Saliva glucose test strip with extended room temperature storage time, and preparation method therefor, test method thereof and use thereof

Abstract

Provided are a saliva glucose test strip with extended room temperature storage time, and a preparation method therefor, a test method thereof and a use thereof. The saliva glucose test strip comprises: glucose oxidase, horseradish peroxidase, a vitamin C oxidase solution, a chromogenic agent, an enzyme / protein protectant, an enzyme stabilizer, and a sodium alginate solution. The storage time of the test strip at room temperature is extended by adding reagents. The saliva glucose test strip has important clinical significance in early prevention, preliminary diagnosis, treatment, and like of diabetes and oral diseases.

Description

Saliva glucose test strips with extended shelf life at room temperature, their preparation, detection methods and applications Technical Field

[0001] This invention belongs to the field of medical devices, specifically relating to a saliva glucose test strip that extends its shelf life at room temperature, its preparation and detection methods, and its applications. Background Technology

[0002] Saliva contains a range of biomarkers, including DNA, RNA, proteins, and metabolites, making it a valuable diagnostic medium for a variety of diseases, including respiratory infections, sexually transmitted infections, tropical diseases, hormonal imbalances, and cancer. Furthermore, saliva collection is simple, non-invasive, technically insensitive, and readily accepted by patients, giving it a natural advantage as a diagnostic sample. In recent years, the application of saliva testing in global medical research has been growing at a rate of approximately 10% annually.

[0003] Point-of-Care Tests (POCT), also known as point-of-care testing, represent the forefront of modern healthcare, providing a rapid and convenient solution for early disease detection and monitoring. Saliva testing is a significant advancement in POCT diagnostics in recent years, potentially making testing more convenient and efficient. Several saliva-based POCT technologies have been developed and used for large-scale epidemiological screening and routine health monitoring, including HIV saliva test strips, Helicobacter pylori saliva test kits, and drug saliva test strips. Saliva glucose has gained increasing attention from researchers due to its close association with diabetes and periodontal disease. Test strips are a simple analytical tool with advantages such as low cost, biodegradability, and portability. Test strips that allow for direct visual reading of results meet the needs of point-of-care testing. Previously, we successfully developed saliva glucose test strips; however, because the added enzymes are biological enzymes, the test strips could not be stored for long periods at room temperature, limiting their application. Therefore, we developed a saliva glucose test strip that can be stored at room temperature for extended periods.

[0004] Summary of the Invention

[0005] The purpose of this invention is to provide a salivary glucose test strip with extended shelf life at room temperature, its preparation method, and its applications. The test strip's shelf life at room temperature is extended by adding reagents. The synthesized salivary glucose test strip has significant clinical value for the early prevention, preliminary diagnosis, and treatment of diabetes and oral diseases.

[0006] Before describing the content of this invention, the following terms are defined as follows:

[0007] The term "AEP-HBC" refers to a protein protectant used for enzyme protection, anti-precipitation, solubilization, and efficacy enhancement. It is suitable for enzyme and protein liquid systems. Its main component has the molecular formula C2. 45 H 76 O 36 -[CH2CH(OH)CH3] n pH value: 5.0~8.0.

[0008] The term "TY" refers to a protein protectant that reduces background noise and improves stability. Its main components (mass fraction) are: protein content >92.0%, sodium chloride ≤0.5%, amino acids ≥3.5%, and pH value: 7.0~7.5.

[0009] The term "GH" refers to a protein protectant used to protect and stabilize enzymes and proteins. It reduces background noise and enhances stabilization. Its main components and content are: protein content >95.0%, water ≤5%, arsenic ≤0.5 mg / kg, lead ≤1 mg / kg, pH: 7.0–7.5.

[0010] The term "PS01" refers to a compound liquid protein protectant that significantly improves the storage and thermal stability of proteins and other bioactive molecules. Its main components are amino acids, and representative manufacturers include, but are not limited to, Duoruiste (Shenzhen) Medical Technology Co., Ltd.

[0011] The term "PS02" refers to a compound liquid protein protectant that significantly improves the storage and thermal stability of proteins and other bioactive molecules. Its main components are amino acids, and representative manufacturers include, but are not limited to, Duoruiste (Shenzhen) Medical Technology Co., Ltd.

[0012] The term "PS03" refers to a compound liquid protein protectant that significantly improves the storage and thermal stability of proteins and other bioactive molecules. Its main components are amino acids, and representative manufacturers include, but are not limited to, Duoruiste (Shenzhen) Medical Technology Co., Ltd.

[0013] The term "PS04" refers to a compound liquid protein protectant that significantly improves the storage and thermal stability of proteins and other bioactive molecules. Its main components are amino acids, and representative manufacturers include, but are not limited to, Duoruiste (Shenzhen) Medical Technology Co., Ltd.

[0014] The term "PS05" refers to a compound liquid protein protectant that significantly improves the storage and thermal stability of proteins and other bioactive molecules. Its main components are amino acids, and representative manufacturers include, but are not limited to, Duoruiste (Shenzhen) Medical Technology Co., Ltd.

[0015] The term "PS06" refers to a compound liquid protein protectant that significantly improves the storage and thermal stability of proteins and other bioactive molecules. Its main components are amino acids, and representative manufacturers include, but are not limited to, Duoruiste (Shenzhen) Medical Technology Co., Ltd.

[0016] The term "PS07" refers to a compound liquid protein protectant that significantly improves the storage and thermal stability of proteins and other bioactive molecules. Its main components are amino acids, and representative manufacturers include, but are not limited to, Duoruiste (Shenzhen) Medical Technology Co., Ltd.

[0017] The term "PS08" refers to a compound liquid protein protectant that significantly improves the storage and thermal stability of proteins and other bioactive molecules. Its main components are amino acids, and representative manufacturers include, but are not limited to, Duoruiste (Shenzhen) Medical Technology Co., Ltd.

[0018] The term "PS09" refers to a compound liquid protein protectant that significantly improves the storage and thermal stability of proteins and other bioactive molecules. Its main components are amino acids, and representative manufacturers include, but are not limited to, Duoruiste (Shenzhen) Medical Technology Co., Ltd.

[0019] The term "PS10" refers to a compound liquid protein protectant that significantly improves the storage and thermal stability of proteins and other bioactive molecules. Its main components are amino acids, and representative manufacturers include, but are not limited to, Duoruiste (Shenzhen) Medical Technology Co., Ltd.

[0020] The term "PS11" refers to a compound liquid protein protectant that significantly improves the storage and thermal stability of proteins and other bioactive molecules. Its main components are amino acids, and representative manufacturers include, but are not limited to, Duoruiste (Shenzhen) Medical Technology Co., Ltd.

[0021] The term "ADP" refers to a broad-spectrum complex enzyme protectant that also has unique properties that can improve enzyme activity and sterilization, thereby conserving the original enzyme and improving efficacy. Its English name is Enzyme protectant ADP. Representative manufacturers include, but are not limited to, Shanghai Shifeng Biotechnology Co., Ltd., Xibao Biotechnology (Shanghai) Co., Ltd., Shanghai Yuanye Biotechnology Co., Ltd., Shanghai Jinpan Biotechnology Co., Ltd., and Shanghai Huicheng Biotechnology Co., Ltd.

