fluorescence signal value was read using an immunofluorescence analyzer, and the T / C value was calculated. The results are shown in Table 2.

[0125] Table 2:

[0126]

[0127] The results in Table 2 show that when the test paper of Example 1 of the present invention is used for detection, the T / C value is less than 1 when the concentration of (1-3)-β-D-glucan is 20 pg / mL, which is positive, and the minimum detection limit is 20 pg / mL. The T / C value of the test strip of Comparative Example 1-3 is less than 1 at a concentration of 40 pg / mL, which is positive, and the minimum detection limit is 40 pg / mL, indicating that the human body fluid fungus detection test strip of the present invention has high sensitivity.

[0128] Experimental Example 4: Clinical negative and positive samples were randomly divided into two groups, each with 32 cases. 50 ng / mL of mannan was added to each sample in one group, and 5 mg / mL of triglyceride was added to the other group. The change rate of clinical compliance rate before and after the addition of interference was calculated. The results are shown in Table 3.

[0129] Table 3:

[0130]

[0131] The results in Table 3 show that the change rate of the clinical compliance rate of Example 2 is lower than that of Comparative Examples 1-3, indicating that the human body fluid fungus detection test paper of the present invention has strong anti-interference ability.

[0132] Experimental Example 5: The microsphere-antibody solution prepared in Example 3 and Comparative Example 1 was stored at 4°C for 6 months, and the state of the microsphere-antibody solution was observed. Figure 3 As shown, no precipitation occurred in Example 1 group, while a large amount of precipitation occurred at the bottom of Comparative Example 2 group, indicating that the reconstituted storage solution of the conjugate of the present invention can effectively improve the stability and extend the shelf life.

[0133] During the test, if the C band of the test strip does not develop color, it means that the test strip has expired and the result is invalid. The test strips of Examples 1-3 and Comparative Examples 1-3 were stored at 30°C, and the time when the C band did not develop color was recorded. The results showed that the C band of Examples 1-3 did not develop color after 24 months of storage, Comparative Examples 1 and 3 did not develop color after 21 months of storage, and Comparative Example 2 did not develop color after 20 months of storage. This shows that the test strip of the present invention has good stability and can extend the service life.

Claims

1. A human body fluid fungus detection test strip, characterized in that: Including PVC base plate, sample pad, fluorescent pad, reaction membrane and water absorbent pad; The method for preparing the sample pad comprises the following steps: (1) heating ethylene glycol methyl ether to 160° C., adding dropwise a mixed solution of N-vinyl pyrrolidone, N,N-dimethylacrylamide, and di-tert-butyl peroxide, and maintaining the temperature after the addition, cooling to 70° C., and stirring to obtain a polyvinyl pyrrolidone copolymer; (2) adding N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to the polyvinylpyrrolidone copolymer obtained in step (1), stirring, adding inulin, continuing stirring, dialyzing, centrifuging, and freeze-drying the supernatant to obtain a composite polymer; (3) adding the composite polymer obtained in step (2), casein and Tween-20 to PBS buffer and mixing evenly to obtain treatment solution A; (4) soaking the glass fiber membrane in the treatment solution A obtained in step (3) and drying it to obtain a sample pad; The preparation method of the fluorescent pad comprises the following steps: (a) Mixing the fluorescent microspheres with MES buffer, sonicating, centrifuging to remove the supernatant, re-adding MES buffer, sonicating, and obtaining a fluorescent microsphere mixed solution; (b) using MES buffer to prepare NHS solution and EDAC solution respectively, and adding them dropwise to the fluorescent microsphere mixed solution of step (a) to make the final concentrations of NHS and EDAC be 0.8 mg / mL and 0.3 mg / mL, respectively, and sonicate, react at 37°C in the dark for 1 h to obtain a mixed solution A; (c) centrifuging the mixed solution A in step (b), washing the precipitate, adding the fungal (1-3)-β-D glucan antibody solution, sonicating, reacting in the dark, adding the bovine serum albumin solution, sonicating for 1 min, rotating in the dark, and centrifuging to obtain a microsphere-antibody conjugate precipitate; (d) Dissolve trehalose and oxalic anhydride in acetonitrile, add NaOH, stir, concentrate under reduced pressure, and recrystallize in a mixed solution of ethanol and ether to obtain carboxylated trehalose; dissolve ascorbic acid in dichloromethane, add thionyl chloride at 0°C, stir at 25°C, concentrate under reduced pressure, wash, and dry. Dissolve the dried product in 1wt% acetic acid solution, add 3,4-dihydroxybenzaldehyde, stir, add sodium borohydride and continue stirring, and filter to obtain a filter cake; mix the filter cake with carboxylated trehalose and deionized water to obtain a mixed solution B, add 3 mol / L hydrochloric acid solution and distilled water, mix well, stir, centrifuge, filter the supernatant, and freeze-dry to obtain a trehalose-ascorbic acid modified complex; (e) adding the trehalose-ascorbic acid modified complex obtained in step (d), glycine and dithiothreitol to the HEPES buffer solution and mixing them evenly to obtain a conjugate reconstitution storage solution; washing the microsphere-antibody conjugate precipitate obtained in step (c) with PBS buffer, adding it to the conjugate reconstitution storage solution, resuspending and mixing, and obtaining a microsphere-antibody solution; (f) adding glucose and borax to a 4% w / v bovine serum albumin solution to obtain a treatment solution B, soaking the polyester film in the treatment solution B and then drying it to obtain a pretreated polyester film; (g) diluting the microsphere-antibody solution obtained in step (e) with PBS buffer, spraying it on the pretreated polyester film obtained in step (f), and drying it to obtain a fluorescent pad; The method for preparing the reaction film comprises the following steps: (i) mixing (1-3)-β-D glucan and deionized water to obtain a 20 mg / L glucan solution, adding the glucan solution to a 10 wt % hydrogen peroxide solution, stirring, adding diethylene glycol, continuing to stir, ultrafiltering, and freeze-drying to obtain oxidized glucan; (ii) mixing equal volumes of phosphate buffer and acetate buffer to obtain a mixed buffer, adding BSA and the oxidized dextran obtained in step (i) to the mixed buffer, reacting at 4° C., centrifuging, and dialyzing the supernatant to obtain a (1-3)-β-D-glucan-BSA conjugate; (iii) adding the composite polymer obtained in step (2) and sodium chloride to deionized water to obtain a treatment solution C, soaking the nitrocellulose membrane in the treatment solution C and drying it to obtain a pre-treated reaction membrane; (iv) diluting the (1-3)-β-D-glucan-BSA conjugate to 0.33 mg / mL and the goat anti-mouse IgG to 0.5 mg / mL with PBS, spraying them on the pretreated reaction membrane in step (iii) to draw lines as the detection line and quality control line, respectively, and drying to obtain a reaction membrane.

