CN118914550B - A human fluid fungus detection test paper and preparation method thereof - Google Patents
A human fluid fungus detection test paper and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biological detection, in particular to human body liquid fungus detection test paper and a preparation method thereof. Wherein, the fluorescent pad is coated with fluorescent microsphere marked antibody, the reaction film is provided with a detection line and a quality control line, the detection line is coated with (1-3) -beta-D glucan-bovine serum albumin conjugate, and the quality control line is coated with goat anti-mouse IgG secondary antibody. The test strip provided by the invention can be used for rapidly detecting the (1-3) -beta-D glucan, and has the advantages of high sensitivity, good accuracy and strong repeatability and stability.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to human body fluid fungus detection test paper and a preparation method thereof.
Background
The (1-3) -beta-D glucan is widely existing in fungus cell walls and accounts for more than 50% of the mass of fungus cell stems, all fungus cell wall components except for the conjugant (mainly rhizopus and mucor) contain the (1-3) -beta-D glucan, the yeast-like fungus content is highest, and human cells, viruses, prokaryotes and the like do not contain the component, so that the method is an ideal marker for detecting deep fungus infection. When the fungus invades human blood or deep tissues and causes deep infection, after phagocytic digestion of the fungal cells by phagocytes, (1-3) -beta-D glucan is released from the cell wall, resulting in a significant increase in this component in blood and body fluids. The (1-3) -beta-D glucan has 2 existing forms, one is a lower structure in a single-chain form, the other is a higher structure (1-3) -beta-D glucan in a double-strand spiral or triple-strand spiral form, is neutral, contains a small amount of beta-1, 6 glycosidic bond connecting structure, forms a compact triple-spiral structure under the action of polyhydroxy in a molecule, and has the characteristics of high immunocompetence and low solubility. Therefore, (1-3) -beta-D glucan quantitative detection is a powerful basis for diagnosing deep fungal infections and can be used for identifying invasive mycoses (Invasive Fungal Disease, IFD). Because of the high risk of IFD and high incidence, it is becoming an important factor for health hazard, especially for immunocompromised patients or hospitalized patients with severe basic diseases. Therefore, rapid and accurate early diagnosis of deep fungal infections is of great importance. At present, a plurality of dextran chromogenic detection kits based on a limulus reagent have been developed, and the kit has the advantages of rapidness, sensitivity, strong specificity, good repeatability and the like, and has been widely applied clinically. The disadvantages are that the detection time is long, and the limulus reagent produced by the conventional method is easily interfered by endotoxin, thus causing false positive. Horseshoe crab is a national secondary protective animal, and thus, development of a kit for detecting glucose based on immunology is urgent. Such as magnetic particle chemiluminescence technology, but has the disadvantages of being easily interfered by biotin in a sample and low detection accuracy. The fluorescence immunochromatography is applied to IFD detection, has the advantages of simple operation, rapid detection, low equipment requirement, card-inserting type reading, easy judgment of results and great shortening of detection time. Provides an effective auxiliary means for detecting susceptible people. In summary, there is currently no immunochromatographic test paper for detecting fungi (1-3) -beta-D glucan, and the existing detection method and kit have low detection accuracy and low sensitivity. How to provide an immunochromatographic test paper for detecting (1-3) -beta-D glucan and a preparation method thereof, which do not need heating treatment and improve the accuracy and the sensitivity of detection, is one of the problems to be solved by the technicians in the field.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides human body liquid fungus detection test paper and a preparation method thereof.
The invention is realized by the following technical scheme:
A human body liquid fungus detection test paper comprises a PVC base plate, a sample pad, a fluorescent pad, a reaction membrane and a water absorption pad, wherein the fluorescent pad is coated with an antibody marked by fluorescent microspheres, the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with a (1-3) -beta-D glucan-bovine serum albumin conjugate, and the quality control line is coated with a goat anti-mouse IgG secondary antibody.
Further, the preparation method of the sample pad comprises the following steps:
(1) Heating ethylene glycol methyl ether to 160 ℃ in a nitrogen environment, dropwise adding a mixed solution consisting of N-vinyl pyrrolidone, N-dimethylacrylamide and di-tert-butyl peroxide, keeping the temperature at 160 ℃ for 1h after the dropwise adding, cooling to 70 ℃, and stirring for 1h at 180 rpm to obtain a polyvinylpyrrolidone copolymer;
(2) Adding N, N' -dicyclohexylcarbodiimide and 4-dimethylaminopyridine into the polyvinylpyrrolidone copolymer obtained in the step (1), stirring at 60 ℃ and 100 rpm for 1:1 h, adding inulin, continuously stirring for 24: 24 h, placing into a dialysis bag with a molecular weight cutoff of 7000 Da, continuously dialyzing with deionized water for 48: 48 h, collecting a product in the dialysis bag, centrifuging at 4000: 4000 rpm for 10: 10 min, and freeze-drying the supernatant to obtain a composite polymer;
(3) Adding the composite polymer obtained in the step (2), casein and Tween-20 into 20 mM PBS buffer solution with pH of 7.4, and uniformly mixing to obtain a treatment solution A;
(4) And (3) soaking the glass fiber membrane in the treatment liquid A obtained in the step (3) for 2 hours, and drying at 37 ℃ to obtain the sample pad.
Further, the mass ratio of the ethylene glycol methyl ether, the N-vinyl pyrrolidone, the N, N-dimethylacrylamide and the di-tert-butyl peroxide in the step (1) is 40:20:3:2.7.
Further, the dropping speed of the mixed solution in the step (1) is 1.5 g/min.
Further, the mass ratio of the polyvinylpyrrolidone copolymer in the step (2) to the N, N' -dicyclohexylcarbodiimide, 4-dimethylaminopyridine and inulin is 50:15:1:20.
Further, the dosage ratio of the PBS buffer solution, the composite polymer, the Tween-20 and the casein in the step (3) is 1000 mL:10 g:10 mL:1 g.