[0022] The term "HP" refers to a complex protein protectant suitable for liquid and lyophilized systems with a pH of 5-9. It is mainly used for the stability of thrombin, glycosidase, ribooxidase, esterase, hexokinase, thrombin, APTT reagent for hemostasis, serum proteins, etc. Its English name is Enzyme Protectant HP. Representative manufacturers include, but are not limited to, Xibao Biotechnology (Shanghai) Co., Ltd., Shanghai Shifeng Biotechnology Co., Ltd., Shanghai Yixin Biotechnology Co., Ltd., Shanghai Jinpan Biotechnology Co., Ltd., Shanghai Chenyi Biotechnology Co., Ltd., Shanghai Huicheng Biotechnology Co., Ltd., Beijing Qisong Biotechnology Co., Ltd., and Shanghai Lianmai Bioengineering Co., Ltd.

[0023] The term "HPD" refers to a broad-spectrum enzyme, protein, and antibody protectant that can be used in vaccines, diagnostic reagents, etc. It is mainly used for the stabilization of liquids, especially suitable for the stabilization of various oxidases (such as 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, purine enzyme, etc. Its English name is Protein protectant HPD. Representative manufacturers include but are not limited to Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai Jinpan Biotechnology Co., Ltd., Seebio Technology (Shanghai) Co., Ltd., Shanghai Huicheng Biotechnology Co., Ltd., Shanghai Yuanye Biotechnology Co., Ltd.

[0024] The term "EC01" refers to a compound liquid enzyme protectant that can significantly improve the storage stability and thermal stability of various enzyme molecules. Chinese name: Enzyme protectant EC01, English name: Enzyme protectant 01. Its main components are amino acids and polyhydroxy protectants. Representative manufacturers include but are not limited to Duoruisite (Shenzhen) Medical Technology Co., Ltd.

[0025] The term "EC02" refers to a compound liquid enzyme protectant that can significantly improve the storage stability and thermal stability of various enzyme molecules. Chinese name: Enzyme protectant EC02, English name: Enzyme protectant 02. Its main components are amino acids and polyhydroxy protectants. Representative manufacturers include but are not limited to Duoruisite (Shenzhen) Medical Technology Co., Ltd.

[0026] The term "EC03" refers to a compound liquid enzyme protectant that can significantly improve the storage stability and thermal stability of various enzyme molecules. Chinese name: Enzyme protectant EC03, English name: Enzyme protectant 03. Its main components are amino acids and polyhydroxy protectants. Representative manufacturers include but are not limited to Duoruisite (Shenzhen) Medical Technology Co., Ltd.

[0027] The term "EC04" refers to a compound liquid enzyme protectant that can significantly improve the storage stability and thermal stability of various enzyme molecules. Chinese name: Enzyme protectant EC04, English name: Enzyme protectant 04. Its main components are amino acids and polyhydroxy protectants. Representative manufacturers include but are not limited to Duoruisite (Shenzhen) Medical Technology Co., Ltd.

[0028] The term "EC05" refers to a compound liquid enzyme protectant that can significantly improve the storage stability and thermal stability of various enzyme molecules. Chinese name: Enzyme protectant EC05, English name: Enzyme protectant 05. The main components are amino acids and polyhydroxy protectants. Representative manufacturers include but are not limited to Duoruisite (Shenzhen) Medical Technology Co., Ltd.

[0029] The term "EC06" refers to a compound liquid enzyme protectant that can significantly improve the storage stability and thermal stability of various enzyme molecules. Chinese name: Enzyme protectant EC06, English name: Enzyme protectant 06. The main components are amino acids and polyhydroxy protectants. Representative manufacturers include but are not limited to Duoruisite (Shenzhen) Medical Technology Co., Ltd.

[0030] The term "EC07" refers to a compound liquid enzyme protectant that can significantly improve the storage stability and thermal stability of various enzyme molecules. Chinese name: Enzyme protectant EC07, English name: Enzyme protectant 07. The main components are amino acids and polyhydroxy protectants. Representative manufacturers include but are not limited to Duoruisite (Shenzhen) Medical Technology Co., Ltd.

[0031] The term "EC08" refers to a compound liquid enzyme protectant that can significantly improve the storage stability and thermal stability of various enzyme molecules. Chinese name: Enzyme protectant EC08, English name: Enzyme protectant 08. The main components are amino acids and polyhydroxy protectants. Representative manufacturers include but are not limited to Duoruisite (Shenzhen) Medical Technology Co., Ltd.

[0032] The term "EC09" refers to a compound liquid enzyme protectant that can significantly improve the storage stability and thermal stability of various enzyme molecules. Chinese name: Enzyme protectant EC09, English name: Enzyme protectant 09. The main components are amino acids and polyhydroxy protectants. Representative manufacturers include but are not limited to Duoruisite (Shenzhen) Medical Technology Co., Ltd.

[0033] The term "EC010" refers to a compound liquid protein protectant that can significantly improve the storage stability and thermal stability of various enzyme molecules. Chinese name: Enzyme protectant EC10, English name: Enzyme protectant 10. The main components are amino acids and polyhydroxy protectants. Representative manufacturers include but are not limited to Duoruisite (Shenzhen) Medical Technology Co., Ltd.

[0034] The term "EC011" refers to a compound liquid protein protectant that can significantly improve the storage stability and thermal stability of various enzyme molecules. Chinese name: Enzyme protectant EC11, English name: Enzyme protectant 11. Its main components are amino acids and polyhydroxy protectants. Representative manufacturers include but are not limited to Duoruisite (Shenzhen) Medical Technology Co., Ltd.

[0035] The term "AES" refers to a broad-spectrum enzyme, antigen, and antibody stabilizer that saves the original enzyme and improves efficiency; it is mainly used for the stabilization of liquid enzymes and working enzymes, especially applicable to the diluents containing enzymes and proteins in diagnostic reagents, various oxidases, glycerol phosphate oxidase, and uric acid oxidase. English name: Enzyme stabilizer AES. It must be present in more than 2% animal protein, 10 - 30% serum, 5 - 8% sugar, 4% NaCl, 0.1M PBS, other protective components such as protein protectant SHE-50, etc., preservatives (non-enzyme inhibitors), after aseptic configuration or sterile filtration, add 0.3 - 1% enzyme stabilizer. Representative manufacturers include but are not limited to Seebio Technology (Shanghai) Co., Ltd., Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai Jinpan Biotechnology Co., Ltd., Shanghai Huzheng Biotechnology Co., Ltd., Shanghai Huicheng Biotechnology Co., Ltd., Beijing Keyue Zhongkai Biotechnology Co., Ltd., Shanghai Xinyu Biotechnology Co., Ltd., Shanghai Hushi Pharmaceutical Technology Co., Ltd., Shanghai Chunyou Biotechnology Co., Ltd., Shanghai Guchen Biotechnology Co., Ltd.

