Test strip for detecting uric acid in saliva, preparation and detection method therefor and use thereof

Abstract

A test strip for detecting uric acid in saliva that meets the requirement for room-temperature preservation, a preparation and detection method therefor and the use thereof. The test strip for detecting uric acid in saliva comprises a uricase, a peroxidase, a vitamin C oxidase, a bilirubin oxidase, a color developing agent, and a protective film coated on the test strip for detecting uric acid in saliva. To meet the needs of a target population for long-term monitoring and real-time tracking of changes in their uric acid levels, by means of agent addition, the test strip for detecting uric acid in saliva has prolonged preservation time at room temperature, enables non-invasive, efficient and convenient monitoring of a health status at any time, and timely detection and treatment, allows intuitive visual identification of uric acid levels in saliva by a subject, and meets the needs of a subject for long-term preservation at room temperature.

Description

Saliva uric acid detection test strip, preparation and detection method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of medical devices, and particularly relates to a saliva uric acid detection test strip, a preparation and detection method and application thereof. BACKGROUND

[0002] In recent years, hyperuricemia has shown a clear upward trend and a younger trend, and hyperuricemia has become the "fourth high" after diabetes, hypertension and hyperlipidemia. At the same time, hyperuricemia has no obvious symptoms in the early stage, so patients cannot detect and treat it in time, and it can lead to gout, hypertension, kidney stones and other diseases in the later stage. Therefore, it is necessary to early warn and diagnose hyperuricemia and gout. Especially for high-risk groups, clinical recommendations for lifelong monitoring and regular screening.

[0003] Since Michaels in 1901 and Kirk in 1903 first applied saliva analysis as a diagnostic specimen, so far, more and more medical workers have realized the importance of saliva analysis. From the initial basic scientific research to today's clinical application and commercial products, the development of saliva detection technology has made remarkable progress in the past few decades, showing a sustained growth trend. Data shows that the uric acid in saliva is significantly positively correlated with the uric acid concentration in blood. Therefore, saliva uric acid can be used as a marker for clinical diagnosis of hyperuricemia and gout. In the previous work, although the saliva uric acid rapid test paper was studied, the color gradient of the test paper was relatively fuzzy in identifying the uric acid concentration range, and the room temperature storage problem limited the test paper to meet the long-term home monitoring requirement. Based on this, the present application develops a rapid saliva uric acid test paper which can easily identify the high and low uric acid concentration and meet the room temperature storage requirement.

[0004] SUMMARY

[0005] The purpose of the present application is to provide a saliva uric acid detection test strip, a preparation and detection method and application thereof. In order to meet the long-term monitoring requirement of the required population and the requirement of grasping the uric acid content change of oneself at any time, the saliva uric acid detection test paper of the present application can non-invasively, efficiently and conveniently view the uric acid level of oneself at any time, detect and treat in time, and easily identify the high and low uric acid concentration and meet the room temperature storage requirement.

[0006] Before the content of the present application is described, the terms used herein are defined as follows:

[0007] The term "SHE 50" refers to a broad-spectrum composite enzyme stabilizer with a molecular formula (CH2CHOHCH3) nMolecular weight: 65000-80000, heavy metal: <0.001%, sulfate: <0.006%. Prevents the rapid precipitation of enzymes from 3,3',5,5'-tetramethylbenzidine and biochemical substrates, and prevents solution precipitation.

[0008] The term "Phos-E" refers to a stabilizing protective agent for primary enzymes, coenzymes, substrates, and proteins. The English name is Stabilizer protectant Phos-E. The main component is a phosphate compound and other auxiliary emulsifiers. It is a colorless transparent liquid with a relative density of 1.2448 (20°C, water = 1).

[0009] The term "AES" refers to a broad-spectrum enzyme stabilizer. The English name is Enzyme Stabilizer AES. It is mainly used for the stabilization of liquid enzymes and working enzymes, especially suitable for the dilution of enzymes and proteins in diagnostic reagents, various oxidases, glycerol phosphate oxidase, and uric acid oxidase. Representative manufacturers include but are not limited to Shanghai Xibao Biotechnology Co., Ltd., Shanghai Yuan Ye Biotechnology Co., Ltd., Shanghai Jin'an Biotechnology Co., Ltd., and Shanghai Guchen Biotechnology Co., Ltd.

[0010] The term "ADG" refers to a broad-spectrum enzyme stabilizer. The English name is Enzyme Stabilizer ADG. It has anti-precipitation, solubilization, and efficiency promotion. It also has unique properties of improving enzyme activity and sterilization. It can be used in vaccines, diagnostic reagents, etc. Representative manufacturers include but are not limited to Shanghai Xinmin Life Science and Technology Co., Ltd., and Shanghai Jin'an Biotechnology Co., Ltd.

[0011] The term "DPD" refers to a broad-spectrum enzyme stabilizer. The English name is Enzyme Stabilizer DPD. It is mainly used for the stabilization of liquid enzymes and working enzymes, suitable for enzyme dilution in various types of reactions, and stabilization of liquid enzymes. Representative manufacturers include but are not limited to Shanghai Xibao Biotechnology Co., Ltd., and Shanghai Shifeng Biotechnology Co., Ltd.

[0012] The term "DPPD" refers to a broad-spectrum enzyme stabilizer. The English name is Enzyme Stabilizer DPPD. It is mainly used for the stabilization of liquid enzymes and working enzymes, especially suitable for enzyme-linked immunosorbent reaction horseradish peroxidase dilution, various oxidases, and glycerol phosphate oxidase liquid enzyme stabilization. Representative manufacturers include but are not limited to Shanghai Jin'an Biotechnology Co., Ltd., and Shanghai Chenyi Biotechnology Co., Ltd.

[0013] The term "ES01" refers to: a complex enzyme stabilizer, broad-spectrum enhancement of enzyme stability, English name Enzyme Stabilizer ES01, mainly used for the stabilization of liquid and lyophilized enzymes, especially suitable for the stabilization of liquid and lyophilized enzymes of urease, uric acid oxidase, glycerol phosphate oxidase, representative manufacturers include but are not limited to Shanghai Yixin Biotechnology Co., Ltd., Shanghai Jinpan Biotechnology Co., Ltd.

[0014] The term "ES02" refers to: a complex enzyme stabilizer, broad-spectrum enhancement of enzyme stability, English name Enzyme Stabilizer ES02, mainly used for the stabilization of liquid and lyophilized enzymes, especially suitable for the stabilization of liquid and lyophilized enzymes of various oxidases (cholesterol oxidase, uric acid oxidase, creatinine oxidase, glycerol phosphate oxidase, lactate oxidase, etc.), lipoprotein esterase, cholesterol esterase, hexokinase, thrombin, 6-phosphogluconate dehydrogenase, representative manufacturers include but are not limited to Shanghai Yixin Biotechnology Co., Ltd., Shanghai Jinpan Biotechnology Co., Ltd.

[0015] The term "ES03" refers to: a complex enzyme stabilizer, broad-spectrum enhancement of enzyme stability, English name Enzyme Stabilizer ES03, mainly used for the stabilization of liquid and lyophilized enzymes, especially suitable for the stabilization of liquid and lyophilized enzymes of various lactate dehydrogenases, malate dehydrogenases, serum enzymes or proteins, peroxidases, 6-phosphogluconate dehydrogenases, hexokinases, ureases, glutamate dehydrogenases, nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate, nicotinamide adenine dinucleotide phosphate, representative manufacturers include but are not limited to Shanghai Yixin Biotechnology Co., Ltd., Shanghai Jinpan Biotechnology Co., Ltd.