2. The human body fluid fungus detection test strip according to claim 1, characterized in that: The mass ratio of ethylene glycol methyl ether, N-vinyl pyrrolidone, N,N-dimethylacrylamide and di-tert-butyl peroxide in step (1) is 40:20:3:2.7; the dropping speed of the mixed solution in step (1) is 1.5 g / min.

3. The human body fluid fungus detection test strip according to claim 2, characterized in that: The mass ratio of the polyvinyl pyrrolidone copolymer to N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and inulin in step (2) is 50:15:1:20; the amount ratio of the PBS buffer, composite polymer, Tween-20 and casein in step (3) is 1000 mL:10 g:10 mL:1 g.

4. The human body fluid fungus detection test paper according to claim 3, characterized in that: The fluorescent microspheres in step (a) include time-resolved fluorescent microspheres; the surface modification functional groups of the fluorescent microspheres include hydroxyl groups or carboxyl groups; the material of the fluorescent microspheres is polystyrene, polymethyl methacrylate or silica; the concentration of the fluorescent microspheres in the fluorescent microsphere mixture in step (a) is 12.5 mg / mL.

5. The human body fluid fungus detection test strip according to claim 4, characterized in that: The mass ratio of the washed precipitate to the fungus (1-3)-β-D glucan antibody in step (c) is 10:1; the dosage ratio of the bovine serum albumin solution to the washed precipitate in step (c) is 1 mL:0.1 g.

6. The human body fluid fungus detection test strip according to claim 5, characterized in that: The amount ratio of trehalose, oxalic anhydride, acetonitrile and sodium hydroxide in step (d) is 0.2 mol:0.1 mol:200 mL:0.3 mol; the volume ratio of ethanol and ether in the ethanol and ether mixed solution in step (d) is 1:3; the amount ratio of ascorbic acid, dichloromethane and dichloride in step (d) is 0.12 mol:200 mL:0.1 mL; the amount ratio of the dried product, acetic acid solution, 3,4-dihydroxybenzaldehyde and sodium borohydride in step (d) is 15 g:1000 mL:1 g:0.5 g; the amount ratio of carboxylated trehalose, filter cake and deionized water in step (d) is 1 g:10 g:50 mL; the volume ratio of mixed solution B, hydrochloric acid solution and distilled water in step (d) is 25:1:

1.

7. The human body fluid fungus detection test strip according to claim 6, characterized in that: The amount ratio of the HEPES buffer solution, trehalose-ascorbic acid modified complex, glycine, and dithiothreitol in step (e) is 100 mL: 2 g: 1 g: 0.5 g; the amount ratio of the microsphere antibody conjugate precipitate after cleaning and the conjugate re-dissolution storage solution in step (e) is 10 mg: 1 mL; the amount ratio of the bovine serum albumin solution, glucose, and borax in step (f) is 100 mL: 3 g: 2 mmol.

8. The human body fluid fungus detection test strip according to claim 7, characterized in that: The volume ratio of the dextran solution, hydrogen peroxide solution and diethylene glycol in step (i) is 10:1:1; the dosage ratio of BSA, oxidized dextran and mixed buffer in step (ii) is 1 g:2 g:100 mL; and the dosage ratio of deionized water, composite polymer and sodium chloride in step (iii) is 100 mL:0.3 g:1 g.

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