Further, the preparation method of the fluorescent pad comprises the following steps:
(a) Mixing the fluorescent microsphere with 50 mmol/L MES buffer with pH 6.0, performing ultrasonic treatment for 1 min at 150W, centrifuging for 20min at 8-10 ℃ at 12000 and g, removing supernatant, adding 50 mmol/L MES buffer with pH 6.0 again, and performing ultrasonic treatment for 1 min again to obtain fluorescent microsphere mixed solution;
(b) Preparing 10 mg/mL of NHS solution and 5 mg/mL of EDAC solution respectively by using 50 mM of MES buffer solution with pH of 6.0, dropwise adding the mixed solution into the fluorescent microsphere mixed solution obtained in the step (a) until the final concentration of NHS and EDAC is 0.8 mg/mL and 0.3 mg/mL, carrying out ultrasonic treatment for 1 min at 150W, and carrying out light-shielding reaction at 37 ℃ for 1h to obtain a mixed solution A;
(c) Centrifuging the mixed solution A in the step (b) at 8 ℃ and 12000 g for 20 min, discarding the supernatant, washing the precipitate for 2 times by using a 50 mM pH 8.0 HEPES buffer solution, adding a fungus (1-3) -beta-D glucan antibody solution, performing ultrasonic treatment for 150W for 1 min, performing light-proof reaction for 1h at 37 ℃, adding a bovine serum albumin solution at 20 mg/mL, performing ultrasonic treatment for 1 min, performing light-proof rotation for 4 ℃ and 60 rpm for 8 hours, performing centrifugal treatment for 12000 g for 20 min, and discarding the supernatant to obtain microsphere antibody conjugate precipitate;
(d) Dissolving ascorbic acid in methylene dichloride, adding thionyl chloride under the condition of 0 ℃ ice water bath, stirring for 0.5h, 0.01 MPa, 60 ℃ for 10 h, washing with deionized water, drying at 80 ℃, dissolving the dried product in 1wt% acetic acid solution, adding 3, 4-dihydroxybenzaldehyde, stirring for 3-4 h at 30 ℃ and 180 rpm, adding sodium borohydride, continuously stirring for 1h, carrying out suction filtration to obtain a filter cake, uniformly mixing the filter cake with carboxylated trehalose and deionized water to obtain a mixed solution B, adding 3 mol/L hydrochloric acid solution and distilled water, uniformly mixing, stirring for 6-10 h at 70-90 ℃, centrifuging at 8000rpm for 30min, filtering the supernatant with a 0.22 mu m filter membrane, and drying to obtain a modified seaweed compound;
(e) Adding the trehalose-ascorbic acid modified compound obtained in the step (d) into 10 mmol/L HEPES buffer solution, and uniformly mixing glycine and dithiothreitol to obtain conjugate redissolved storage solution; washing the microsphere antibody conjugate precipitate obtained in the step (c) with a PBS buffer solution with the pH of 7.4 mM for 2 times, adding the conjugate into a conjugate re-dissolution storage solution, and re-suspending the conjugate solution uniformly to obtain microsphere-antibody liquid;
(f) Adding glucose and borax into 4% w/v bovine serum albumin solution to obtain a treatment solution B, soaking a polyester film in the treatment solution B for 2h ℃, and drying at 37 ℃ to obtain a pretreated polyester film;
(g) Diluting the microsphere-antibody solution prepared in the step (e) to 1 mg/mL by using 50 mM PBS buffer solution, uniformly spraying the microsphere-antibody solution on the pretreated polyester film prepared in the step (f) at a flow rate of 3 mu L/cm by using a quantitative film spraying instrument, and drying at 37 ℃ to obtain the fluorescent pad.
Further, the fluorescent microsphere in the step (a) comprises a time-resolved fluorescent microsphere, the surface modification functional group of the fluorescent microsphere comprises hydroxyl or carboxyl, and the fluorescent microsphere is made of polystyrene, polymethyl methacrylate or silicon dioxide.
Further, the concentration of the fluorescent microspheres in the fluorescent microsphere mixed solution in the step (a) is 12.5 mg/mL.
Further, the mass ratio of the washed precipitate to the fungal (1-3) - β -D glucan antibody of step (c) is 10:1.
Further, the ratio of the bovine serum albumin solution of step (c) to the amount of precipitate after washing was 1 mL:0.1 g.
Further, the dosage ratio of trehalose, oxalic anhydride, acetonitrile and sodium hydroxide in the step (d) is 0.2 mol:0.1 mol:200 mL:0.3 mol.
Further, the volume ratio of the ethanol to the diethyl ether in the ethanol and diethyl ether mixed solution in the step (d) is 1:3.
Further, the dosage ratio of the ascorbic acid, the dichloromethane and the thionyl chloride in the step (d) is 0.12 mol:200 mL:0.1 mL.
Further, the dried product of step (d), acetic acid solution, 3, 4-dihydroxybenzaldehyde and sodium borohydride are used in a ratio of 15 g:1000 mL:1 g:0.5 g.
Further, the dosage ratio of carboxylated trehalose, filter cake and deionized water in step (d) is 1 g to 10 g to 50 mL.
Further, the volume ratio of the mixed solution B, the hydrochloric acid solution and the distilled water in the step (d) is 25:1:1.
Further, the HEPES buffer solution, the trehalose-ascorbic acid modified complex, glycine and dithiothreitol in the step (e) are used in an amount ratio of 100 mL:2 g:1 g:0.5 g.
Further, the ratio of the amount of the washed microsphere antibody conjugate precipitate to the conjugate reconstitution stock solution in step (e) is 10 mg/1 mL.
Further, the usage ratio of the bovine serum albumin solution, glucose and borax in the step (f) is 100 mL:3 g:2 mmol.
Further, the preparation method of the reaction membrane comprises the following steps:
(i) Uniformly mixing (1-3) -beta-D glucan with deionized water to obtain a glucan solution of 20 mg/L, adding the glucan solution into a hydrogen peroxide solution of 10wt%, stirring 120-rpm for 18-20 h, adding diethylene glycol, continuously stirring for 2-h, placing the mixture in a 10 kDa ultrafiltration membrane for filtration, and freeze-drying to obtain oxidized glucan;
(ii) Uniformly mixing an equal volume of 0.1 mol/L phosphate buffer with 0.1 mol/L acetate buffer with pH 5.0 to obtain a mixed buffer, adding Bovine Serum Albumin (BSA) and oxidized dextran obtained in the step (i) into the mixed buffer, reacting for 14-16 h at 4 ℃, centrifuging at 4000 rpm for 10min, placing the supernatant in a dialysis bag, dialyzing with PBS buffer with pH 7.4 and 50mM at 4 ℃ for 96 h to obtain (1-3) -beta-D glucan-BSA conjugate;
(iii) Adding the composite polymer obtained in the step (2) and sodium chloride into deionized water to obtain a treatment solution C, soaking a nitrocellulose membrane in the treatment solution C for 15 to min, and drying at 37 ℃ to obtain a pretreatment reaction membrane;
(iv) Diluting the (1-3) -beta-D glucan-BSA conjugate to 0.33 mg/mL by using PBS with the pH of 7.4 mM, diluting the goat anti-mouse IgG to 0.5 mg/mL, spraying the diluted solution on the pretreatment reaction membrane in the step (iii) at the flow rate of 3 mu L/cm by using a spot film instrument to mark lines, respectively serving as a detection line (T line) and a quality control line (C line), and drying at 37 ℃ to obtain the reaction membrane.
Further, the volume ratio of the dextran solution, the hydrogen peroxide solution and the diglycol in the step (i) is 10:1:1.
Further, the ratio of the BSA, oxidized dextran, and mixing buffer in step (ii) is 1 g:2 g:100 mL.
Further, the dosage ratio of deionized water, the composite polymer and sodium chloride in step (iii) is 100 mL:0.3 g:1 g.
Further, the water absorbing pad is water absorbing paper.
The invention also provides a preparation method of the human body liquid fungus detection test paper, which comprises the following steps of sequentially adhering a sample pad, a fluorescent pad, a reaction film and a water absorption pad to a substrate by taking a PVC substrate as a back, and cutting a detection line on the reaction film near one end of the fluorescent pad into strips with the width of 3.00 mm by a cutting machine to obtain the human body liquid fungus detection test paper.