[0016] The term "ES04" refers to: a complex enzyme stabilizer, broad-spectrum enhancement of enzyme stability, English name Enzyme Stabilizer ES04, mainly used for the stabilization of liquid and lyophilized enzymes, especially suitable for the stabilization of various oxidases (glycerol phosphate oxidase, cholesterol oxidase, peroxidase, sarcosine oxidase, uric acid oxidase, vitamin C oxidase, etc.), serum protein components or enzymes, cholesterol esterase, creatine imino hydrolase, creatinine amino hydrolase, fructose dehydrogenase, phosphoenolpyruvate carboxylase, representative manufacturers include but are not limited to Shanghai Yixin Biotechnology Co., Ltd., Shanghai Jinpan Biotechnology Co., Ltd.

[0017] The term "ES05" refers to a complex enzyme stabilizer that enhances the stability of enzymes in a broad spectrum. Its English name is Enzyme Stabilizer ES05, and it is mainly used for the stabilization of liquid and lyophilized enzymes. It is particularly suitable for the stabilization of cholesterol oxidase, sarcosine oxidase, serum protein components or enzymes, pyruvate oxidase, glycerol phosphate oxidase, pyruvate dehydrogenase, acetyl-CoA, NADPH, diaphorase, etc. Representative manufacturers include but are not limited to Shanghai Yixin Biotechnology Co., Ltd. and Shanghai Jinpan Biotechnology Co., Ltd.

[0018] The term "ES06" refers to a complex enzyme stabilizer that enhances the stability of enzymes in a broad spectrum. Its English name is Enzyme Stabilizer ES06, and it is mainly used for the stabilization of liquid and lyophilized enzymes. It is particularly suitable for the stabilization of glycerol phosphate oxidase, glucose oxidase, sarcosine oxidase, pyruvate oxidase, cholesterol oxidase, ribose oxidase, serum proteins, etc. Representative manufacturers include but are not limited to Shanghai Yixin Biotechnology Co., Ltd. and Shanghai Jinpan Biotechnology Co., Ltd.

[0019] The term "ES07" refers to a complex enzyme stabilizer that enhances the stability of enzymes in a broad spectrum. Its English name is Enzyme Stabilizer ES07, and it is mainly used for the stabilization of liquid and lyophilized enzymes. It is particularly suitable for the stabilization of nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate. Representative manufacturers include but are not limited to Shanghai Yixin Biotechnology Co., Ltd. and Shanghai Jinpan Biotechnology Co., Ltd.

[0020] The term "ES08" refers to a complex enzyme stabilizer that enhances the stability of enzymes in a broad spectrum. Its English name is Enzyme Stabilizer ES08, and it is mainly used for the stabilization of liquid and lyophilized enzymes. It is particularly suitable for the stabilization of nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate. Representative manufacturers include but are not limited to Shanghai Yixin Biotechnology Co., Ltd. and Shanghai Jinpan Biotechnology Co., Ltd.

[0021] The term "MP" refers to a stabilizer widely used for proteins, enzymes, and antibodies, etc. It is generally in liquid or lyophilized form. Its English name is Enzyme protectant MP. Representative manufacturers include but are not limited to Shanghai Xibao Biotechnology Co., Ltd. and Shanghai Kehansheng Biotechnology Co., Ltd.

[0022] The term "HP" refers to: a broad-spectrum enzyme and antigen, antibody stabilizer, English name Enzyme protectant HP, suitable for PH 5-9 liquid and lyophilized system; mainly used for the stabilization of thrombin, glycosidase, ribose oxidase, esterase, hexokinase, thrombin reagent, serum protein, etc. Representative manufacturers include but are not limited to Beijing Mayrda Technology Co., Ltd., Shanghai Guangrui Biological Technology Co., Ltd., Shanghai Jingkang Biological Engineering Co., Ltd., Shanghai Xibao Biological Technology Co., Ltd.

[0023] The term "AFP" refers to: a broad-spectrum enzyme lyophilized enzyme and protein stabilizer, diluent, stabilizing various oxidases, superoxide dismutase, glycosidase, etc. Liquid, lyophilized agent, especially lyophilized enzymes, English name Enzyme protectant AFP, representative manufacturers include but are not limited to Shanghai Shifeng Biological Technology Co., Ltd., Shanghai Xibao Biological Technology Co., Ltd.

[0024] The term "ADP" refers to: a broad-spectrum enzyme and antibody stabilizer, English name Enzyme protectant ADP, also has the unique performance of improving enzyme activity and sterilization, saving the original enzyme and improving the performance, representative manufacturers include but are not limited to Shanghai Yuan Ye Biological Technology Co., Ltd., Shanghai Jinpan Biological Technology Co., Ltd., Shanghai Xibao Biological Technology Co., Ltd.

[0025] The term "AFD" refers to: a broad-spectrum enzyme and antibody stabilizer, English name Enzyme protectant AFD, mainly used for the stabilization of liquid enzymes, especially suitable for various lactic acid dehydrogenase, malate dehydrogenase, serum enzyme or protein, peroxidase, 6-phosphoric acid dehydrogenase, hexokinase, urease, glutamic acid dehydrogenase, NADH, NAD, NADPH, NADP, liquid and lyophilized enzyme stabilization, representative manufacturers include but are not limited to Shanghai Xinmin Life Science and Technology Co., Ltd., Shanghai Jinpan Biological Technology Co., Ltd.

[0026] The term "DBP" refers to: a complex enzyme stabilizer, English name Enzyme protectant DBP, used for the stabilization of antigens, antibodies (except for bovine origin) and enzymes, representative manufacturers include but are not limited to Shanghai Yuan Ye Biological Technology Co., Ltd., Xibao Biological Technology Co., Ltd.

[0027] The term "DB" refers to: a complex enzyme stabilizer, English name Enzyme protectant DB, used for the stabilization of antigens, antibodies (except for bovine origin) and enzymes, representative manufacturers include but are not limited to Shanghai Yuan Ye Biological Technology Co., Ltd., Shanghai Xibao Biological Technology Co., Ltd.

[0028] The term "S15" refers to a protein protectant, with the English name Protein Protectant S15, mainly used for stabilizing and protecting the biological activity of proteins. It can be used in enzyme diluent to effectively protect the biological activity of enzyme-labeled antibodies and enzymes and improve their stability. It can also be used in liquid standard buffer systems to effectively protect the biological activity of standard substances. It can also be added to animal serum such as bovine serum to protect a wide range of biological active substances in the serum. Representative manufacturers include but are not limited to Shanghai Xibao Biotechnology Co., Ltd. and Wuhan Keyin Century Technology Co., Ltd.

[0029] The term "HPD" refers to a broad-spectrum enzyme, protein, and antibody protectant, with the English name Protein protectant HPD. It can be used in vaccines, diagnostic reagents, etc., and is mainly used for the stabilization of liquids. It is especially suitable for the stabilization of various oxidative enzymes (glycerol phosphate oxidase, cholesterol oxidase, peroxidase, sarcosine oxidase, uric acid oxidase, vitamin C oxidase, etc.), serum protein components or enzymes, cholesterol esterase, creatine imino hydrolase, creatinine amino hydrolase, fructose dehydrogenase, phosphoenolpyruvate carboxylase, dehydrogenase, and purine enzymes. Representative manufacturers include but are not limited to Shanghai Jinpan Biotechnology Co., Ltd., Shanghai Xibao Biotechnology Co., Ltd., Shanghai Yuanye Biotechnology Co., Ltd., and Shanghai Enzyme Link Biotechnology Co., Ltd.

[0030] The term "GH" refers to a protein protectant, with the English name Protein protectant GH, used for the protection and stabilization of enzymes and proteins. It is a coating, blocking, and stabilizing agent for enzyme-linked immunosorbent assay and a protein protectant that can reduce background and improve stability. Representative manufacturers include but are not limited to Shanghai Jiwei Biotechnology Co., Ltd., Shanghai Xibao Biotechnology Co., Ltd., Qiyi Biotechnology (Shanghai) Co., Ltd., and Shanghai Fusheng Industry Co., Ltd.