[0036] The term "DPD" refers to a broad-spectrum enzyme and protein stabilizer, mainly used for the stabilization of liquid enzymes and working enzymes, applicable to enzyme diluents for various types of reactions and the stabilization of liquid enzymes. English name: Enzyme stabilizer DPD. Representative manufacturers include but are not limited to Seebio Technology (Shanghai) Co., Ltd., Shanghai Xuanya Biotechnology Co., Ltd., Shanghai Chunshi Biotechnology Co., Ltd., Shanghai Jinpan Biotechnology Co., Ltd., Shanghai Huicheng Biotechnology Co., Ltd., Shanghai Yansheng Industry Co., Ltd., Shanghai Yiyan Biotechnology Co., Ltd., Shanghai Yuanye Bio-Technology Co., Ltd.

[0037] The term "SD" refers to a protein stabilizer, ready-to-use liquid, which extends the shelf life of proteins in solution; improves protein activity; avoids protein inactivation caused by dilution; avoids protein hydrolysis; avoids protein aggregation; avoids protein denaturation. English name: Stability SD. Representative manufacturers include but are not limited to Seebio Technology (Shanghai) Co., Ltd., Shanghai Jinpan Biotechnology Co., Ltd.

[0038] The term "HRP" refers to a horseradish peroxidase (HRP) enzyme-labeled diluent / stabilizer that provides strong protection for HRP enzyme-labeled reagents, ensuring that diluted HRP enzyme-labeled reagents are stable at room temperature for more than 12 months at extremely low concentrations. Its main components are: Tris-based buffer, containing BSA, pH 7.2±0.2, and containing an inhibitor to effectively eliminate interference and cross-reactions. It is used to dilute HRP labels / conjugates to prepare enzyme-labeled diluents / working solutions for long-term stable storage.

[0039] The term "DCE0061A" refers to: primarily used for the stabilization and protection of antibody-labeled horseradish peroxidase, delaying enzyme activity decay and improving product stability. English name: Enzyme stabilizer DCE0061A. Main components: 2% or more animal protein, 10-30% serum, 5-8% sugar, 4% NaCl, 0.1M PBS, protein protectant SHE-50, preservative (non-enzyme inhibitor).

[0040] The term "SHE-50" refers to an enzyme stabilizer that prevents rapid precipitation and solution precipitation of enzyme-immunized substrates such as 3,3',5,5'-tetramethylbenzidine and other biochemical substrates. Its English name is Stability HE50. Its molecular formula is (CH2CHOHCH3). n Molecular weight: 65,000–80,000.

[0041] The term "DP4" refers to a broad-spectrum enzyme complex protein protectant. Its main active ingredient is 5-methylpyrazine-2-carbonitrile.

[0042] The term "DSP50" refers to a complex protein protectant that effectively protects enzyme activity and improves enzyme stability. Its main components include various proteins, amino acids, and sugars, which effectively prevent enzyme inactivation and denaturation, thereby enhancing enzyme stability and activity.

[0043] The term "DP7" refers to a broad-spectrum complex protein protectant. Its main chemical component is diethylamino hydroxybenzoyl hexyl benzoate.

[0044] The term "AFP" refers to a broad-spectrum complex enzyme protectant and diluent. Its main components are proteins, buffers, humectants, protectants, antioxidants, and dispersants. The English name is Enzyme Protectant AFP. It can be described as either a protectant or a stabilizer, reflecting its functional nature.

[0045] The term "MP" refers to a broad-spectrum enzyme and antigen / antibody protectant. It is widely used as a protectant for proteins, enzymes, and antibodies, as well as in liquids and lyophilized solutions. Its English name is Enzyme Protectant MP. Representative manufacturers include, but are not limited to, Xibao Biotechnology (Shanghai) Co., Ltd., Shanghai Jinpan Biotechnology Co., Ltd., and Shanghai Kehansheng Biotechnology Co., Ltd.

[0046] The term "Proclin 300" refers to ProClin, a liquid biological preservative used in IVD production. TM The preservative is a mixture of two isothiazolinones, namely 5-Chloro-2-methyl-4-isothiazolin-3-one (CMIT) and 2-Methyl-4-isothiazolin-3-one (MIT), in a ratio of 3:1, and the content of this mixture is 3.0-3.6%.

[0047] The term "Proclin 950" refers to ProClin 950 used in IVD production. TM The preservative, whose main active ingredient is a 9.5% solution of 2-methyl-4-isothiazolin-3-one.

[0048] The term "KroVin 300" refers to a broad-spectrum, low-toxicity, and environmentally friendly preservative. Its antibacterial active ingredient is CMIT / MIT (chloromethylisothiazolinone / methylisothiazolinone), and the product composition contains alkyl carboxylate and solvent (modified propylene glycol).

[0049] The term "KroVin 950" refers to a stable, low-toxicity, and environmentally friendly preservative whose active ingredient is 2-methyl-4-isothiazolin-3-one (MIT).

[0050] The term "Rozone S16" refers to a compound bactericidal, bacteriostatic, and preservative agent. Its Chinese name is "Broad-spectrum and highly effective preservative Rozone S16". Its main components are usually isothiazolinone compounds, one of which is 2-methyl-4-isothiazolinone (MI).

[0051] The term "prediabetes" refers to the transitional stage before the onset of diabetes, encompassing impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and a mixed state of both (IFG+IGT). It represents an intermediate hyperglycemic state between normal blood glucose and diabetes. The definition and diagnostic criteria for prediabetes are adopted from the World Health Organization (WHO) 1999 standard.

[0052] The term "high-risk group for diabetes" refers to individuals who possess one or more risk factors for diabetes. Due to certain characteristics associated with the onset of diabetes, their risk of developing diabetes is higher than that of non-high-risk groups, making them a key focus of diabetes prevention and control efforts. Criteria for high-risk groups for diabetes include: 1. Age 45 and above, body mass index (BMI) 24 and above; IGT (impaired glucose tolerance, postprandial blood glucose between 7.0-11.1 mmol / L) and IGF (impaired fasting glucose, fasting blood glucose between 5.7-7.0 mmol / L). 2. History of diabetes. 3. Dyslipidemia, with decreased high-density lipoprotein cholesterol (HDL) and increased triglycerides. 4. Hypertension (140 / 90 mmHg), cardiovascular disease, or cerebrovascular disease. 5. Pregnant women aged 30 or older; those with a history of gestational diabetes; those who have given birth to large babies (≥4kg); those with unexplained prolonged labor; women with polycystic ovary syndrome; 6. Those whose work involves a lack of exercise and prolonged sitting; 7. Those using certain medications, such as glucocorticoids or diuretics. If any one of these seven criteria is met, the individual is considered high-risk. Based on past experience, without intervention, 10% of these individuals develop diabetes annually. High-risk individuals can also be defined as those with "early-stage diabetes." The definition follows the "2022 Expert Consensus on Screening and Intervention for High-Risk Groups of Diabetes."