Compared with the prior art, the invention has the following beneficial effects:
The nitrocellulose membrane and the glass fiber which are materials used in the fluorescence immunochromatography contain (1-3) -beta-D glucan, and the fluorescence immunochromatography is used for respectively carrying out secondary treatment on the materials by using different treatment liquids, so that the interference of the (1-3) -beta-D glucan in the materials is eliminated, and the sensitivity of the fluorescence immunochromatography detection is enhanced. The sample treatment fluid and the treatment mode are also optimized, and compared with the sample treatment of the dextran chromogenic detection kit based on the limulus reagent, which is heated and incubated after alkali treatment (the sample treatment in the kit of the enzyme-linked immunosorbent assay and the magnetic particle chemiluminescence method usually needs to be incubated), the sample treatment fluid and the treatment mode do not need heating treatment, are simple and convenient to operate, and improve the sensitivity and the specificity of fluorescence immunochromatography detection. The composite polymer can effectively reduce the nonspecific adsorption of antibodies, thereby improving the detection sensitivity of the test strip, increasing the hydrophilicity of the sample pad, simultaneously, the aqueous solution has suspension dispersion function, can be used for protecting antigens in a sample, is favorable for detecting the antigens in the sample and the antibodies in the test strip to react rapidly, has good hydrophobicity, can be well used in sol, can not be dissolved in water along with the solution flow, and reduces the background rise caused by the use environment. The treatment fluid of the reaction membrane is added with a composite polymer, so that the background interference can be reduced, the occurrence of non-specific binding is reduced, the detection accuracy and sensitivity are improved, the hydrophilicity is improved, the absorption efficiency of a sample on the membrane is improved, the sensitivity of the reaction is enhanced, the binding capacity of an antibody is improved, and the binding force of the antibody and the membrane is enhanced, thereby improving the detection specificity and sensitivity, the reaction speed and the reaction time. According to the invention, the trehalose-ascorbic acid modified compound is prepared, the biological activities of trehalose and ascorbic acid are reserved by a way of preparing the trehalose-ascorbic acid modified compound, the stability and antibacterial and antioxidant effects of the ascorbic acid are improved, the water solubility is further improved by introducing the trehalose, the toxicity is reduced, the obtained trehalose-ascorbic acid modified compound is added into conjugate redissolution storage liquid, the sterilization and antioxidant effects can be effectively realized, the stability of microsphere antibody conjugate is improved, the storage life is prolonged, the aggregation and precipitation of antibodies can be prevented, the activity and the function of the antibodies are maintained, and the detection sensitivity and accuracy are effectively improved.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only of the invention and that other drawings can be obtained from them without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the human body fluid fungus test paper according to the present invention;
FIG. 2 is a graph showing the reproducibility of test strips according to examples 1-3 and comparative examples 1-3 of the present invention;
FIG. 3 is a photograph of the microsphere-antibody liquid of example 3 and comparative example 2 of the present invention after storage.
Detailed Description
The present invention will be further described in detail with reference to the following specific examples, but the present invention is not limited to the following examples, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.
Unless otherwise specified, the chemical reagents involved in the present invention are all commercially available.
Embodiment 1. The embodiment provides a human body liquid fungus test paper, which comprises a PVC bottom plate, a sample pad, a fluorescent pad, a reaction film and a water absorption pad, wherein the water absorption pad is water absorption paper.
The preparation method of the sample pad comprises the following steps:
(1) Heating ethylene glycol methyl ether to 160 ℃ in a nitrogen environment, dropwise adding a mixed solution consisting of N-vinyl pyrrolidone, N-dimethylacrylamide and di-tert-butyl peroxide at the speed of 1.5 g/min, wherein the mass ratio of the ethylene glycol methyl ether to the N-vinyl pyrrolidone to the N, N-dimethylacrylamide to the di-tert-butyl peroxide is 40:20:3:2.7, preserving heat at 160 ℃ for 1 h after the dropwise adding is completed, cooling to 70 ℃, and stirring for 1 h at 180 rpm to obtain a polyvinylpyrrolidone copolymer;
(2) Adding N, N' -dicyclohexylcarbodiimide and 4-dimethylaminopyridine into the polyvinylpyrrolidone copolymer obtained in the step (1), stirring at 60 ℃ and 100 rpm for 1: 1h, adding inulin, continuously stirring at 24: 24 h, placing into a dialysis bag with a molecular weight cutoff of 7000 Da, continuously dialyzing with deionized water for 48 h, collecting the product in the dialysis bag, centrifuging at 4000 rpm for 10: 10min, taking supernatant, and freeze-drying to obtain a composite polymer;
(3) Adding the composite polymer obtained in the step (2), casein and Tween-20 into 20mM PBS buffer with pH of 7.4, and uniformly mixing, wherein the dosage ratio of the PBS buffer to the composite polymer to the Tween-20 to the casein is 1000 mL:10 g:10 mL:1 g, so as to obtain a treatment solution A;
(4) And (3) soaking the glass fiber membrane in the treatment liquid A obtained in the step (3) for 2 hours, and drying at 37 ℃ to obtain the sample pad.
The preparation method of the fluorescent pad comprises the following steps:
(a) Mixing the hydroxyl polystyrene time-resolved fluorescent microsphere with 50 mmol/L MES buffer with pH 6.0, carrying out ultrasonic treatment on the mixture for 1 min at 150W, centrifuging the mixture for 20 min at 10 ℃ at 12000 and g, removing supernatant, adding 50 mmol/L MES buffer with pH 6.0 again, carrying out ultrasonic treatment on the mixture for 1 min again, and obtaining fluorescent microsphere mixed solution, wherein the concentration of fluorescent microsphere in the fluorescent microsphere mixed solution is 12.5 mg/mL;
(b) Preparing 10 mg/mL of NHS solution and 5 mg/mL of EDAC solution respectively by using 50 mM of MES buffer solution with pH of 6.0, dropwise adding the mixed solution into the fluorescent microsphere mixed solution obtained in the step (a) until the final concentration of NHS and EDAC is 0.8 mg/mL and 0.3 mg/mL, carrying out ultrasonic treatment for 1 min at 150W, and carrying out light-shielding reaction at 37 ℃ for 1h to obtain a mixed solution A;
(c) Centrifuging the mixed solution A in the step (b) at 8 ℃ and 12000 g for 20 min, discarding supernatant, washing the precipitate for 2 times by using 50 mM pH 8.0 HEPES buffer solution, adding a fungus (1-3) -beta-D glucan antibody solution, performing ultrasonic treatment for 1 min at 37 ℃ with the mass ratio of the washed precipitate to the fungus (1-3) -beta-D glucan antibody being 10:1, performing light-shielding reaction for 1h at 37 ℃, adding 20 mg/mL of bovine serum albumin solution, performing rotation for 8h with the use ratio of the bovine serum albumin solution to the washed precipitate being 1 mL:0.1 g, performing ultrasonic treatment for 1 min, performing light-shielding for 4 ℃, performing rotation for 60 rpm h at 8 ℃, and performing centrifugal treatment for 20 min with 12000 g to obtain microsphere antibody conjugate precipitate;
(d) Dissolving trehalose and oxalic anhydride in acetonitrile, adding NaOH, stirring for 7 h at the temperature of 70 ℃, stirring for 7 h at the temperature of 120 rpm, concentrating for 6h at the temperature of 0.01 MPa and 60 ℃ under reduced pressure, recrystallizing in a mixed solution of ethanol and diethyl ether (volume ratio of 1:3) to obtain carboxylated trehalose, dissolving ascorbic acid in dichloromethane, adding thionyl chloride under the condition of 0 ℃ ice water bath, stirring for reacting for 0.5 h at the temperature of 0.12 mol:200 mL:0.1 mL,25 ℃ in the ratio of the ascorbic acid, dichloromethane and thionyl chloride, stirring for reacting for 100 rpm h, stirring for 10 h at the temperature of 0.01 MPa and 60 ℃, washing with deionized water, drying at the temperature of 80 ℃, dissolving the dried product in a 1wt% acetic acid solution, adding 3, 4-dihydroxybenzaldehyde, stirring for 4h at the temperature of 30 ℃ and 180 ℃, adding sodium borohydride, continuously stirring for 1h, stirring for the dried product, acetic acid solution, 3, 4-dihydroxybenzaldehyde and sodium borohydride at the volume ratio of 15 g:1000 mL:1 g:0.5 g, filtering to obtain a filter cake, uniformly mixing the filter cake with the carboxylated trehalose, stirring for 1:8000 g of the mixed solution of the seaweed with distilled water, stirring for obtaining a mixed solution of hydrochloric acid and water at the volume ratio of 1:25 g, and 80:50 g, stirring for obtaining a mixed solution of hydrochloric acid and hydrochloric acid, and mixed solution after stirring for 80 g, and stirring for 1:50 g, and stirring for obtaining a mixed solution of hydrochloric acid solution, and distilled solution after stirring and stirring for 1:1:10:10:10:1;
(e) Adding the trehalose-ascorbic acid modified complex obtained in the step (d) and glycine and dithiothreitol into 10 mmol/L HEPES buffer solution, and uniformly mixing, wherein the dosage ratio of the HEPES buffer solution to the trehalose-ascorbic acid modified complex to the glycine to the dithiothreitol is 100 mL:2 g:1 g:0.5 g, so as to obtain conjugate redissolution storage solution; washing the microsphere antibody conjugate precipitate obtained in the step (c) for 2 times by using a PBS buffer solution with the pH of 7.4 mM, adding the mixture into a conjugate re-dissolution storage solution, and re-suspending the mixture uniformly, wherein the dosage ratio of the washed microsphere antibody conjugate precipitate to the conjugate re-dissolution storage solution is 10 mg/1 mL, so as to obtain microsphere-antibody liquid;
(f) Adding glucose and borax into 4% w/v bovine serum albumin solution to obtain a treatment solution B, wherein the dosage ratio of the bovine serum albumin solution to the glucose to the borax is 100 mL:3 g:2 mmol, soaking a polyester film in the treatment solution B for 2 h, and drying at 37 ℃ to obtain a pretreated polyester film;
(g) Diluting the microsphere-antibody solution prepared in the step (e) to 1 mg/mL by using 50 mM PBS buffer solution, uniformly spraying the microsphere-antibody solution on the pretreated polyester film prepared in the step (f) at a flow rate of 3 mu L/cm by using a quantitative film spraying instrument, and drying at 37 ℃ to obtain the fluorescent pad.