[0031] The term "YT" refers to a protein protectant, with the English name Protein protectant TY. It is a coating, blocking, and stabilizing agent for enzyme-linked immunosorbent assay and a protein protectant that can reduce background and improve stability. Representative manufacturers include but are not limited to Shanghai Yeyuan Biotechnology Co., Ltd., Shanghai Yanwei Commercial and Trade Co., Ltd., and Shanghai Xibao Biotechnology Co., Ltd.

[0032] The term "AEP-HBC" refers to a protein protectant, with the English name Protein protectant AEP-HBC. It is an enzyme protectant, anti-precipitation, solubilization, and efficiency enhancer suitable for enzyme and protein liquid systems, with a density of 0.85 g / cm 3, water solubility (20℃) ~ 75g / 100ml, representative manufacturers include but not limited to Shanghai source leaf biotechnology Co., Ltd., Shanghai Fusheng Industry Co., Ltd., Shanghai Xibaobao Biotechnology Co., Ltd.

[0033] The term "DP4" refers to: a protein protectant, English name Protein protectant DP4, broad-spectrum enzyme and antigen, antibody stabilizer, diluent (small molecules), representative manufacturers include but not limited to Shanghai Yanzun Biotechnology Co., Ltd., Shanghai Guyan Industry Co., Ltd., Shanghai Yuli Ke Life Science Co., Ltd., Shanghai Yian Biotechnology Co., Ltd., Shanghai Chenglin Biotechnology Co., Ltd.

[0034] The term "DP7" refers to: a protein protectant, English name Protein protectant DP7, broad-spectrum enzyme and antigen, antibody stabilizer, diluent (small molecules), representative manufacturers include but not limited to Shanghai Yanwei Biotechnology Co., Ltd., Shanghai Yanzun Biotechnology Co., Ltd., Beijing Solaybao Technology Co., Ltd.

[0035] The term "DPS50" refers to: a protein protectant, English name Protein protectant DPS50, mainly used for the stabilization of liquid and freeze-dried enzymes, especially suitable for the stabilization of urease, uric acid oxidase, glycerol phosphate oxidase and other liquid and freeze-dried enzymes, representative manufacturers include but not limited to Shanghai Hu Zheng Biotechnology Co., Ltd., Shanghai Guyan Industry Co., Ltd.

[0036] The term "PS01" refers to: a complex liquid protein protectant, English name Protein protectant 01, transparent liquid, can significantly improve the storage stability and thermal stability of protein and other active molecules, representative manufacturers include but not limited to Shenzhen Duoruisite Medical Technology Co., Ltd.

[0037] The term "PS03" refers to: a complex liquid protein protectant, English name Protein protectant 03, transparent liquid, can significantly improve the storage stability and thermal stability of protein and other active molecules, PS03 is widely used in protein and antibody stabilization, representative manufacturers include but not limited to Shenzhen Duoruisite Medical Technology Co., Ltd.

[0038] The term "PS04" refers to: a complex liquid protein protectant, English name Protein protectant 04, can significantly improve the storage stability and thermal stability of protein and other active molecules, PS04 is widely used in protein and antibody stabilization, representative manufacturers include but not limited to Shenzhen Duoruisite Medical Technology Co., Ltd.

[0039] The term "PS05" refers to a compounded liquid protein protectant, English name Protein protectant 05, transparent liquid, can significantly improve the storage stability and thermal stability of protein and other active molecules, PS05 is widely used in protein and antibody stability, representative manufacturers include but are not limited to Shenzhen Duoruisite Medical Technology Co., Ltd.

[0040] The term "PS06" refers to a compounded liquid protein protectant, English name Protein protectant 06, transparent liquid, can significantly improve the storage stability and thermal stability of protein and other active molecules, representative manufacturers include but are not limited to Shenzhen Duoruisite Medical Technology Co., Ltd.

[0041] The term "PS07" refers to a compounded liquid protein protectant, English name Protein protectant 07, transparent liquid, can significantly improve the storage stability and thermal stability of protein and other active molecules, representative manufacturers include but are not limited to Shenzhen Duoruisite Medical Technology Co., Ltd.

[0042] The term "PS09" refers to a compounded liquid protein protectant, English name Protein protectant 09, transparent liquid, can significantly improve the storage stability and thermal stability of protein and other active molecules, PS09 is widely used in protein and antibody stability, representative manufacturers include but are not limited to Shenzhen Duoruisite Medical Technology Co., Ltd.

[0043] The term "PS10" refers to a compounded liquid protein protectant, English name Protein protectant 10, transparent liquid, can significantly improve the storage stability and thermal stability of protein and other active molecules, representative manufacturers include but are not limited to Shenzhen Duoruisite Medical Technology Co., Ltd.

[0044] The term "PS11" refers to a compounded liquid protein protectant, English name Protein protectant 11, transparent liquid, can significantly improve the storage stability and thermal stability of protein and other active molecules, representative manufacturers include but are not limited to Shenzhen Duoruisite Medical Technology Co., Ltd.

[0045] The term "KroVin 950" refers to a stable, low-toxicity, environmentally friendly preservative, the effective ingredient is 2-methyl-4-isothiazolin-3-one (MIT).

[0046] The term "KroVin 500" refers to: a broad-spectrum, stable preservative, the active ingredient is a 40% aqueous solution of pyridine sodium salt.

[0047] The term "KroVin 300" refers to: a broad-spectrum, low-toxicity, environmentally friendly preservative, the bacteriostatic active ingredient is CMIT / MIT (chloromethyl isothiazolinone / methyl isothiazolinone), and the product composition contains alkyl carboxylate and solvent (modified glycol).

[0048] The term "KroVin 100" refers to: a high-efficiency, broad-spectrum, stable, low-toxicity preservative, English name: KroVin100, which can inhibit and even eliminate a large dose of gram-positive and negative microorganisms through its antimicrobial activity, representative manufacturers include but are not limited to Shanghai Jinpan Biotechnology Co., Ltd. and Shanghai Xibao Biotechnology Co., Ltd.

[0049] The term "Britton-Robinson buffer solution" refers to: a mixture of phosphoric acid, boric acid and acetic acid (H3PO4-HAc-H3BO3), adding different amounts of sodium hydroxide to it can form a wide range of pH buffer solutions, pH 1.8-11.9, which belongs to a wide range of buffer solutions.

[0050] The term "Tris-boric acid-EDTA buffer solution" refers to: the buffer solution contains two components, Tris buffer is a commonly used stable, non-toxic, non-hygroscopic buffer, in addition, ethylenediaminetetraacetic acid (EDTA), a metal chelating agent, binds to cationic metal ions, further inactivates DNA enzymes that require metal ion activation, is a fast and convenient ready-to-use buffer that minimizes workload-no need to weigh and adjust pH.

[0051] The term "PBS" refers to: a buffer solution widely used in biochemical research, the main components are Na2HPO4, KH2PO4, NaCl and KCl, which is generally used as a solvent to dissolve and protect reagents.

[0052] The term "Tirs-HCl buffer solution" refers to: Tris-Hydroxymethyl Aminomethane Hydrochloride buffer solution, English name: Tris(Hydroxymethyl)Aminomethane Hydrochloride, in powder form, commonly used to prepare buffer solutions, effective buffer range pH 7.0-9.0, commonly used as a solvent for nucleic acids and proteins in molecular biology.

[0053] The term "Tetronic 1307" refers to: a non-ionic surfactant, with high hydrophilicity and good dispersion performance, it is a high molecular compound arranged by polyoxyethylene and polypropylene alcohol chain alternately and repeatedly, used for surface activity modification of liquid system or solid surface, has excellent wetting, dispersion, emulsification and antistatic performance for various substances, can improve the fluidity and wettability of products, make the particles uniformly dispersed, enhance the stability and reduce the viscosity.

[0054] The term "Triton X-100" refers to: a non-ionic surfactant, Chinese name Triton X-100, used in molecular biology and immunology to promote the dissolution of lipids and proteins, can be used in various experiments, such as cell lysis, protein extraction, enzyme storage solution and preparation of other protein stabilizing solution.