[0053] The term "undiagnosed diabetes population" refers to individuals who have not yet been diagnosed with diabetes by a clinician.

[0054] A first aspect of the present invention provides a salivary glucose test strip with extended shelf life at room temperature, the salivary glucose test strip comprising: glucose oxidase, horseradish peroxidase, vitamin C oxidase solution, chromogenic agent, enzyme / protein protectant, enzyme stabilizer, and sodium alginate solution; wherein,

[0055] The enzyme / protein protectant is selected from one or more of the following: complex protein protectant, protein protectant, complex enzyme protectant, and enzyme protectant;

[0056] The colorimetric agent is selected from one or more of the following: 2,4,6-tribromo-3-hydroxybenzoic acid, 4-aminoantipyrine, 2,4-dichlorophenol, 3,3',5,5'-tetramethylbenzidine, preferably 2,4,6-tribromo-3-hydroxybenzoic acid and / or 4-aminoantipyrine.

[0057] According to the first aspect of the present invention, a salivary glucose test strip is provided, wherein...

[0058] The composite protein protectant is selected from one or more of the following: HPD, DP4, DSP50, DP7, preferably selected from one or more of the following: HPD, DP4, DSP50, more preferably HPD or DP4;

[0059] The protein protectant is selected from one or more of the following: AEP-HBC, TY, GH, PS01, PS02, PS03, PS04, PS05, PS06, PS07, PS08, PS09, PS10, PS11, preferably selected from one or more of the following: AEP-HBC, TY, GH, PS01, PS02, PS03, PS05, PS06, PS08, PS10, PS11, more preferably selected from one or more of the following: AEP-HBC, TY, GH, PS01, PS10;

[0060] The complex enzyme protectant is selected from one or more of the following: ADP, HP, AFP, MP, preferably selected from one or more of the following: ADP, HP, MP, more preferably ADP or HP;

[0061] The enzyme protectant is preferably selected from one or more of the following: CE01, CE02, CE03, CE04, CE05, CE06, CE07, CE08, CE09, CE10, CE11; more preferably selected from one or more of the following: CE01, CE02, CE04, CE05, CE07, CE08, CE09, CE11; and more preferably selected from one or more of the following: CE01, CE04, CE05, CE08, CE09; and / or

[0062] The enzyme stabilizer is selected from one or more of the following: AES, DPD, SD, HRP, DCE0061A, SHE-50, preferably from one or more of the following: AES, SD, HRP, DCE0061A, SHE-50, and more preferably from one or more of the following: AES, SD, HRP, SHE-50.

[0063] According to a first aspect of the present invention, a salivary glucose test strip further includes: a protective film coated on the salivary glucose test strip; wherein,

[0064] The protective film is selected from one or more of the following materials: polyvinyl alcohol, dopamine, tannic acid, 3-aminopropyltriethoxysilane, hyperbranched polyethyleneimine, polyvinylpyrrolidone, polydimethylsiloxane, preferably polyvinyl alcohol or hyperbranched polyethyleneimine, most preferably polyvinyl alcohol; and / or

[0065] The salivary glucose test strip also includes: chitosan, acetic acid, non-enzyme activator, polyethylene glycol, citral, and bovine serum albumin;

[0066] Preferably, the non-enzyme activator is selected from one or more of the following: halogenated resorcinol, sodium hydroxybenzenesulfonate, palladium chloride, cobalt chloride, and more preferably halogenated resorcinol and / or sodium hydroxybenzenesulfonate;

[0067] Preferably, the halogenated resorcinol is selected from one or more of the following: 2,4-dichlorophenol, 2,4-dibromophenol, 2,4-difluorophenol, 2,4-diiodophenol, 4-chlororesorcinol, 4-bromoresorcinol, 5-substituted resorcinol, more preferably 4-chlororesorcinol or 4-bromoresorcinol, and most preferably 4-chlororesorcinol; and / or

[0068] Preferably, the sodium hydroxybenzenesulfonate is selected from one or more of the following: sodium 3,5-dichloro-2-hydroxybenzenesulfonate, sodium 4-hydroxybenzenesulfonate, sodium 3-amino-4-hydroxybenzenesulfonate, sodium p-hydroxybenzenesulfonate, sodium o-hydroxybenzenesulfonate, sodium methylhydroxybenzenesulfonate, sodium ethylhydroxybenzenesulfonate, more preferably sodium 3,5-dichloro-2-hydroxybenzenesulfonate or sodium 3-amino-4-hydroxybenzenesulfonate, and most preferably sodium 3,5-dichloro-2-hydroxybenzenesulfonate.

[0069] According to the first aspect of the present invention, the salivary glucose test strip for extending the storage time at room temperature further includes: a preservative, gelatin, and a desiccant;

[0070] The preservative is selected from one or more of the following: Proclin 300, Proclin 950, KroVin 300, KroVin 950, Rozone S16, carbamate, dimethyl sulfide, Na2EDTA+NaF, sodium thiosulfate, preferably selected from one or more of the following: Proclin 300, Proclin 950, KroVin 300, KroVin 950, Rozone S16, more preferably selected from one or more of the following: Proclin 300, Proclin 950, KroVin 300, KroVin 950; and / or

[0071] The desiccant is selected from one or more of the following: silica gel, activated carbon, molecular sieve, WiseFlex+ composite drying filter membrane, WiseSheet polymer drying sheet, preferably WiseFlex+ composite drying filter membrane and / or WiseSheet polymer drying sheet, more preferably WiseSheet polymer drying sheet.

[0072] A second aspect of the present invention provides a method for preparing the salivary glucose test strip described in the first aspect, the method comprising the following steps:

[0073] (1) Prepare an impregnation solution containing glucose oxidase, horseradish peroxidase, vitamin C oxidase solution, color reagent, enzyme / protein protectant and enzyme stabilizer and cool it for later use.

[0074] (2) Immerse the test paper in the impregnation solution prepared in step (1), dry it and coat it with a protective film.

[0075] According to a second aspect of the method of the present invention, wherein step (1), preparing the impregnation solution includes the following steps:

[0076] a) Pre-prepared mixed solution;

[0077] b) Add glucose oxidase, horseradish peroxidase, vitamin C oxidase solution, bovine serum albumin, polyethylene glycol, enzyme / protein protectant, enzyme stabilizer, preservative, and chromogenic agent to a petri dish, disperse by sonication, then add citral and the pre-prepared mixed solution from step a), allow to stand, and filter; and

[0078] c) Add the gelatin solution and sodium alginate solution cooled to room temperature, stir well, and the impregnation solution is obtained.

[0079] According to the method of the second aspect of the present invention, wherein,

[0080] In step a), the mixed solution includes: a mixed solution of acetic acid and chitosan and a non-enzyme activator solution; the mass ratio of chitosan to acetic acid is preferably 1:50-500, more preferably 1:100-300, and most preferably 1:160.