The preparation method of the reaction membrane comprises the following steps:
(i) Uniformly mixing (1-3) -beta-D glucan and deionized water to obtain a glucan solution of 20 mg/L, adding the glucan solution into a hydrogen peroxide solution of 10wt%, stirring 20h by 120 rpm, adding diethylene glycol, continuously stirring 2h, filtering by using an ultrafiltration membrane of 10 kDa, and freeze-drying to obtain oxidized glucan, wherein the volume ratio of the glucan solution to the hydrogen peroxide solution to the diethylene glycol is 10:1:1;
(ii) Uniformly mixing an equal volume of 0.1 mol/L phosphate buffer with 0.1 mol/L acetate buffer with pH 5.0 to obtain a mixed buffer, adding BSA and oxidized dextran obtained in the step (i) into the mixed buffer, reacting the BSA, the oxidized dextran and the mixed buffer for 16 hours at 4 ℃ at a dosage ratio of 1 g to 2 g to 100 mL, centrifuging at 10 min at 4000 rpm, placing the supernatant in a dialysis bag at 4 ℃ and dialyzing at 96 h with PBS buffer with pH 7.4 mM to obtain (1-3) -beta-D dextran-BSA conjugate;
(iii) Adding the composite polymer and sodium chloride obtained in the step (2) into deionized water, wherein the dosage ratio of the deionized water to the composite polymer to the sodium chloride is 100 mL:0.3 g:1 g, so as to obtain a treatment solution C, soaking the nitrocellulose membrane in the treatment solution C for 15min ℃ and then drying at 37 ℃ so as to obtain a pretreatment reaction membrane;
(iv) Diluting the (1-3) -beta-D glucan-BSA conjugate to 0.33 mg/mL by using PBS with the pH of 7.4 mM, diluting the goat anti-mouse IgG to 0.5 mg/mL, spraying the diluted solution on the pretreatment reaction membrane in the step (iii) at the flow rate of 3 mu L/cm by using a spot film instrument to mark lines, respectively serving as a detection line (T line) and a quality control line (C line), and drying at 37 ℃ to obtain the reaction membrane.
The embodiment also provides a preparation method of the human body liquid fungus detection test paper, which comprises the following steps of sequentially adhering a sample pad, a fluorescent pad, a reaction film and a water absorption pad to a substrate by taking a PVC substrate as a back, cutting a detection line on the reaction film near one end of the fluorescent pad into strips with the width of 3.00 mm by using a cutting machine, and obtaining the human body liquid fungus detection test paper, wherein the structure schematic diagram is shown in figure 1.
Embodiment 2 the fungus test paper for human body fluid comprises a PVC bottom plate, a sample pad, a fluorescent pad, a reaction film and a water absorption pad, wherein the water absorption pad is water absorption paper.
The preparation method of the sample pad comprises the following steps:
(1) Heating ethylene glycol methyl ether to 160 ℃ in a nitrogen environment, dropwise adding a mixed solution consisting of N-vinyl pyrrolidone, N-dimethylacrylamide and di-tert-butyl peroxide at the speed of 1.5 g/min, wherein the mass ratio of the ethylene glycol methyl ether to the N-vinyl pyrrolidone to the N, N-dimethylacrylamide to the di-tert-butyl peroxide is 40:20:3:2.7, preserving heat at 160 ℃ for 1 h after the dropwise adding is completed, cooling to 70 ℃, and stirring for 1 h at 180 rpm to obtain a polyvinylpyrrolidone copolymer;
(2) Adding N, N' -dicyclohexylcarbodiimide and 4-dimethylaminopyridine into the polyvinylpyrrolidone copolymer obtained in the step (1), stirring at 60 ℃ and 100 rpm for 1: 1h, adding inulin, continuously stirring at 24: 24 h, placing into a dialysis bag with a molecular weight cutoff of 7000 Da, continuously dialyzing with deionized water for 48 h, collecting the product in the dialysis bag, centrifuging at 4000 rpm for 10: 10min, taking supernatant, and freeze-drying to obtain a composite polymer;
(3) Adding the composite polymer obtained in the step (2), casein and Tween-20 into 20mM PBS buffer with pH of 7.4, and uniformly mixing, wherein the dosage ratio of the PBS buffer to the composite polymer to the Tween-20 to the casein is 1000 mL:10 g:10 mL:1 g, so as to obtain a treatment solution A;
(4) And (3) soaking the glass fiber membrane in the treatment liquid A obtained in the step (3) for 2 hours, and drying at 37 ℃ to obtain the sample pad.