[0055] The term "Tween-20" refers to: a non-ionic surfactant derived from sorbitan ester, polysorbate-20, easily miscible in water.

[0056] The term "Tween-80" refers to: a non-ionic surfactant and emulsifier, made from sorbitan and oleic acid by ethoxylation, often used as an emulsifier in food, Tween 80 is a amber oil liquid, easily soluble in water, its hydrophilic group is polyoxyethylene group, which is a polymer of ethylene oxide.

[0057] The term "BSA" refers to: bovine serum albumin, generally used as a stabilizer in the storage solution and reaction solution of restriction enzyme or modified enzyme to stabilize enzyme activity and prevent enzyme decomposition and non-specific adsorption.

[0058] The term "PEG" refers to: a high molecular polymer, the chemical formula is HO(CH2CH2O) n H, non-irritating, slightly bitter taste, good water solubility, and good compatibility with many organic components. It has excellent lubricity, moisturizing, dispersing, and adhesive properties, at the same time, polyethylene glycol is also a common enzyme stabilizer.

[0059] The term "sodium alginate" refers to: a natural polysaccharide, which exhibits viscous properties in aqueous solution, sodium alginate is used as thickening agent, gelling agent, stabilizer and emulsifier, etc., to extend the shelf life, and has good heat resistance and stability, in the medical field, sodium alginate is often used as a filler, stabilizer and film coating agent for preparations.

[0060] The first aspect of the present application provides a saliva uric acid detection test strip, the saliva uric acid detection test strip comprises: uricase, peroxidase, vitamin C oxidase, bilirubin oxidase, color developing agent and protective film coated on the saliva uric acid detection test strip; wherein:

[0061] the color developer comprises o-phenylenediamine hydrochloride and a color developer component selected from one or more of 3,3,5,5-tetramethylbenzidine, 4-aminoantipyrine, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, 3,5-dichloro-2-hydroxybenzenesulfonic acid sodium, preferably a color developer component selected from one or more of 4-aminoantipyrine, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, 3,5-dichloro-2-hydroxybenzenesulfonic acid sodium, more preferably 4-aminoantipyrine and / or N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt.

[0062] The saliva uric acid test strip according to the first aspect of the present application, wherein the saliva uric acid test strip further comprises: a composite enzyme stabilizer and a protein protective agent;

[0063] the composite enzyme stabilizer is selected from one or more of MP, HP, AFP, ADP, DBP, DB, SHE-50, Phos-E, AES, ADG, DPD, DPPD, AFD, ES01, ES02, ES03, ES04, ES05, ES06, ES07, ES08, preferably selected from one or more of HP, ADP, AES, ADG, DPD, ES01, ES03, ES04, ES07, more preferably selected from one or more of ADP, AES, ADG, DPD, ES01, ES03, ES04; and / or

[0064] the protein protective agent is selected from one or more of S15, HPD, GH, YT, AEP-HBC, DP4, DP7, DPS50, PS01, PS03, PS04, PS05, PS06, PS07, PS09, PS10, PS1, preferably selected from one or more of HPD, AEP-HBC, DP4, DPS50, PS04, PS05, PS09, PS10, more preferably selected from one or more of HPD, AEP-HBC, DPS50, PS04, PS09.

[0065] The saliva uric acid test strip according to the first aspect of the present application, wherein,

[0066] the peroxidase is horseradish peroxidase or catalase; and / or

[0067] the protective film is selected from one or more of polyvinyl alcohol, dopamine, tannic acid, 3-aminopropyl triethoxysilane, hyperbranched polyethyleneimine, polyvinylpyrrolidone, polydimethylsiloxane, preferably polyvinyl alcohol or hyperbranched polyethyleneimine, most preferably polyvinyl alcohol.

[0068] The saliva uric acid detection test strip according to the first aspect of the present application, wherein the saliva uric acid detection test strip further comprises one or more components selected from the group consisting of a liquid diffusion control agent, a buffer base solution, bovine serum albumin, sodium alginate, gelatin, a preservative and a desiccant; wherein:

[0069] Preferably, the liquid diffusion control agent is selected from one or more of polyethylene glycol 200, Tetronic 1307, Triton X-100, Tween-20, Tween-80, polyvinylpyrrolidone, more preferably from one or more of ethylene glycol 200, Triton X-100, Tween-20, Tween-80, polyvinylpyrrolidone, most preferably polyethylene glycol 200;

[0070] Preferably, the buffer base solution is selected from one or more of a Britton-Robinson buffer solution, a Tris-boric acid-EDTA buffer solution, PBS, Tirs-HCl, more preferably a Britton-Robinson buffer solution or a Tris-boric acid-EDTA buffer solution, most preferably a Britton-Robinson buffer solution;

[0071] Preferably, the preservative is selected from one or more of KroVin 950, KroVin 500, KroVin 300, KroVin 100, more preferably from one or more of KroVin 950, KroVin 500, KroVin 300, further preferably KroVin 950 or KroVin 300; and / or

[0072] Preferably, the desiccant is selected from one or more of silica gel, activated carbon, molecular sieve, WiseFlex+ composite dry filter membrane, WiseSheet polymer desiccant sheet, more preferably from one or more of silica gel, WiseFlex+ composite dry filter membrane, WiseSheet polymer desiccant sheet, further preferably silica gel or WiseSheet polymer desiccant sheet.

[0073] The second aspect of the present application provides a method for preparing the saliva uric acid detection test strip according to the first aspect, the method comprising the following steps:

[0074] (1) preparing an impregnation solution containing uricase, peroxidase, vitamin C oxidase, bilirubin oxidase, chromogenic agent and sodium alginate, and mixing well for use;

[0075] (2) soaking the test paper in the impregnation solution prepared in step (1), plating a protective film and drying.

[0076] According to the method of the second aspect of the present application, wherein, in the step (1), the preparation of the immersion solution comprises the following steps:

[0077] a) adding a buffer base into a soaking dish, and then adding uricase, peroxidase, vitamin C oxidase, bilirubin oxidase, bovine serum albumin, a liquid diffusion control agent, a color developing agent, and a complex enzyme stabilizer, a protein protective agent, and a preservative in sequence, fully dissolving and mixing, and then adding the protein protective agent and the preservative; and

[0078] b) adding a sodium alginate and gelatin solution cooled to room temperature, and stirring uniformly to obtain the immersion solution.

[0079] According to the method of the second aspect of the present application, wherein, in the step a):

[0080] The amount of the buffer base is 3-15 mL, preferably 3-10 mL, and more preferably 5-8 mL;

[0081] The amount of the uricase is 10-500 U, preferably 20-300 U, and more preferably 30-100 U;

[0082] The amount of the peroxidase is 1000-3000 U, preferably 1000-2500 U, and more preferably 1500-2000 U;

[0083] The amount of the vitamin C oxidase is 5-50 U, preferably 5-30 U, and more preferably 5-20 U;

[0084] The amount of the bilirubin oxidase is 2-50 U, preferably 2-20 U, and more preferably 2-10 U;

[0085] The amount of the bovine serum albumin is 30-300 mg, preferably 50-200 mg, and more preferably 50-100 mg;

[0086] The amount of the liquid diffusion control agent is 0.25-3 mL, preferably 0.25-2 mL, and more preferably 0.5-1 mL;

[0087] The amount of the protein protective agent (in mass volume percentage, unless otherwise specified, the mass volume percentage unit herein is mg / mL) is 0.5%-5%, preferably 0.5%-3%, and more preferably 1%-2.5%;

[0088] The amount of the complex enzyme stabilizer (in volume percentage) is 0.2%-5%, preferably 0.3%-3%, and more preferably 0.5%-2%; and / or

[0089] The amount of the preservative (in volume percentage or mass volume percentage, wherein when mass volume percentage is used, the unit of mass volume percentage herein is mg / mL) is 0.5% to 3%, preferably 0.5% to 2%, more preferably 1% to 2%.