[0081] In steps b) and c), the vitamin C oxidase solution and the gelatin solution are prepared 24–36 hours in advance, preferably 24 hours in advance; and / or

[0082] In step c), the amount of sodium alginate solution used is 0-5 mL, preferably 0-3 mL, more preferably 0-2 mL, and most preferably 1 mL.

[0083] According to the method of the second aspect of the present invention, in step b):

[0084] The amount of glucose oxidase added is 5-50 mg, preferably 5-30 mg, more preferably 5-20 mg, and even more preferably 5-10 mg;

[0085] The amount of horseradish peroxidase added is 2.5-30 mg, preferably 2.5-20 mg, more preferably 2.5-10 mg, and even more preferably 5.0-10 mg;

[0086] The amount of vitamin C oxidase solution added is 5-50 μL, preferably 10-50 μL, more preferably 10-40 μL / mL, and even more preferably 10-30 μL;

[0087] The amount of bovine serum albumin added is 30-300 mg, preferably 50-250 mg, more preferably 50-200 mg, and even more preferably 50-100 mg;

[0088] The amount of polyethylene glycol added is 0.25-3 mL, preferably 0.25-2 mL, more preferably 0.25-1 mL, and even more preferably 0.5-1 mL;

[0089] The amount of the enzyme / protein protectant added is 0.1% to 5%, preferably 0.3% to 3%, more preferably 0.5% to 2%, and even more preferably 1% to 2%.

[0090] The amount of enzyme stabilizer added is 0.5% to 5%, preferably 0.5% to 3%, more preferably 0.8% to 2%, and even more preferably 1% to 1.5%.

[0091] The amount of the preservative added is 0.1% to 3%, preferably 0.3% to 2.5%, more preferably 0.5% to 2%, and even more preferably 1% to 1.5%.

[0092] The amount of 2,4,6-tribromo-3-hydroxybenzoic acid added in the colorimetric reagent is 10-60 mg, preferably 20-60 mg, more preferably 30-60 mg, further preferably 40-60 mg, and most preferably 50 mg;

[0093] The amount of 4-aminoantipyrine added in the colorimetric reagent is 100-400 mg, preferably 100-300 mg, more preferably 150-250 mg, further preferably 150-200 mg, and most preferably 150 mg;

[0094] The amount of citral added is 3-12 μL, preferably 5-10 μL, more preferably 5-8 μL, and even more preferably 5-7 μL;

[0095] The amount of the acetic acid and chitosan mixed solution added is 0.3-3 mL, preferably 0.3-2 mL, more preferably 0.3-1 mL, further preferably 0.5-0.8 mL, and most preferably 0.5 mL;

[0096] The amount of the non-enzyme activator solution added is 50-500 μL, preferably 50-400 μL, more preferably 50-300 μL, even more preferably 50-200 μL, and most preferably 100 μL.

[0097] The ultrasonic dispersion time is 2–10 min, preferably 3–6 min, more preferably 3–5 min; and / or

[0098] The settling time is 2 to 20 minutes, preferably 5 to 15 minutes, and more preferably 5 to 10 minutes.

[0099] According to a second aspect of the present invention, step (2) includes the following steps: placing the rough side of the filter paper down, immersing it in the impregnation solution in an oven and drying it, then immersing the rough side of the filter paper down in a protective film solution, and drying it to obtain the salivary glucose test strip; wherein,

[0100] The temperature of the oven is 20-50℃, preferably 20-40℃, and most preferably 30℃;

[0101] The soaking time in the impregnation solution is 20-36 hours, more preferably 24-36 hours, and most preferably 24 hours;

[0102] The immersion time in the protective film solution is 1.5 to 5 minutes, more preferably 1.5 to 3 minutes, and most preferably 2 minutes; and / or

[0103] The protective film solution must completely cover the test paper.

[0104] According to a second aspect of the present invention, the method further includes:

[0105] (3) Cut the test strip prepared in step (2), store it at room temperature and in a vacuum seal to obtain the salivary glucose test strip with extended room temperature storage time.

[0106] A third aspect of the present invention provides the use of the salivary glucose test strip described in the first aspect, which has an extended shelf life at room temperature, in the preparation of products for diagnosing blood glucose disorders or oral diseases; wherein,

[0107] The room temperature storage time is 0 to 12 months, preferably 1 to 10 months, more preferably 2 to 8 months, and even more preferably 6 months;

[0108] Preferably, the blood glucose disorder is selected from one or more of the following: diabetes mellitus, hypoglycemia, prediabetes, insulin resistance, obesity, insulinoma, thyroid dysfunction, Cushing's syndrome; more preferably, it is selected from one or more of the following: diabetes mellitus, hypoglycemia, prediabetes; and most preferably, diabetes mellitus; and / or

[0109] Preferably, the oral disease is selected from one or more of the following: periodontitis, dental caries, gingivitis, dental plaque, thrush; more preferably, it is selected from one or more of the following: periodontitis, dental caries, gingivitis, dental plaque; and even more preferably, dental caries or dental plaque.

[0110] A fourth aspect of the present invention provides a method for diagnosing blood glucose disorders, the method comprising using a saliva glucose test strip with an extended room temperature storage time as described in the first aspect for diagnosis.

[0111] A fifth aspect of the present invention provides a method for diagnosing oral diseases, the method comprising using a saliva glucose test strip with an extended room temperature storage time as described in the first aspect for diagnosis.

[0112] A sixth aspect of the present invention provides a method for detecting salivary glucose, the method comprising testing a saliva sample or a subject in need using the salivary glucose test strip described in the first aspect.

[0113] The seventh aspect of the present invention provides a salivary glucose detection product or device, the detection product or device comprising the salivary glucose test strip with extended room temperature storage time as described in the first aspect;

[0114] Preferably, the detection product is a detection kit.

[0115] According to a specific embodiment of the present invention, the working principle of the present invention is as follows: under the catalysis of glucose oxidase, glucose in the sample reacts with oxygen to generate hydrogen peroxide, and then under the action of peroxidase, hydrogen peroxide reacts with a colorimetric agent to generate a visible red imine substance.

[0116] The test strip is prepared as follows:

[0117] (1) Preparation of solutions 1-5

[0118] Solution 1: 4.1 mg Vitamin C oxidase + 1 mL distilled water.

[0119] Solution No. 2: 1.5 mL acetic acid + 10 mg chitosan + 50 mL distilled water.

[0120] Solution No. 3: 10 mg 4-chlororesorcinol + 10 mg sodium 3,5-dichloro-2-hydroxybenzenesulfonate + 10 mL distilled water.

[0121] Solution #4: 1.5g gelatin + 10mL distilled water.

[0122] Solution No. 5: 1g sodium alginate + 60-80mL water.

[0123] Solution No. 6: Dissolve 500mg polyvinyl alcohol by heating at 150℃ on a hot plate.

[0124] Of these, numbers 2, 3, and 5 are prepared and used immediately; numbers 1 and 4 are prepared 24 hours in advance.

[0125] Before using No. 4 and No. 5, heat them on a hot table at 150°C to dissolve them, and then add them to the impregnation solution after they have cooled to room temperature.