The preparation method of the fluorescent pad comprises the following steps:
(a) Mixing the hydroxyl polystyrene time-resolved fluorescent microsphere with 50 mmol/L MES buffer with pH 6.0, carrying out ultrasonic treatment on the mixture for 1 min at 150W, centrifuging the mixture for 20min at 8 ℃ at 12000 and g, removing supernatant, adding 50 mmol/L MES buffer with pH 6.0 again, carrying out ultrasonic treatment on the mixture for 1 min again, and obtaining fluorescent microsphere mixed solution, wherein the concentration of fluorescent microsphere in the fluorescent microsphere mixed solution is 12.5 mg/mL;
(b) Preparing 10 mg/mL of NHS solution and 5 mg/mL of EDAC solution respectively by using 50 mM of MES buffer solution with pH of 6.0, dropwise adding the mixed solution into the fluorescent microsphere mixed solution obtained in the step (a) until the final concentration of NHS and EDAC is 0.8 mg/mL and 0.3 mg/mL, carrying out ultrasonic treatment for 1 min at 150W, and carrying out light-shielding reaction at 37 ℃ for 1h to obtain a mixed solution A;
(c) Centrifuging the mixed solution A in the step (b) at 8 ℃ and 12000 g for 20 min, discarding supernatant, washing the precipitate for 2 times by using 50 mM pH 8.0 HEPES buffer solution, adding a fungus (1-3) -beta-D glucan antibody solution, performing ultrasonic treatment for 1 min at 37 ℃ with the mass ratio of the washed precipitate to the fungus (1-3) -beta-D glucan antibody being 10:1, performing light-shielding reaction for 1h at 37 ℃, adding 20 mg/mL of bovine serum albumin solution, performing rotation for 8h with the use ratio of the bovine serum albumin solution to the washed precipitate being 1 mL:0.1 g, performing ultrasonic treatment for 1 min, performing light-shielding for 4 ℃, performing rotation for 60 rpm h at 8 ℃, and performing centrifugal treatment for 20 min with 12000 g to obtain microsphere antibody conjugate precipitate;
(d) Dissolving trehalose and oxalic anhydride in acetonitrile, adding NaOH, stirring the trehalose, the oxalic anhydride, acetonitrile and sodium hydroxide for 7 h at 70 ℃, concentrating the mixture under reduced pressure for 5 h at 60 ℃ under 120 rpm, continuously stirring for 1h by adding sodium borohydride, continuously stirring for 1:3 by volume ratio, dissolving ascorbic acid in dichloromethane, adding thionyl chloride under 0 ℃ ice water bath condition, stirring for 0.5 h by using the ratio of the ascorbic acid, the dichloromethane and the thionyl chloride for 0.12 mol:200 mL:0.1 mL,25 ℃, stirring for 100 rpm, stirring for reacting for 10 h at 0.01 MPa, concentrating the mixture under 60 ℃ under reduced pressure for 10 h, washing with deionized water, drying at 80 ℃, dissolving the dried product in 1wt% of acetic acid solution, adding 3, 4-dihydroxybenzaldehyde, stirring for 3h at 30 ℃, adding sodium borohydride, continuously stirring for 1h, stirring for the product after drying, acetic acid solution, 3, 4-dihydroxybenzaldehyde and sodium borohydride for 15 g:1000 mL:1 g:0.5 g, filtering to obtain a filter cake, uniformly stirring for 1:8000 g of the mixture with the trehalose, stirring for 1:50 g by volume ratio of the mixture with the aqueous solution of the ascorbic acid and the sodium borohydride, stirring for obtaining a solution after the mixture is mixed solution, and the solution is mixed with distilled water for 1:50 g, and the solution is mixed with hydrochloric acid for 1:50 g, and the solution is mixed with distilled water for 1:50 g of hydrochloric acid, and the solution is prepared by stirring for 80 g, and mixed solution is prepared by stirring for 1:50 g;
(e) Adding the trehalose-ascorbic acid modified complex obtained in the step (d) and glycine and dithiothreitol into 10 mmol/L HEPES buffer solution, and uniformly mixing, wherein the dosage ratio of the HEPES buffer solution to the trehalose-ascorbic acid modified complex to the glycine to the dithiothreitol is 100 mL:2 g:1 g:0.5 g, so as to obtain conjugate redissolution storage solution; washing the microsphere antibody conjugate precipitate obtained in the step (c) for 2 times by using a PBS buffer solution with the pH of 7.4 mM, adding the mixture into a conjugate re-dissolution storage solution, and re-suspending the mixture uniformly, wherein the dosage ratio of the washed microsphere antibody conjugate precipitate to the conjugate re-dissolution storage solution is 10 mg/1 mL, so as to obtain microsphere-antibody liquid;
(f) Adding glucose and borax into 4% w/v bovine serum albumin solution to obtain a treatment solution B, wherein the dosage ratio of the bovine serum albumin solution to the glucose to the borax is 100 mL:3 g:2 mmol, soaking a polyester film in the treatment solution B for 2 h, and drying at 37 ℃ to obtain a pretreated polyester film;
(g) Diluting the microsphere-antibody solution prepared in the step (e) to 1 mg/mL by using 50 mM PBS buffer solution, uniformly spraying the microsphere-antibody solution on the pretreated polyester film prepared in the step (f) at a flow rate of 3 mu L/cm by using a quantitative film spraying instrument, and drying at 37 ℃ to obtain the fluorescent pad.
The preparation method of the reaction membrane comprises the following steps:
(i) Uniformly mixing (1-3) -beta-D glucan and deionized water to obtain a glucan solution of 20 mg/L, adding the glucan solution into a hydrogen peroxide solution of 10wt%, stirring 18 h by 120 rpm, adding diethylene glycol, continuously stirring 2h, filtering by using an ultrafiltration membrane of 10 kDa, and freeze-drying to obtain oxidized glucan, wherein the volume ratio of the glucan solution to the hydrogen peroxide solution to the diethylene glycol is 10:1:1;
(ii) Uniformly mixing an equal volume of 0.1 mol/L phosphate buffer with 0.1 mol/L acetate buffer with pH 5.0 to obtain a mixed buffer, adding BSA and oxidized dextran obtained in the step (i) into the mixed buffer, reacting the BSA, the oxidized dextran and the mixed buffer for 14 h at 4 ℃ with the dosage ratio of 1 g to 2 g to 100 mL, centrifuging at 10 min at 4000 rpm, placing the supernatant in a dialysis bag at 4 ℃ and dialyzing at 96 h with PBS buffer with pH 7.4 mM to obtain (1-3) -beta-D dextran-BSA conjugate;
(iii) Adding the composite polymer and sodium chloride obtained in the step (2) into deionized water, wherein the dosage ratio of the deionized water to the composite polymer to the sodium chloride is 100 mL:0.3 g:1 g, so as to obtain a treatment solution C, soaking the nitrocellulose membrane in the treatment solution C for 15min ℃ and then drying at 37 ℃ so as to obtain a pretreatment reaction membrane;
(iv) Diluting the (1-3) -beta-D glucan-BSA conjugate to 0.33 mg/mL by using PBS with the pH of 7.4 mM, diluting the goat anti-mouse IgG to 0.5 mg/mL, respectively spraying the diluted solution on the pretreatment reaction membrane in the step (iii) at the flow rate of 3 mu L/cm by using a spot film instrument to mark, respectively serving as a detection line and a quality control line, and drying at 37 ℃ to obtain the reaction membrane.
The embodiment also provides a preparation method of the human body liquid fungus detection test paper, which comprises the following steps of sequentially adhering a sample pad, a fluorescent pad, a reaction film and a water absorption pad to a PVC bottom plate serving as a back, wherein a detection line on the reaction film is close to one end of the fluorescent pad, and cutting the detection line into strips with the width of 3.00 mm by a cutting machine to obtain the human body liquid fungus detection test paper.
Embodiment 3 the fungus test paper for human body fluid comprises a PVC bottom plate, a sample pad, a fluorescent pad, a reaction film and a water absorption pad, wherein the water absorption pad is water absorption paper.