[0090] According to the method of the second aspect of the present application, in the step a), when the color developing agent is 4-aminoantipyrine, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt and o-phenylenediamine hydrochloride:

[0091] The mass ratio of the 4-aminoantipyrine, the N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt and the o-phenylenediamine hydrochloride is 1 to 10: 5 to 25: 1 to 8, preferably 2 to 8: 5 to 15: 1 to 5, more preferably 2 to 5: 8 to 12: 2 to 3.

[0092] According to the method of the second aspect of the present application, in the step a), the vitamin C oxidase solution and the gelatin solution are prepared 24 to 36 hours in advance, preferably 24 hours in advance.

[0093] In the steps a) and b), the vitamin C oxidase solution and the gelatin solution are prepared 24 to 36 hours in advance, preferably 24 hours in advance.

[0094] In the step b), the amount of the gelatin in the sodium alginate solution is 0.1 to 3 mL, preferably 0.5 to 2 mL, most preferably 1 mL; and / or

[0095] In the step b), the amount of the gelatin in the gelatin solution is 0.1 to 3 mL, preferably 0.5 to 2 mL, most preferably 1 mL.

[0096] According to the method of the second aspect of the present application, in the step (2), the following steps are included: the rough surface of the filter paper is immersed in the soaking solution and dried, and then the rough surface of the filter paper is immersed in the protective film solution and dried to obtain the saliva uric acid detection test strip; wherein,

[0097] The time for the immersion in the soaking solution is 20 to 36 hours, more preferably 24 to 36 hours, most preferably 24 hours;

[0098] The time for the immersion in the protective film solution is 1.5 to 5 minutes, more preferably 1.5 to 3 minutes, most preferably 2 minutes; and / or

[0099] The protective film solution must completely cover the test paper.

[0100] Preferably, in the step (2), after the immersion in the protective film solution, the following steps are further included:

[0101] (3) The test paper plated with the protective film and dried in step (2) is cut and placed in a desiccator at room temperature, and then vacuum packaged to obtain the saliva uric acid detection test paper strip.

[0102] The third aspect of the present application provides use of the saliva uric acid detection test paper strip of the first aspect in the preparation of a product for diagnosing hyperuricemia diseases.

[0103] The fourth aspect of the present application provides a method for diagnosing hyperuricemia diseases, which comprises using the saliva uric acid detection test paper strip of the first aspect for diagnosis.

[0104] The fifth aspect of the present application provides a method for detecting saliva uric acid, which comprises using the saliva uric acid detection test paper strip of the first aspect to detect saliva samples or subjects in need.

[0105] According to the use or method of the third aspect, the fourth aspect or the fifth aspect of the present application, the hyperuricemia disease is selected from one or more of the following: hyperuricemia, gout, hypouricemia, urinary tract stones, uric acid nephropathy, renal dysfunction, urinary tract infection, preferably selected from one or more of the following: hyperuricemia, gout, hypouricemia, more preferably hyperuricemia and / or gout.

[0106] The sixth aspect of the present application provides a saliva uric acid detection product or device, which comprises the saliva uric acid detection test paper strip of the first aspect.

[0107] Preferably, the detection product is a detection kit.

[0108] According to a specific embodiment of the present application, the preparation method of the saliva rapid uric acid detection test paper of the present application is as follows:

[0109] Raw materials: whatman test paper, uric acid, uricase, horseradish peroxidase, bilirubin oxidase, lithium carbonate, 4-aminoantipyrine, N-ethyl-N-(3-sulfopropyl)-3-methyl aniline sodium salt, o-phenylenediamine hydrochloride, sodium alginate purchased from Macklin, vitamin C oxidase, polyethylene glycol-200 purchased from Shanghai Yuan Ye Biological Technology Co., Ltd., bovine serum albumin, gelatin purchased from sigma, Britton-Robinson pH = 8.0 purchased from Qingdao Jetseikang Biological Technology Co., Ltd., polyvinyl alcohol purchased from Shanghai Mayreer Chemical Technology Co., Ltd., composite enzyme stabilizer AES, protein protective agent HPD and preservative KroVin 950 purchased from Shanghai Xibaobao Biological Technology Co., Ltd. Desiccant silica gel purchased from Shangwan Weisheng Technology Development Co., Ltd.

[0110] The chromogenic substance 4-aminoantipyrine and N-ethyl-N-(3-sulfopropyl)-3-methyl aniline sodium salt can form a very stable purple dye through oxidative coupling reaction, and at the same time, the chromogenic agent o-phenylenediamine hydrochloride (OPD) can also generate a yellow product 2,2-diaminoazobenzene in the presence of hydrogen peroxide and horseradish peroxidase. Through the competitive reaction of different chromogenic agents in the hydrogen peroxide and horseradish peroxidase environment, the test paper can not be colored under the normal saliva uric acid concentration of 100 mu M, and can be colored purple under the high saliva uric acid concentration of 325 mu M. Therefore, the high and low of the saliva uric acid content can be directly judged by whether the test paper is colored or not.

[0111] In order to improve the differentiation degree of the test paper coloration corresponding to the high and low of the saliva uric acid concentration, the chromogenic agent further comprises one or more of the following chromogenic agent components: 3,3,5,5-tetramethylbenzidine, o-phenylenediamine hydrochloride, 4-aminoantipyrine, N-ethyl-N-(3-sulfopropyl)-3-methyl aniline sodium salt, 3,5-dichloro-2-hydroxybenzenesulfonic acid sodium.

[0112] The saliva uric acid detection test paper strip of the application does not color under the normal saliva uric acid concentration, and the coloration under the high concentration is convenient for the tester to intuitively distinguish the high and low of the uric acid content, and the room temperature storage time is up to half a year.

[0113] The enzyme stabilizer ES series product is a compound broad-spectrum enzyme stabilizer, which can reduce the risk of enzyme inactivation due to changes in external conditions such as temperature, pH value, ion concentration, prevent the direct impact of enzymes under adverse conditions, inhibit the inactivation of enzymes, thereby enhancing the storage life of enzymes, increasing the stability of enzyme molecules, prolonging the storage life of enzymes, and keeping them active for a longer time. The enzyme protection components of the ES series do not interfere with enzyme activity. 01-08 mainly differs in components, and the applicable enzymes are also different.

[0114] The protein protection agent PS01 series product is a compound liquid protein protection agent, which is specially designed for adding to customer's own prepared antigen or antibody standard, calibration or quality control diluent, and can also be used for the preparation of conjugate diluent. These products are designed to significantly improve the storage stability and thermal stability of protein active molecules such as working solution, thereby prolonging the shelf life of the kit. The protein protection agent PS series has strong stabilizing effect, and provides a reliable reagent storage solution for customers.

[0115] Take 5 mL Britton-Robinson buffer solution with a pipette into the soaking dish, add 50 U uricase, 1800 U horseradish peroxidase, 10 U vitamin C oxidase, 5 U bilirubin oxidase, 50 mg bovine serum albumin, 500 μL polyethylene glycol-200, 4.06 mg 4-aminoantipyrine, 11.8 mg N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, 3 mg o-phenylenediamine hydrochloride into the soaking dish in turn, fully dissolve and mix, then add 4.44 mg AES, 87.5 μL HPD, 15 μL KroVin 950, then add 1 mL sodium alginate (1 g sodium alginate is added to 80 ml water, heated to dissolve and then placed at room temperature), 1 mL gelatin (1.5 g gelatin is added to 10 mL distilled water, soaked overnight, heated to dissolve before use and then placed at room temperature), then immerse the test paper with the front side down in the solution, after 24 h in a 30℃ water bath, dry with an oven at 30℃. The above dried test paper is soaked in 50 mL 10 mg / mL polyvinyl alcohol solution, and after drying completely in a 30℃ oven again, the test paper is cut into 1 cm x 1.5 cm test paper for detection of salivary uric acid. Unused test paper is vacuum packaged after adding desiccant silica gel and stored at room temperature in the dark.