[0126] Before using No. 6, the solution needs to be cooled to room temperature before immersing it in the filter paper.

[0127] (2) Preparation of impregnation solution

[0128] Pipette 5 mL of enzyme reagent into a petri dish (60 mm in diameter, 30 mm in height). Add 10 mg glucose oxidase, 7.5 mg horseradish peroxidase, 20 μL of solution 1, 50 mg bovine serum albumin, 500 μL polyethylene glycol-200, 120 μL of HPD complex protein protectant, 100 mg of AES complex enzyme stabilizer, 7 μL of KroVin300 preservative, 50 mg of 2,4,6-tribromo-3-hydroxybenzoic acid, and 150 mg of 4-aminoantipyrine. Mix well and sonicate for 3-5 min. Then add 5 μL of citral, 500 μL of solution 2, and 100 μL of solution 3. Let stand for 5-10 min and filter with a clean cloth. Add 1 mL of gelatin solution (cooled to room temperature) and 1 mL of sodium alginate solution (cooled to room temperature) to the filtrate and stir well.

[0129] (3) Soaking and drying

[0130] Place the filter paper with the rough side down in an oven at 30°C and soak it (during operation, every 2 hours, remove the filter paper, stir the soaking solution with a glass rod until it is evenly mixed, and then immerse the filter paper again). After 24 hours, remove the filter paper, place it with the rough side down on a clean cloth, and dry it at 30°C for about 30-50 minutes.

[0131] Note: Only one filter paper can be immersed in one petri dish.

[0132] (4) Applying a protective film

[0133] Immerse the test paper in (3) with the rough side down in solution No. 6 for 2 minutes, then take it out and place it on a clean cloth with the rough side down to dry at 30°C for about 30-50 minutes.

[0134] (5) Test strip preservation

[0135] Cut the test strips into 1*1cm squares, vacuum seal them, protect them from light, and store them at room temperature.

[0136] The salivary glucose test strip of the present invention, which extends the shelf life at room temperature, may have, but is not limited to, the following beneficial effects:

[0137] 1. By adding sodium alginate, a protective film, an enzyme / protein protectant, an enzyme stabilizer, and a preservative, the shelf life of the salivary glucose test strip at room temperature has been extended. At the same time, adding a desiccant can extend the shelf life of the test strip at room temperature by more than 6 months, and the color development effect of the test strip after 6 months is comparable to that when it was first synthesized.

[0138] 2. The salivary glucose test strip of the present invention, which extends the storage time at room temperature, significantly increases the rate of enzymatic reaction by using chitosan and acetic acid, enabling complete color development in 30 seconds, while maintaining the advantages of high specificity and mild conditions of enzymatic glucose detection.

[0139] 3. The test strip synthesized in this invention, which can shorten the detection time of saliva glucose, has important clinical significance for the early prevention, preliminary diagnosis, and treatment of diabetes and oral diseases.

[0140] Brief description of the attached figures

[0141] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0142] Figure 1 shows the color change of saliva glucose test strips with glucose concentration when sodium alginate, protective film, enzyme / protein protectant, enzyme stabilizer, and preservative are added to extend the storage time at room temperature.

[0143] Figure 2 shows a comparison of the preservation effects of the salivary glucose test strip of the present invention with extended room temperature storage time and the test strip of the prior art.

[0144] The best way to implement an invention

[0145] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for more detailed and specific illustration and should not be construed as limiting the present invention in any way.

[0146] This section provides a general description of the materials and testing methods used in the experiments of this invention. While many of the materials and methods of operation used to achieve the objectives of this invention are well known in the art, the invention is still described in as much detail as possible herein. It will be apparent to those skilled in the art that, unless otherwise stated in the context, the materials and methods of operation used in this invention are well known in the art.

[0147] The following examples will help to further understand the present invention, but do not limit the scope of the invention.

[0148] The reagents and instruments used in the following examples are as follows:

[0149] Material:

[0150] Whatman No. 3 filter paper, purchased from Whatman.

[0151] Reagents:

[0152] Enzyme reagent (blood glucose assay kit, application type, serial number 1003) was purchased from Baoding Great Wall Clinical Reagent Co., Ltd.; glucose oxidase, horseradish peroxidase, polyethylene glycol-200, 4-aminoantipyrine, 2,4,6-tribromo-3-hydroxybenzoic acid, and citral were purchased from Macklin Biotechnology Co., Ltd.; bovine serum albumin was purchased from Beijing Keerhui Technology Co., Ltd.; gelatin was purchased from Sigma-Aldrich; vitamin C oxidase was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; polyvinyl alcohol was purchased from Shanghai Mairui Chemical Technology Co., Ltd.; chitosan (95%) was purchased from Shandong Aokang Biotechnology Co., Ltd. Acetic acid, purchased from Macklin; 4-chlororesorcinol, purchased from Macklin; sodium 3,5-dichloro-2-hydroxybenzenesulfonate, purchased from Macklin; sodium alginate, purchased from Macklin; enzyme protectant EC01, purchased from Dorest (Shenzhen) Medical Technology Co., Ltd.; enzyme stabilizer HRP, complex protein protectant HPD, complex enzyme stabilizer AES, complex protein protectant DP4, preservatives KroVin300 and KroVin950 were all purchased from Xibao Biotechnology (Shanghai) Co., Ltd.

[0153] Example 1

[0154] This embodiment illustrates the salivary glucose test strip of the present invention with extended room temperature storage time and its preparation method.

[0155] (1) Prepare the impregnation solution

[0156] (a) Preparation of solutions 1-6

[0157] Solution 1: 4.1 mg Vitamin C oxidase + 1 mL distilled water.

[0158] Solution No. 2: 1.5 mL acetic acid + 10 mg chitosan + 50 mL distilled water.

[0159] Solution No. 3: 10 mg 4-chlororesorcinol + 10 mg sodium 3,5-dichloro-2-hydroxybenzenesulfonate + 10 mL distilled water.

[0160] Solution #4: 1.5g gelatin + 10mL distilled water.

[0161] Solution No. 5 --- 1g sodium alginate + 80mL water.

[0162] Solution No. 6: Dissolve 500mg polyvinyl alcohol by heating at 150℃ on a hot plate.

[0163] Of these, numbers 2, 3, and 5 are prepared and used immediately; numbers 1 and 4 are prepared 24 hours in advance.

[0164] Before using No. 4 and No. 5, heat them on a hot table at 150°C to dissolve them, and then add them to the impregnation solution after they have cooled to room temperature.

[0165] Before using No. 6, the solution needs to be cooled to room temperature before immersing it in the filter paper.