The preparation method of the sample pad comprises the following steps:
(1) Heating ethylene glycol methyl ether to 160 ℃ in a nitrogen environment, dropwise adding a mixed solution consisting of N-vinyl pyrrolidone, N-dimethylacrylamide and di-tert-butyl peroxide at the speed of 1.5 g/min, wherein the mass ratio of the ethylene glycol methyl ether to the N-vinyl pyrrolidone to the N, N-dimethylacrylamide to the di-tert-butyl peroxide is 40:20:3:2.7, preserving heat at 160 ℃ for 1 h after the dropwise adding is completed, cooling to 70 ℃, and stirring for 1 h at 180 rpm to obtain a polyvinylpyrrolidone copolymer;
(2) Adding N, N' -dicyclohexylcarbodiimide and 4-dimethylaminopyridine into the polyvinylpyrrolidone copolymer obtained in the step (1), stirring at 60 ℃ and 100 rpm for 1: 1h, adding inulin, continuously stirring at 24: 24 h, placing into a dialysis bag with a molecular weight cutoff of 7000 Da, continuously dialyzing with deionized water for 48 h, collecting the product in the dialysis bag, centrifuging at 4000 rpm for 10: 10min, taking supernatant, and freeze-drying to obtain a composite polymer;
(3) Adding the composite polymer obtained in the step (2), casein and Tween-20 into 20mM PBS buffer with pH of 7.4, and uniformly mixing, wherein the dosage ratio of the PBS buffer to the composite polymer to the Tween-20 to the casein is 1000 mL:10 g:10 mL:1 g, so as to obtain a treatment solution A;
(4) And (3) soaking the glass fiber membrane in the treatment liquid A obtained in the step (3) for 2 hours, and drying at 37 ℃ to obtain the sample pad.
The preparation method of the fluorescent pad comprises the following steps:
(a) Mixing the hydroxyl polystyrene time-resolved fluorescent microsphere with 50 mmol/L MES buffer with pH 6.0, carrying out ultrasonic treatment on the mixture for 1 min at 150W, centrifuging the mixture for 20min at 9 ℃ at 12000 and g, removing supernatant, adding 50 mmol/L MES buffer with pH 6.0 again, carrying out ultrasonic treatment on the mixture for 1 min again, and obtaining fluorescent microsphere mixed solution, wherein the concentration of fluorescent microsphere in the fluorescent microsphere mixed solution is 12.5 mg/mL;
(b) Preparing 10 mg/mL of NHS solution and 5 mg/mL of EDAC solution respectively by using 50 mM of MES buffer solution with pH of 6.0, dropwise adding the mixed solution into the fluorescent microsphere mixed solution obtained in the step (a) until the final concentration of NHS and EDAC is 0.8 mg/mL and 0.3 mg/mL, carrying out ultrasonic treatment for 1 min at 150W, and carrying out light-shielding reaction at 37 ℃ for 1h to obtain a mixed solution A;
(c) Centrifuging the mixed solution A in the step (b) at 8 ℃ and 12000 g for 20 min, discarding supernatant, washing the precipitate for 2 times by using 50 mM pH 8.0 HEPES buffer solution, adding a fungus (1-3) -beta-D glucan antibody solution, performing ultrasonic treatment for 1 min at 37 ℃ with the mass ratio of the washed precipitate to the fungus (1-3) -beta-D glucan antibody being 10:1, performing light-shielding reaction for 1h at 37 ℃, adding 20 mg/mL of bovine serum albumin solution, performing rotation for 8h with the use ratio of the bovine serum albumin solution to the washed precipitate being 1 mL:0.1 g, performing ultrasonic treatment for 1 min, performing light-shielding for 4 ℃, performing rotation for 60 rpm h at 8 ℃, and performing centrifugal treatment for 20 min with 12000 g to obtain microsphere antibody conjugate precipitate;
(d) Dissolving trehalose and oxalic anhydride in acetonitrile, adding NaOH, stirring for 7 h at the temperature of 70 ℃, stirring for 7 h at the temperature of 120 rpm, concentrating for 5.5 h at the temperature of 0.01 MPa and at the temperature of 60 ℃ under reduced pressure, recrystallizing in a mixed solution of ethanol and diethyl ether (volume ratio of 1:3) to obtain carboxylated trehalose, dissolving ascorbic acid in dichloromethane, adding thionyl chloride under the condition of 0 ℃ ice water bath, stirring for reacting for 0.5 h at the temperature of 0.12 mol:200 mL:0.1 mL,25 ℃ at the temperature of 100 rpm, stirring for 10 h at the temperature of 0.01 MPa and at the temperature of 60 ℃, washing with deionized water, drying at the temperature of 80 ℃, dissolving the dried product in a 1wt% acetic acid solution, adding 3, 4-dihydroxybenzaldehyde, stirring for 3.5 h at the temperature of 180 ℃, adding sodium borohydride, continuously stirring for 1:49, stirring for obtaining a product after drying, an acetic acid solution, 3, 4-dihydroxybenzaldehyde and sodium borohydride in the volume ratio of 15 g:1000 mL:1 g:0.5 g, filtering cake, stirring for obtaining a filter cake, uniformly mixing with the carboxylated trehalose and the solution of hydrochloric acid solution of 1:28-60 ℃ to obtain a mixed solution of hydrochloric acid and water of which the volume ratio of water is 1:50 g, stirring for obtaining a mixed solution of the modified trehalose and the water of 50 g, and the solution of hydrochloric acid is mixed solution of the water after stirring for 80g, and the solution is mixed solution of the water is mixed with distilled for 80g at the hydrochloric acid and the water of 1:1:1:7L, and the water is mixed solution is mixed with the distilled for 80:370mL;
(e) Adding the trehalose-ascorbic acid modified complex obtained in the step (d) and glycine and dithiothreitol into 10 mmol/L HEPES buffer solution, and uniformly mixing, wherein the dosage ratio of the HEPES buffer solution to the trehalose-ascorbic acid modified complex to the glycine to the dithiothreitol is 100 mL:2 g:1 g:0.5 g, so as to obtain conjugate redissolution storage solution; washing the microsphere antibody conjugate precipitate obtained in the step (c) for 2 times by using a PBS buffer solution with the pH of 7.4 mM, adding the mixture into a conjugate re-dissolution storage solution, and re-suspending the mixture uniformly, wherein the dosage ratio of the washed microsphere antibody conjugate precipitate to the conjugate re-dissolution storage solution is 10 mg/1 mL, so as to obtain microsphere-antibody liquid;
(f) Adding glucose and borax into 4% w/v bovine serum albumin solution to obtain a treatment solution B, wherein the dosage ratio of the bovine serum albumin solution to the glucose to the borax is 100 mL:3 g:2 mmol, soaking a polyester film in the treatment solution B for 2 h, and drying at 37 ℃ to obtain a pretreated polyester film;
(g) Diluting the microsphere-antibody solution prepared in the step (e) to 1 mg/mL by using 50 mM PBS buffer solution, uniformly spraying the microsphere-antibody solution on the pretreated polyester film prepared in the step (f) at a flow rate of 3 mu L/cm by using a quantitative film spraying instrument, and drying at 37 ℃ to obtain the fluorescent pad.
The preparation method of the reaction membrane comprises the following steps:
(i) Uniformly mixing (1-3) -beta-D glucan and deionized water to obtain a glucan solution of 20 mg/L, adding the glucan solution into a hydrogen peroxide solution of 10wt%, stirring 19 h by 120 rpm, adding diethylene glycol, continuously stirring 2h, filtering by using an ultrafiltration membrane of 10 kDa, and freeze-drying to obtain oxidized glucan, wherein the volume ratio of the glucan solution to the hydrogen peroxide solution to the diethylene glycol is 10:1:1;
(ii) Uniformly mixing an equal volume of 0.1 mol/L phosphate buffer with 0.1 mol/L acetate buffer with pH 5.0 to obtain a mixed buffer, adding BSA and oxidized dextran obtained in the step (i) into the mixed buffer, reacting the BSA, the oxidized dextran and the mixed buffer for 15 h at 4 ℃ with the dosage ratio of 1 g to 2 g to 100 mL, centrifuging at 4000 rpm for 10 min, taking supernatant, and dialyzing with PBS buffer with pH 7.4 and 50 mM for 96 h at 4 ℃ in a dialysis bag to obtain the (1-3) -beta-D dextran-BSA conjugate;
(iii) Adding the composite polymer and sodium chloride obtained in the step (2) into deionized water, wherein the dosage ratio of the deionized water to the composite polymer to the sodium chloride is 100 mL:0.3 g:1 g, so as to obtain a treatment solution C, soaking the nitrocellulose membrane in the treatment solution C for 15min ℃ and then drying at 37 ℃ so as to obtain a pretreatment reaction membrane;
(iv) Diluting the (1-3) -beta-D glucan-BSA conjugate to 0.33 mg/mL by using PBS with the pH of 7.4 mM, diluting the goat anti-mouse IgG to 0.5 mg/mL, respectively spraying the diluted solution on the pretreatment reaction membrane in the step (iii) at the flow rate of 3 mu L/cm by using a spot film instrument to mark, respectively serving as a detection line and a quality control line, and drying at 37 ℃ to obtain the reaction membrane.