[0116] The working principle of the product: uric acid in the saliva sample can be catalytically oxidized to generate product allantoin, carbon dioxide and hydrogen peroxide under the action of uricase; hydrogen peroxide can further generate red quinone imine compound with color developing substance 4-aminoantipyrine (4-AAP) and N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS) under the action of horseradish peroxidase, at the same time, color developing agent o-phenylenediamine hydrochloride (OPD) can also generate yellow product 2,2-diaminoazobenzene under the condition of hydrogen peroxide and horseradish peroxidase. Through the competitive reaction of different color developing agents in the environment of hydrogen peroxide and horseradish peroxidase, the test paper can not develop color under normal salivary uric acid concentration of 100 μM, and develop purple color under high salivary uric acid concentration of 325 μM. Therefore, the salivary uric acid content can be directly judged by whether the test paper develops color or not. The corresponding reaction process is shown in Figure 5.

[0117] The use method of the product: fresh saliva is directly dropped onto the detection area of the test paper, and the color of the test paper is directly observed by the naked eye after the color is stable, and the color of the test paper is compared with the standard color card to obtain the salivary uric acid content range.

[0118] The application can non-invasively and timely monitor the concentration of uric acid of a patient, is beneficial to the patient to timely control the health state and early discover the disease, and select the best treatment opportunity. The product is simple in manufacturing process and low in cost, and can be mass-produced. The tester can clearly distinguish the high and low of the concentration of saliva uric acid detection, and provides a long-term room temperature storage method. The application provides an efficient, convenient and economical method for monitoring the uric acid content.

[0119] The saliva rapid uric acid detection test paper of the application is convenient for timely monitoring the high and low of the uric acid content, and provides certain guiding significance for the health examination of patients. As a practical product for clinically understanding the uric acid concentration of patients, as an auxiliary tool for clinically evaluating the uric acid concentration of patients, and as a disease screening and detection for large-scale rural residents.

[0120] The saliva rapid uric acid detection test paper of the application can have but is not limited to the following beneficial effects:

[0121] 1. The saliva rapid uric acid detection test paper of the application can clearly distinguish the high and low of the concentration of saliva uric acid detection, and can be stored at room temperature for a long time.

[0122] 2. Convenience and non-invasiveness: the saliva rapid uric acid detection test paper of the application is more convenient and non-invasive than blood sampling, does not need blood drawing, and only needs to collect a small amount of saliva sample for detection, is suitable for personal self-testing or rapid screening at home or in medical institutions, reduces the discomfort and sampling risk of patients, and is particularly suitable for some specific groups of people, such as children, the elderly and needle-phobic people.

[0123] 3. Real-time: the collection and detection process of saliva samples is relatively fast, and the result of the saliva rapid uric acid detection test paper of the application can be obtained quickly, which is helpful for timely understanding of the uric acid level of an individual and improving the treatment and intervention effect.

[0124] 4. Cost saving: compared with the traditional urine or blood detection method, the development of the saliva rapid uric acid detection test paper can be lower in cost, is suitable for large-scale screening and monitoring, and is helpful for reducing the medical cost.

[0125] 5. Popularity: the collection of saliva samples is relatively simple, the saliva rapid uric acid detection test paper of the application can be used in different environments and resource-limited areas, and is helpful for improving the coverage and popularity of uric acid monitoring, especially in remote areas and developing countries.

[0126] 6. Privacy protection: the saliva collection process is more private and concealed, the saliva rapid uric acid detection test paper of the application does not involve blood collection and personal privacy exposure, and is more in line with the privacy protection needs of patients.

[0127] Brief description of the drawings

[0128] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings. In the following description, well-known functions or constructions are not described in detail because they would obscure the application in unnecessary detail. Also, the same reference numerals and signs are used to the same elements throughout the several drawings.

[0129] FIG. 1 shows a preservation effect of the saliva uric acid test strip of the present application prepared in Example 1.

[0130] FIG. 2 shows a preservation effect of the saliva uric acid test strip of the present application prepared in Example 2.

[0131] FIG. 3 shows a preservation effect of the saliva uric acid test strip of the present application prepared in Example 3.

[0132] FIG. 4 shows a comparison of the saliva uric acid test strip of the present application with other components in the experiment optimization in Examples 1-3.

[0133] FIG. 5 shows a reaction process of the saliva uric acid test strip of the present application.

[0134] Best Mode for Carrying Out the Invention

[0135] The present application will be further described by way of specific examples, but it is to be understood that these examples are only by way of illustration and should not be construed to limit the present application in any form.

[0136] This section describes the materials and methods used in the experiments of the present application in general. Although many materials and methods used to achieve the purpose of the present application are well known in the art, the present application is described as much as possible in detail. It is clear to those skilled in the art that, in the context, if not specifically stated, the materials and methods used in the present application are well known in the art.

[0137] The following examples will help to further understand the present application, but do not limit the content of the present application.

[0138] The reagents and instruments used in the following examples are as follows:

[0139] Materials:

[0140] Filter paper Whatman No. 3, purchased from Whatman Company.

[0141] Reagents:

[0142] Uric acid, uricase, horseradish peroxidase, bilirubin oxidase, lithium carbonate, 4-aminoantipyrine, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, o-phenylenediamine hydrochloride, sodium alginate were purchased from Macklin. Vitamin C oxidase, polyethylene glycol-200 were purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. Bovine serum albumin, gelatin were purchased from Sigma. Polyvinyl alcohol was purchased from Shanghai Mayre Chemical Technology Co., Ltd. Britton-Robinson pH = 8.0 was purchased from Qingdao Jetsekang Biological Technology Co., Ltd. Compound enzyme stabilizer ES03, ES04 were purchased from Shanghai Jinpan Biological Technology Co., Ltd. Protein protective agent HPD, AEP-HBC and preservative KroVin300, KroVin950 were purchased from Shanghai Xibao Biological Technology Co., Ltd. Desiccant silica gel was purchased from Shangwan Weisheng Technology Development Co., Ltd.

[0143] Example 1

[0144] This example is used to illustrate the saliva uric acid test strip and the preparation method thereof.

[0145] (1) Take 5 mL of Britton-Robinson buffer solution into the soaking dish with a pipette, and then add 50 U of uricase, 1800 U of horseradish peroxidase, 10 U of vitamin C oxidase, 5 U of bilirubin oxidase, 50 mg of bovine serum albumin, 500 μL of polyethylene glycol-200, 4.06 mg of 4-aminoantipyrine, 11.8 mg of N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, and 3 mg of o-phenylenediamine hydrochloride into the soaking dish in turn, fully dissolve and mix, then add 3.5 mg of ES04, 175 μL of HPD, and 20 μL of KroVin300.

[0146] (2) Then add 1 mL of sodium alginate (1 g of sodium alginate is added to 80 ml of water, heated to dissolve, and then placed at room temperature), 1 ml of gelatin (3 g of gelatin is added to 10 mL of distilled water, soaked overnight, heated to dissolve before use, and then placed at room temperature), then immerse the test paper with the front side facing down in the solution, and after 24 hours in a 30°C water bath, dry completely in a 30°C oven for 30 minutes.

[0147] (3) Soak the above dried test paper in 50 mL of 10 mg / mL polyvinyl alcohol solution for 2 minutes, and then dry completely in a 30°C oven for 30 minutes. Cut the test paper into 1 cm x 1.5 cm test paper for detection of saliva uric acid. Unused test paper is vacuum packaged with desiccant silica gel and stored at room temperature in the dark.

[0148] Example 2

[0149] This example is used to illustrate the saliva uric acid detection test strip and its preparation method of the present application.