[0166] (b) Preparation of impregnation solution

[0167] Pipette 5 mL of enzyme reagent into a culture dish (60 mm in diameter, 30 mm in height). Add 10 mg glucose oxidase, 7.5 mg horseradish peroxidase, 20 μL of solution 1, 50 mg bovine serum albumin, 500 μL polyethylene glycol-200, 60 μL HRP enzyme stabilizer, 60 μL enzyme protectant EC01, 2.5 μL KroVin300 preservative, 50 mg 2,4,6-tribromo-3-hydroxybenzoic acid, and 150 mg 4-aminoantipyrine. Mix well and sonicate for 5 min. Then add 5 μL citral, 500 μL solution 2, and 100 μL solution 3. Let stand for 5 min and filter with a clean cloth. Add 1 mL gelatin solution (cooled to room temperature) and 1 mL sodium alginate solution (cooled to room temperature) to the filtrate and stir well.

[0168] (2) Soaking and drying

[0169] Place the filter paper with the rough side down in an oven at 30°C and soak it (during operation, every 2 hours, remove the filter paper, stir the soaking solution with a glass rod until it is evenly mixed, and then immerse the filter paper again). After 24 hours, remove the filter paper, place it with the rough side down on a clean cloth, and dry it at 30°C for about 30-50 minutes.

[0170] Note: Only one filter paper can be immersed in one petri dish.

[0171] (3) Applying a protective film

[0172] Immerse the test paper in (2) completely in solution No. 6 with the rough side facing down for 2 minutes, then take it out and place it on a clean cloth with the rough side facing down to dry at 30°C for about 30-50 minutes.

[0173] (4) Test strip preservation

[0174] Cut the test strips into 1*1cm squares, vacuum seal them, protect them from light, and store them at room temperature.

[0175] Example 2

[0176] This embodiment illustrates the salivary glucose test strip of the present invention with extended room temperature storage time and its preparation method.

[0177] (1) Prepare the impregnation solution

[0178] (a) Preparation of solutions 1-6

[0179] Solution No. 1: 3 mg Vitamin C oxidase + 1 mL distilled water.

[0180] Solution No. 2: 2 mL hydrochloric acid + 20 mg chitosan + 50 mL distilled water.

[0181] Solution No. 3: 10 mg 4-chlororesorcinol + 10 mg sodium 3,5-dichloro-2-hydroxybenzenesulfonate + 10 mL distilled water.

[0182] Solution #4: 2g gelatin + 10mL distilled water.

[0183] Solution No. 5 --- 1g sodium alginate + 60mL water.

[0184] Solution No. 6: Dissolve 500mg polyvinylpyrrolidone in 50mL distilled water by heating at 150℃ on a hot plate.

[0185] Of these, numbers 2, 3, and 5 are prepared and used immediately; numbers 1 and 4 are prepared 24 hours in advance.

[0186] Before using No. 4 and No. 5, heat them on a hot table at 150°C to dissolve them, and then add them to the impregnation solution after they have cooled to room temperature.

[0187] Before using No. 6, the solution needs to be cooled to room temperature before immersing it in the filter paper.

[0188] (b) Preparation of impregnation solution

[0189] Pipette 5 mL of enzyme reagent into a culture dish (60 mm in diameter, 30 mm in height). Add 8 mg of glucose oxidase, 7 mg of horseradish peroxidase, 25 μL of solution 1, 70 mg of bovine serum albumin, 600 μL of polyethylene glycol-200, 120 μL of HPD complex protein protectant, 100 mg of AES complex enzyme stabilizer, 7 μL of KroVin300 preservative, 50 mg of 2,4,6-tribromo-3-hydroxybenzoic acid, and 150 mg of 4-aminoantipyrine. Mix well and sonicate for 3 min. Then add 6 μL of citral, 500 μL of solution 2, and 100 μL of solution 3. Let stand for 8 min and filter with a clean cloth. Add 1 mL of gelatin solution (cooled to room temperature) and 1 mL of sodium alginate solution (cooled to room temperature) to the filtrate and stir well.

[0190] (2) Soaking and drying

[0191] Place the filter paper with the rough side down in an oven at 30°C and soak it (during operation, every 2 hours, remove the filter paper, stir the soaking solution with a glass rod until it is evenly mixed, and then immerse the filter paper again). After 24 hours, remove the filter paper, place it with the rough side down on a clean cloth, and dry it at 30°C for about 30-50 minutes.

[0192] Note: Only one filter paper can be immersed in one petri dish.

[0193] (3) Applying a protective film

[0194] Immerse the test paper in (2) with the rough side facing down completely in solution No. 6 for 5 minutes, then remove it and place it on a clean cloth with the rough side facing down to dry at 30°C for about 30-50 minutes.

[0195] (4) Test strip preservation

[0196] Cut the test strips into 1*1cm squares, vacuum seal them, protect them from light, and store them at room temperature.

[0197] Example 3

[0198] This embodiment illustrates the salivary glucose test strip of the present invention with extended room temperature storage time and its preparation method.

[0199] (1) Prepare the impregnation solution

[0200] (a) Preparation of solutions 1-6

[0201] Solution 1: 4.1 mg Vitamin C oxidase + 1 mL distilled water.

[0202] Solution No. 2: 1.5 mL acetic acid + 10 mg chitosan + 50 mL distilled water.

[0203] Solution No. 3: 10 mg 4-chlororesorcinol + 10 mg sodium 3,5-dichloro-2-hydroxybenzenesulfonate + 10 mL distilled water.

[0204] Solution #4: 1.5g gelatin + 10mL distilled water.

[0205] Solution No. 5 --- 1g sodium alginate + 70mL water.

[0206] Solution No. 6: Dissolve 500mg polyvinyl alcohol by heating at 150℃ on a hot plate.

[0207] Of these, numbers 2, 3, and 5 are prepared and used immediately; numbers 1 and 4 are prepared 24 hours in advance.

[0208] Before using No. 4 and No. 5, heat them on a hot table at 150°C to dissolve them, and then add them to the impregnation solution after they have cooled to room temperature.

[0209] Before using No. 6, the solution needs to be cooled to room temperature before immersing it in the filter paper.

[0210] (b) Preparation of impregnation solution

[0211] Pipette 5 mL of enzyme reagent into a culture dish (60 mm in diameter, 30 mm in height). Add 6 mg of glucose oxidase, 10 mg of horseradish peroxidase, 10 μL of solution 1, 95 mg of bovine serum albumin, 800 μL of polyethylene glycol-200, 60 μL of DP4 protein protectant, 60 μL of HRP enzyme stabilizer, 7 μL of KroVin950 preservative, 50 mg of 2,4,6-tribromo-3-hydroxybenzoic acid, and 150 mg of 4-aminoantipyrine. Mix well and sonicate for 4 min. Then add 7 μL of citral, 500 μL of solution 2, and 100 μL of solution 3. Let stand for 10 min and filter with a clean cloth. Add 1 mL of gelatin solution (cooled to room temperature) and 1 mL of sodium alginate solution (cooled to room temperature) to the filtrate and stir well.

[0212] (2) Soaking and drying

[0213] Place the filter paper with the rough side down in an oven at 30°C and soak it (during operation, every 2 hours, remove the filter paper, stir the soaking solution with a glass rod until it is evenly mixed, and then immerse the filter paper again). After 24 hours, remove the filter paper, place it with the rough side down on a clean cloth, and dry it at 30°C for about 30-50 minutes.