The embodiment also provides a preparation method of the human body liquid fungus detection test paper, which comprises the following steps of sequentially adhering a sample pad, a fluorescent pad, a reaction film and a water absorption pad to a PVC bottom plate serving as a back, wherein a detection line on the reaction film is close to one end of the fluorescent pad, and cutting the detection line into strips with the width of 3.00 mm by a cutting machine to obtain the human body liquid fungus detection test paper.
Comparative example 1 differs from example 1 only in that polyvinylpyrrolidone was used instead of the complex polymer in the treatment liquid a.
Comparative example 2 differs from example 1 only in that the trehalose-ascorbic acid modified complex is replaced by ascorbic acid.
Comparative example 3 differs from example 1 only in that the composite polymer was replaced with polyvinyl alcohol in the treatment liquid C.
Experimental example 1 (1-3) -beta-D-glucan standard 1 count is taken, sterilized by 75% alcohol, then opened, the standard is taken out, added into non-heat source water for dissolution, placed into a vortex mixer for mixing 5min, prepared into 100 pg/mL (1-3) -beta-D-glucan solution reference, dripped onto sample pads of test papers obtained in examples 1-3 and comparative examples 1-3, subjected to standing chromatography, and accuracy and repeatability detection are carried out on the reference papers by using the test papers obtained in examples 1-3 and comparative examples 1-3, and the results are shown in FIG. 2.
The results of FIG. 2 show that the test paper of the invention is used for carrying out repeated detection on the reference product for 10 times, the repeatability CV of the test paper detection reference product prepared in the examples 1-3 is less than 10 percent, and the repeatability of the kit prepared in the comparative examples 1-3 is inferior to that of the test paper prepared in the examples 1-3, so that the test paper of the invention has high detection accuracy and good repeatability. In addition, the test strips of the groups 1-3 were stored for 18 months at 30 ℃ and the CV still satisfied <10%, 4.12%, 4.15% and 4.21%, respectively, indicating good stability of the test strips of the present invention.
The test example 2:T shows that the fluorescence intensity of the T line is stronger or stronger than that of the C line, the fluorescence detector detects the fluorescence values of the T line and the C line to obtain the T/C value which is more than or equal to 1, the T line is negative, the T line does not have 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 to obtain the T/C value which is less than 1, the T/C value is positive, and the C line does not have fluorescence, the T/C value is invalid.
According to the test paper of the invention examples 1-3 and comparative examples 1-3, 200 clinical negative samples (serum of healthy adults without liver, kidney and respiratory tract diseases and without any antibiotics in recent years) are detected simultaneously, 200 positive samples (serum 100, urine 50, hydrothorax 30 and bronchial lavage liquid 20) are detected simultaneously, and the sensitivity and the specificity are calculated according to the yin-yang of the detection result, wherein the sensitivity (%) = (number of detected positive examples/number of detected positive examples) ×100%, and the specificity (%) = (number of detected negative examples/number of detected negative examples) ×100%. Clinical compliance= (number of cases/total number of samples where the detected result and the clinical result agree) ×100%. When the number of simultaneous readouts is stable, the detection time is recorded and the results are shown in table 1.
Table 1:
The results in Table 1 show that the test paper of the embodiment 1-3 is used for detecting clinical samples, the clinical coincidence rate can reach 99%, the sensitivity is high, the specificity is good, the effect is better than that of the test paper of the comparative embodiment 1-3, the sensitivity and the specificity of the test paper for detecting human body liquid fungi are strong, the accuracy is high, and the detection speed is high.
Experimental example 3 (1-3) -beta-D-glucan standard 1 is taken, sterilized by 75% alcohol, then opened, the standard is taken out, the standard is added with non-heat source water, and the mixture is placed in a vortex mixer to be mixed with 5min, so that 100 pg/mL of (1-3) -beta-D-glucan solution is prepared, and the solution is diluted to 80 pg/mL, 40 pg/mL, 20 pg/mL and 10 pg/mL by using the non-heat source water gradient to obtain a calibrator. The calibrator was dropped onto the sample pads of the test papers obtained in example 1 and comparative examples 1 to 3, respectively, and subjected to stationary chromatography, and fluorescence signal values were read using an immunofluorescence analyzer, and T/C values were calculated, and the results are shown in Table 2.
Table 2:
The results in Table 2 show that when the test paper of the embodiment 1 of the invention is used for detection, the T/C value of the test paper of the comparative example 1-3 is less than 1 under the condition that the concentration of (1-3) -beta-D-glucan is 20 pg/mL, the test paper is positive, the lowest detection limit is 20 pg/mL, the test paper of the comparative example 1-3 is less than 1 under the condition that the concentration of the test paper is 40 pg/mL, the test paper is positive, and the lowest detection limit is 40 pg/mL, which indicates that the human body liquid fungus detection test paper of the invention has high sensitivity.
Experimental example 4 clinical negative and positive samples were randomly divided into 2 groups of 32 samples each, with 50 ng/mL of mannan added to each sample in one group and 5 mg/mL of triglyceride added to the other group. The rate of change of clinical compliance before and after addition of the interferents was calculated. The results are shown in Table 3.
Table 3:
The results in Table 3 show that the rate of change of the clinical compliance rate of example 2 is lower than that of comparative examples 1-3, indicating that the human body fluid fungus test paper of the invention has strong anti-interference capability.
Experimental example 5 the microsphere-antibody solution prepared in example 3 and comparative example 1 was stored at 4℃for 6 months, and the microsphere-antibody solution state was observed. As shown in FIG. 3, the group 1 of the example has no precipitation, and the group 2 of the comparative example has a large amount of precipitation at the bottom, which shows that the conjugate redissolved storage solution of the invention can effectively improve the stability and prolong the shelf life.
In the detection process, if the test strip C is not developed, the test strip is invalid, the test strips 1-3 and the test strips 1-3 are stored at 30 ℃, the time is recorded when the test strip C is not developed, and the result shows that the test strips C are not developed after the test strips 1-3 are stored for 24 months, the test strips 1 and the test strips 3 are not developed after the test strips are stored for 21 months, and the test strips 2 are not developed after the test strips are stored for 20 months. The test paper has good stability and prolonged service life.