[0150] (1) Take 5 mL Britton-Robinson buffer solution into the soaking dish with a pipette gun, and then add 50 U uricase, 1800 U horseradish peroxidase, 10 U vitamin C oxidase, 5 U bilirubin oxidase, 50 mg bovine serum albumin, 500 μL polyethylene glycol-200, 4.06 mg 4-aminoantipyrine, 11.8 mg N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, 3 mg o-phenylenediamine hydrochloride, 3.5 mg ES04, 33.6 μL AEP-HBC, and 75 μL KroVin 950 into the soaking dish in sequence, and then fully dissolve and mix.

[0151] (2) Then add 1 mL of sodium alginate (1 g of sodium alginate is added to 80 ml of water, heated to dissolve, and then placed at room temperature), and then add 1 mL of gelatin (3 g of gelatin is added to 10 mL of distilled water, soaked overnight, heated to dissolve before use, and then placed at room temperature), and then immerse the test paper with the front face downward in the solution, and then place in a 30°C water bath for 24 hours, and then dry completely in a 30°C oven for 30 minutes.

[0152] (3) Soak the above dried test paper in 50 mL of 10 mg / mL polyvinyl alcohol solution for 2 minutes, and then dry completely in a 30°C oven for 30 minutes, and then cut the test paper into 1 cm x 1.5 cm test paper for saliva uric acid detection. Unused test paper is vacuum packaged with desiccant silica gel and stored at room temperature in the dark.

[0153] Example 3

[0154] This example is used to illustrate the saliva uric acid detection test strip and its preparation method of the present application.

[0155] (1) Take 5 mL Britton-Robinson buffer solution into the soaking dish with a pipette gun, and then add 50 U uricase, 1800 U horseradish peroxidase, 10 U vitamin C oxidase, 5 U bilirubin oxidase, 50 mg bovine serum albumin, 500 μL polyethylene glycol-200, 4.06 mg 4-aminoantipyrine, 11.8 mg N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, 3 mg o-phenylenediamine hydrochloride, 17.5 mg ES03, 99 μL AEP-HBC, and 30 μL KroVin 950 into the soaking dish in sequence, and then fully dissolve and mix.

[0156] (2) Then 1 mL of sodium alginate (1 g of sodium alginate was added to 80 mL of water, heated to dissolve and then left to room temperature) and 1 mL of gelatin (3 g of gelatin was added to 10 mL of distilled water, soaked overnight, heated to dissolve and then left to room temperature before use) were added, and then the test paper was immersed in the solution with the front side facing down. After 24 h in a 30°C water bath, the test paper was dried completely in a 30°C oven for 30 min.

[0157] (3) The dried test paper was immersed in 50 mL of a 10 mg / mL polyvinyl alcohol solution for 2 min, and then dried completely in a 30°C oven for 30 min. The test paper was then cut into 1 cm x 1.5 cm test papers for use in the detection of salivary uric acid. Unused test papers were vacuum-sealed with desiccant silica gel and stored in the dark at room temperature.

[0158] Test Example 1

[0159] This test example was used to illustrate the storage effect of the salivary uric acid detection test paper strip of the present application.

[0160] The salivary uric acid detection test paper strips prepared in Examples 1-3 were used in this test example.

[0161] A 1600 μmol / L uric acid standard stock solution was prepared by dissolving 60 mg of lithium carbonate in 40 mL of distilled water in a 60°C water bath to completely dissolve the lithium carbonate. 26.9 mg of uric acid was accurately weighed and dissolved in the hot lithium carbonate solution, which was then cooled to room temperature and transferred to a 100 mL volumetric flask. Distilled water was added to the flask to the calibration mark, and then diluted to the corresponding concentration for detection. The solution was stored in a brown bottle for use.

[0162] Uric acid solutions with concentrations of 0, 100, 325, and 750 μM were prepared to test the effect of the prepared test paper strips. The color development area of the obtained test paper strips was immersed in the uric acid solution for 5 s, taken out and left to stand for 30 s, and then the results were read after the color change of the test paper was stable.

[0163] The test paper was vacuum-sealed after being placed in a desiccator at room temperature, and the performance of the test paper was tested every other month.

[0164] Figures 1-3 show the storage effect of the salivary uric acid detection test paper strips of the present application prepared in Examples 1-3, respectively. As can be seen from Figures 1-3, the detection effect of the test paper remained good after being placed at room temperature for 6 months, indicating that the room temperature storage time of the salivary uric acid detection test paper strip of the present application can be as long as half a year.

[0165] Comparative Example 1

[0166] This comparative example was used to compare the effect of the salivary uric acid detection test paper strip of the present application and other components in the experimental optimization.

[0167] Table 1 Effect comparison of saliva uric acid detection test strips of the present application and other components in experimental optimization

[0168] From Table 1, it can be seen that:

[0169] Comparative Example 1 and the test strips prepared in Examples 1-3 of the present application differ only in that the impregnating solution of Comparative Example 1 does not contain o-phenylenediamine hydrochloride, sodium alginate, a composite enzyme stabilizer, a protein protective agent, a preservative and a drying agent.

[0170] Comparative Example 2 and the test strips prepared in Examples 1-3 of the present application differ only in that the impregnating solution of Comparative Example 2 does not contain sodium alginate, a composite enzyme stabilizer, a protein protective agent, a preservative and a drying agent.

[0171] As shown in Figure 4, the room temperature storage time of Comparative Examples 1 and 2 is 46 days and 45 days, respectively, both of which are less than the storage time of the test strips of the present application. The color development of the test strips in Comparative Example 1 cannot intuitively distinguish saliva uric acid content in the normal value / abnormal value range. In Comparative Example 2, although the color development of the test strips is sufficient, if the storage time exceeds the above-mentioned storage time, the color development is not convenient for intuitively distinguishing whether the saliva uric acid content is in the normal value range. It is shown that the present application selects o-phenylenediamine hydrochloride, sodium alginate, a composite enzyme stabilizer, a protein protective agent, a preservative and a drying agent, which greatly improves the color development contrast of the test strips, facilitates the tester to intuitively distinguish the uric acid content, and at the same time realizes the real-time detection of uric acid content for the applicable population.

[0172] Comparative Example 2

[0173] The present comparative example is used to compare the saliva uric acid detection test strips of the present application with other uric acid detection test strips.

[0174] Table 2 Comparison of saliva uric acid detection test strips of the present application and other uric acid detection test strips

[0175] From Table 2, it can be seen that the present application selects color developers 4-aminoantipyrine, N-ethyl-N-(3-sulfopropyl)-3-methyl aniline sodium salt and o-phenylenediamine hydrochloride, which have very stable color response, the addition of vitamin C oxidase and bilirubin oxidase overcomes false positive results, PEG-200, gelatin, a composite enzyme stabilizer and a protein protective agent increase the stability of the enzyme and the uniformity of the system, at the same time, the addition of sodium alginate, a preservative and a drying agent also improves the room temperature storage time of the test strips, and realizes the real-time detection of uric acid content for the applicable population.

[0176] While the application has been described with a certain degree of particularity, it is to be understood that the present application is capable of numerous modifications. It is intended that the scope of the application be limited only by the broadest interpretation of the appended claims to encompass all equivalents.

Claims

1. A saliva uric acid test strip, characterized by, The saliva uric acid detection test strip comprises: uricase, peroxidase, vitamin C oxidase, bilirubin oxidase, color developing agent and protective film coated on the saliva uric acid detection test strip; wherein: The color developing agent comprises o-phenylenediamine hydrochloride, and a color developing agent component selected from one or more of 3,3,5,5-tetramethylbenzidine, 4-aminoantipyrine, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, 3,5-dichloro-2-hydroxybenzenesulfonic acid sodium, preferably a color developing agent component selected from one or more of 4-aminoantipyrine, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, 3,5-dichloro-2-hydroxybenzenesulfonic acid sodium, more preferably 4-aminoantipyrine and / or N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt.

2. The saliva uric acid test strip according to claim 1, characterized in that, The saliva uric acid detection test strip further comprises: a composite enzyme stabilizer and a protein protective agent; The composite enzyme stabilizer is selected from one or more of MP, HP, AFP, ADP, DBP, DB, SHE-50, Phos-E, AES, ADG, DPD, DPPD, AFD, ES01, ES02, ES03, ES04, ES05, ES06, ES07, ES08, preferably selected from one or more of HP, ADP, AES, ADG, DPD, ES01, ES03, ES04, ES07, more preferably selected from one or more of ADP, AES, ADG, DPD, ES01, ES03, ES04; and / or The protein protective agent is selected from one or more of S15, HPD, GH, YT, AEP-HBC, DP4, DP7, DPS50, PS01, PS03, PS04, PS05, PS06, PS07, PS09, PS10, PS1, preferably selected from one or more of HPD, AEP-HBC, DP4, DPS50, PS04, PS05, PS09, PS10, more preferably selected from one or more of HPD, AEP-HBC, DPS50, PS04, PS09.

3. The saliva uric acid detection test strip according to claim 1 or 2, characterized in that: The peroxidase is horseradish peroxidase or catalase; and / or The protective film is selected from one or more of polyvinyl alcohol, dopamine, tannic acid, 3-aminopropyl triethoxysilane, hyperbranched polyethyleneimine, polyvinylpyrrolidone, polydimethylsiloxane, preferably polyvinyl alcohol or hyperbranched polyethyleneimine, most preferably polyvinyl alcohol.

4. The saliva uric acid test strip according to any one of claims 1 to 3, characterized in that, The saliva uric acid detection test strip further comprises one or more components selected from a liquid diffusion control agent, a buffer base solution, bovine serum albumin, sodium alginate, gelatin, a preservative and a desiccant; wherein: Preferably, the liquid diffusion control agent is selected from one or more of polyethylene glycol 200, Triton X-100, Tween-20, Tween-80, polyvinylpyrrolidone, more preferably from one or more of polyethylene glycol 200, Triton X-100, Tween-20, Tween-80, polyvinylpyrrolidone, most preferably polyethylene glycol 200; Preferably, the liquid diffusion control agent is selected from one or more of polyethylene glycol 200, Triton X-100, Tween-20, Tween-80, polyvinylpyrrolidone, more preferably from one or more of polyethylene glycol 200, Triton X-100, Tween-20, Tween-80, polyvinylpyrrolidone, most preferably polyethylene glycol 200; Preferably, the buffer base solution is selected from one or more of Britton-Robinson buffer solution, Tris-boric acid-EDTA buffer solution, PBS, Tirs-HCl, more preferably Britton-Robinson buffer solution or Tris-boric acid-EDTA buffer solution, most preferably Britton-Robinson buffer solution; Preferably, the preservative is selected from one or more of KroVin 950, KroVin 500, KroVin 300, KroVin 100, more preferably from one or more of KroVin 950, KroVin 500, KroVin 300, further preferably KroVin 950 or KroVin 300; and / or Preferably, the drying agent is selected from one or more of silica gel, activated carbon, molecular sieve, WiseFlex+ composite drying filter membrane, WiseSheet polymer drying sheet, more preferably from one or more of silica gel, WiseFlex+ composite drying filter membrane, WiseSheet polymer drying sheet, further preferably silica gel or WiseSheet polymer drying sheet.

5. A method for preparing the saliva uric acid test strip according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: (1) configuring an immersion solution containing uricase, peroxidase, vitamin C oxidase, bilirubin oxidase, color developing agent and sodium alginate, mixing and ready for use; (2) soaking the test paper in the immersion solution configured in step (1), plating a protective film and drying.

6. The method of claim 5, wherein, In step (1), configuring the immersion solution comprises the following steps: a) adding a buffer base solution into a soaking dish, then adding uricase, peroxidase, vitamin C oxidase, bilirubin oxidase, bovine serum albumin, a liquid diffusion control agent, a color developing agent in sequence, fully dissolving and mixing, then adding a composite enzyme stabilizer, a protein protective agent and a preservative; and b) adding a sodium alginate and gelatin solution cooled to room temperature and stirring uniformly to obtain the immersion solution.

7. The method of claim 6, wherein, In step a): The amount of the buffer base solution is 3-15 mL, preferably 3-10 mL, more preferably 5-8 mL; The amount of the uricase is 10-500 U, preferably 20-300 U, more preferably 30-100 U; The amount of the peroxidase is 1000-3000 U, preferably 1000-2500 U, more preferably 1500-2000 U; The amount of the vitamin C oxidase is 5-50 U, preferably 5-30 U, more preferably 5-20 U; The amount of said bilirubin oxidase is 2-50 U, preferably 2-20 U, more preferably 2-10 U; The amount of said bovine serum albumin is 30-300 mg, preferably 50-200 mg, more preferably 50-100 mg; The amount of said liquid diffusion control agent is 0.25-3 mL, preferably 0.25-2 mL, more preferably 0.5-1 mL; The amount of said protein protective agent is 0.5%-5%, preferably 0.5%-3%, more preferably 1%-2.5%; The amount of said complex enzyme stabilizer is 0.2%-5%, preferably 0.3%-3%, more preferably 0.5%-2%; and / or The amount of said preservative is 0.5%-3%, preferably 0.5%-2%, more preferably 1%-2%.

8. The method according to claim 6 or 7, characterized in that, In said step a), when the color developing agent is 4-aminoantipyrine, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt and o-phenylenediamine hydrochloride: The mass ratio of said 4-aminoantipyrine, said N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt and said o-phenylenediamine hydrochloride is 1-10:5-25:1-8, preferably 2-8:5-15:1-5, more preferably 2-5:8-12:2-3.

9. The method of any one of claims 6-8, wherein: In said step a) and step b), said vitamin C oxidase solution and said gelatin solution are prepared 24-36 hours in advance, preferably 24 hours in advance; In said step b), the amount of gelatin in said sodium alginate solution is 0.1-3 mL, preferably 0.5-2 mL, most preferably 1 mL; and / or In said step b), the amount of gelatin in said gelatin solution is 0.1-3 mL, preferably 0.5-2 mL, most preferably 1 mL.

10. The method according to any one of claims 5 to 9, characterized in that, In said step (2), the following steps are included: placing the filter paper with the rough surface facing down, immersing it in said immersion solution and drying, then immersing the filter paper with the rough surface facing down in a protective film solution, and drying to obtain said saliva uric acid test strip; wherein, The time for immersing in said immersion solution is 20-36 hours, more preferably 24-36 hours, most preferably 24 hours; The time for immersing in said protective film solution is 1.5-5 minutes, more preferably 1.5-3 minutes, most preferably 2 minutes; and / or The protective film solution must completely cover the test paper; Preferably, in said step (2), after immersing in said protective film solution, the following steps are further included: (3) cutting the test paper plated with the protective film and dried in step (2), and vacuum packaging after placing in a desiccant at room temperature to obtain said saliva uric acid test strip.

11. Use of the saliva uric acid test strip of any one of claims 1-4 in the preparation of a product for diagnosing hyperuricemia.

12. A method of diagnosing hyperuricemic disease, characterized by, The method comprises using the saliva uric acid test strip of any one of claims 1-4 for diagnosis.

13. A method of detecting salivary uric acid, characterized by, The method comprises using the saliva uric acid test strip of any one of claims 1-4 to detect saliva samples or subjects in need.

14. The use or method of any one of claims 11 to 13, wherein, The hyperuricemic disease is selected from one or more of the following: hyperuricemia, gout, hypouricemia, urinary stone, urate nephropathy, renal dysfunction, urinary tract infection, preferably from one or more of the following: hyperuricemia, gout, hypouricemia, more preferably hyperuricemia and / or gout.

15. A product or device for detecting salivary uric acid, characterized by, The detection product or detection device comprises the saliva uric acid detection test strip according to any one of claims 1 to 4; Preferably, the detection product is a detection kit.

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