Claims (8)
1. The human body liquid fungus detection test paper is characterized by comprising a PVC bottom plate, a sample pad, a fluorescent pad, a reaction membrane and a water absorption pad;
The preparation method of the sample pad comprises the following steps:
(1) Heating ethylene glycol methyl ether to 160 ℃, dropwise adding a mixed solution consisting of N-vinyl pyrrolidone, N-dimethylacrylamide and di-tert-butyl peroxide, preserving heat after the dropwise adding is finished, cooling to 70 ℃, and stirring to obtain a polyvinylpyrrolidone copolymer;
(2) Adding N, N' -dicyclohexylcarbodiimide and 4-dimethylaminopyridine into the polyvinylpyrrolidone copolymer obtained in the 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 the step (2), casein and Tween-20 into PBS buffer solution, and uniformly mixing to obtain a treatment solution A;
(4) Soaking a glass fiber membrane in the treatment liquid A obtained in the step (3), and drying to obtain a sample pad;
the preparation method of the fluorescent pad comprises the following steps:
(a) Mixing fluorescent microspheres with MES buffer, performing ultrasound, centrifuging to remove supernatant, adding the MES buffer again, and performing ultrasound to obtain fluorescent microsphere mixed solution;
(b) Respectively preparing an NHS solution and an EDAC solution by using an MES buffer solution, dripping the NHS solution and the EDAC solution into the fluorescent microsphere mixed solution in the step (a) until the final concentration of the NHS and the EDAC is 0.8 mg/mL and 0.3 mg/mL, carrying out ultrasonic and 37 ℃ light-shielding reaction for 1 h to obtain a mixed solution A;
(c) Centrifuging the mixed solution A in the step (b), washing the precipitate, adding a fungus (1-3) -beta-D glucan antibody solution, performing ultrasonic and light-proof reaction, adding a bovine serum albumin solution, performing ultrasonic 1 min, performing light-proof rotation, and centrifuging to obtain a microsphere antibody conjugate precipitate;
(d) Dissolving the ascorbic acid in methylene dichloride, adding thionyl chloride at 0 ℃, stirring at 25 ℃, concentrating under reduced pressure, washing, drying, dissolving the dried product in 1wt% acetic acid solution, adding 3, 4-dihydroxybenzaldehyde, stirring, adding sodium borohydride, continuing stirring, filtering to obtain a filter cake, uniformly mixing the filter cake with carboxylated trehalose and deionized water to obtain a mixed solution B, adding 3 mol/L hydrochloric acid solution and distilled water, stirring, centrifuging, filtering supernatant, and freeze-drying to obtain a trehalose-ascorbic acid modified compound;
(e) Adding the trehalose-ascorbic acid modified compound obtained in the step (d) into HEPES buffer solution, and uniformly mixing glycine and dithiothreitol to obtain conjugate redissolved storage solution; washing the microsphere antibody conjugate precipitate obtained in the step (c) by using PBS buffer solution, adding the solution into conjugate re-dissolving storage solution, and re-suspending the solution uniformly to obtain microsphere-antibody solution;
(f) Adding glucose and borax into a 4% w/v bovine serum albumin solution to obtain a treatment solution B, soaking a polyester film in the treatment solution B, and drying to obtain a pretreated polyester film;
(g) Diluting the microsphere-antibody liquid prepared in the step (e) with PBS buffer solution, spraying the diluted microsphere-antibody liquid on the pretreated polyester film prepared in the step (f), and drying the pretreated polyester film to obtain a fluorescent pad;
the preparation method of the reaction membrane comprises the following steps:
(i) Uniformly mixing (1-3) -beta-D glucan with deionized water to obtain a glucan solution of 20 mg/L, adding the glucan solution into a 10wt% hydrogen peroxide solution, stirring, adding diethylene glycol, continuously stirring, ultrafiltering, and freeze-drying to obtain oxidized glucan;
(ii) Uniformly mixing an equal volume of phosphate buffer solution and an acetic acid buffer solution to obtain a mixed buffer solution, adding BSA and oxidized glucan obtained in the step (i) into the mixed buffer solution, reacting at 4 ℃, centrifuging, and dialyzing supernatant to obtain a (1-3) -beta-D glucan-BSA conjugate;
(iii) Adding the composite polymer obtained in the step (2) and sodium chloride into deionized water to obtain a treatment solution C, soaking a nitrocellulose membrane in the treatment solution C, and drying to obtain a pretreatment reaction membrane;
(iv) Diluting the (1-3) -beta-D glucan-BSA conjugate to 0.33 mg/mL by using PBS, diluting the goat anti-mouse IgG to 0.5 mg/mL, spraying the mixture on the pretreatment reaction film in the step (iii) for scribing, respectively serving as a detection line and a quality control line, and drying to obtain the reaction film.
2. The human body fluid fungus test paper according to claim 1, wherein the mass ratio of the ethylene glycol methyl ether, the N-vinyl pyrrolidone, the N, N-dimethyl acrylamide and the di-tert-butyl peroxide in the step (1) is 40:20:3:2.7, and the dripping speed of the mixed liquid in the step (1) is 1.5 g/min.
3. The human body fluid fungus test paper according to claim 2, wherein the mass ratio of the polyvinylpyrrolidone copolymer to the N, N' -dicyclohexylcarbodiimide, the 4-dimethylaminopyridine and the inulin in the step (2) is 50:15:1:20, and the dosage ratio of the PBS buffer solution, the composite polymer, the Tween-20 and the casein in the step (3) is 1000 mL:10 g:10 mL:1 g.
4. The human body fluid fungus detection test paper according to claim 3, wherein the fluorescent microsphere in the step (a) comprises time-resolved fluorescent microsphere, the surface modification functional group of the fluorescent microsphere comprises hydroxyl or carboxyl, the fluorescent microsphere is made of polystyrene, polymethyl methacrylate or silicon dioxide, and the concentration of the fluorescent microsphere in the fluorescent microsphere mixed solution in the step (a) is 12.5 mg/mL.
5. The human body fluid fungus test paper according to claim 4, wherein the mass ratio of the precipitate after washing to the fungus (1-3) -beta-D glucan antibody in the step (c) is 10:1, and the dosage ratio of the bovine serum albumin solution in the step (c) to the precipitate after washing is 1 mL:0.1 g.
6. The human body fluid fungus test paper according to claim 5, wherein the dosage ratio of trehalose, oxalic anhydride, acetonitrile and sodium hydroxide in the step (d) is 0.2 mol:0.1 mol:200 mL:0.3 mol, the volume ratio of ethanol to diethyl ether in the ethanol and diethyl ether mixed solution in the step (d) is 1:3, the dosage ratio of ascorbic acid, dichloromethane to thionyl chloride in the step (d) is 0.12 mol:200 mL:0.1 mL, the dosage ratio of dried product, acetic acid solution, 3, 4-dihydroxybenzaldehyde and sodium borohydride in the step (d) is 15 g:1000 mL:1 g:0.5 g, the dosage ratio of carboxylated trehalose, filter cake and deionized water in the step (d) is 1 g:10 g:50 mL, and the volume ratio of the mixed solution B, hydrochloric acid solution and distilled water in the step (d) is 25:1:1.
7. The human body fluid fungus test paper according to claim 6, wherein the dosage ratio of the HEPES buffer solution, the trehalose-ascorbic acid modified complex, the glycine and the dithiothreitol in the step (e) is 100 mL:2 g:1 g:0.5 g, the dosage ratio of the washed microsphere antibody conjugate precipitate to the conjugate redissolved storage solution in the step (e) is 10 mg to 1 mL, and the dosage ratio of the bovine serum albumin solution, the glucose and the borax in the step (f) is 100 mL:3 g:2 mmol.
8. The human fluid fungus test paper according to claim 7, wherein the volume ratio of the dextran solution, the hydrogen peroxide solution and the diglycol in the step (i) is 10:1:1, the dosage ratio of the BSA, the oxidized dextran and the mixed buffer in the step (ii) is 1 g:2 g:100 mL, and the dosage ratio of the deionized water, the composite polymer and the sodium chloride in the step (iii) is 100 mL:0.3 g:1 g.
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