[0214] Note: Only one filter paper can be immersed in one petri dish.

[0215] (3) Applying a protective film

[0216] Immerse the test paper in (2) completely in solution No. 6 with the rough side facing down for 2 minutes, then take it out and place it on a clean cloth with the rough side facing down to dry at 30°C for about 30-50 minutes.

[0217] (4) Test strip preservation

[0218] Cut the test strips into 1*1cm squares, vacuum seal them, protect them from light, and store them at room temperature.

[0219] Experimental Example 1

[0220] This experimental example illustrates the effect of the present invention on the salivary glucose test strip, which extends the storage time at room temperature.

[0221] This embodiment compares the salivary glucose test strip prepared in Example 1 with one prepared in Example 1 with a test strip in which sodium alginate, enzyme / protein protectant, enzyme stabilizer, and preservative were not added to the impregnation solution.

[0222] The only difference between the two is in the preparation of the impregnation solution. The impregnation solution of the saliva glucose test strip for extended room temperature storage in Example 1 contains sodium alginate, enzyme / protein protectant, enzyme stabilizer, and preservative, while the impregnation solution of the other test strip does not contain sodium alginate, enzyme / protein protectant, enzyme stabilizer, and preservative.

[0223] Figure 1 shows the color change of the saliva glucose test strip with glucose concentration after adding sodium alginate, enzyme / protein protectant, enzyme stabilizer, and preservative to extend its shelf life at room temperature. As shown in Figure 1, with the extension of storage time (1 month, 2 months, 3 months, 4 months, 5 months, 6 months), the sensitivity of the test strip to different glucose concentrations did not change significantly. This indicates that the saliva glucose test strip of the present invention, which extends the shelf life at room temperature, extends the shelf life of the saliva glucose test strip to more than 6 months at room temperature by adding sodium alginate, protective film, enzyme / protein protectant, enzyme stabilizer, preservative, and desiccant. Moreover, at 6 months, the color development effect of the test strip is comparable to that when it was first synthesized.

[0224] Experimental Example 2

[0225] This test example is used to compare the salivary glucose test strip of the present invention with the extended room temperature storage time with the test strip of the prior art.

[0226] This experiment compares the salivary glucose test strips prepared in Examples 1-3 with those in the prior art (see Comparative Examples 1-4).

[0227] As shown in Table 1 and Figure 2, the room temperature storage time of Examples 1-3 was significantly longer than that of Comparative Examples 1-4, indicating that enzyme / protein protectants, enzyme stabilizers, preservatives, and sodium alginate can extend the room temperature storage time of the test strips. Furthermore, the color development effect of the test strips after 6 months was comparable to that immediately after synthesis.

[0228] Table 1. Comparison of the salivary glucose test strip of the present invention with the existing test strips for extended room temperature storage.

[0229] Although the invention has been described to a certain extent, it is apparent that appropriate variations can be made to the various conditions without departing from the spirit and scope of the invention. It is understood that the invention is not limited to the described embodiments, but falls within the scope of the claims, which include equivalent substitutions for each of the elements.

Claims

1. A salivary glucose test strip with extended shelf life at room temperature, characterized in that, The salivary glucose test strip comprises: glucose oxidase, horseradish peroxidase, vitamin C oxidase solution, chromogenic agent, enzyme / protein protectant, enzyme stabilizer, and sodium alginate solution; wherein... The enzyme / protein protectant is selected from one or more of the following: complex protein protectant, protein protectant, complex enzyme protectant, and enzyme protectant; The colorimetric agent is selected from one or more of the following: 2,4,6-tribromo-3-hydroxybenzoic acid, 4-aminoantipyrine, 2,4-dichlorophenol, 3,3',5,5'-tetramethylbenzidine, preferably 2,4,6-tribromo-3-hydroxybenzoic acid and / or 4-aminoantipyrine.

2. The salivary glucose test strip according to claim 1, characterized in that: The composite protein protectant is selected from one or more of the following: HPD, DP4, DSP50, DP7, preferably selected from one or more of the following: HPD, DP4, DSP50, more preferably HPD or DP4; The protein protectant is selected from one or more of the following: AEP-HBC, TY, GH, PS01, PS02, PS03, PS04, PS05, PS06, PS07, PS08, PS09, PS10, PS11, preferably selected from one or more of the following: AEP-HBC, TY, GH, PS01, PS02, PS03, PS05, PS06, PS08, PS10, PS11, more preferably selected from one or more of the following: AEP-HBC, TY, GH, PS01, PS10; The complex enzyme protectant is selected from one or more of the following: ADP, HP, AFP, MP, preferably selected from one or more of the following: ADP, HP, MP, more preferably ADP or HP; The enzyme protectant is preferably selected from one or more of the following: CE01, CE02, CE03, CE04, CE05, CE06, CE07, CE08, CE09, CE10, CE11; more preferably selected from one or more of the following: CE01, CE02, CE04, CE05, CE07, CE08, CE09, CE11; and more preferably selected from one or more of the following: CE01, CE04, CE05, CE08, CE09; and / or The enzyme stabilizer is selected from one or more of the following: AES, DPD, SD, HRP, DCE0061A, SHE-50, preferably from one or more of the following: AES, SD, HRP, DCE0061A, SHE-50, and more preferably from one or more of the following: AES, SD, HRP, SHE-50.

3. The salivary glucose test strip according to claim 1 or 2, characterized in that, The salivary glucose test strip further includes: a protective film coated on the salivary glucose test strip; wherein... The protective film is selected from one or more of the following materials: polyvinyl alcohol, dopamine, tannic acid, 3-aminopropyltriethoxysilane, hyperbranched polyethyleneimine, polyvinylpyrrolidone, polydimethylsiloxane, preferably polyvinyl alcohol or hyperbranched polyethyleneimine, most preferably polyvinyl alcohol; and / or The salivary glucose test strip also includes: chitosan, acetic acid, a non-enzyme activator, polyethylene glycol, and citral. Wagyu bovine serum albumin; Preferably, the non-enzyme activator is selected from one or more of the following: halogenated resorcinol, sodium hydroxybenzenesulfonate, palladium chloride, cobalt chloride, and more preferably halogenated resorcinol and / or sodium hydroxybenzenesulfonate; Preferably, the halogenated resorcinol is selected from one or more of the following: 2,4-dichlorophenol, 2,4-dibromophenol, 2,4-difluorophenol, 2,4-diiodophenol, 4-chlororesorcinol, 4-bromoresorcinol, 5-substituted resorcinol, more preferably 4-chlororesorcinol or 4-bromoresorcinol, and most preferably 4-chlororesorcinol; and / or Preferably, the sodium hydroxybenzenesulfonate is selected from one or more of the following: sodium 3,5-dichloro-2-hydroxybenzenesulfonate, sodium 4-hydroxybenzenesulfonate, sodium 3-amino-4-hydroxybenzenesulfonate, sodium p-hydroxybenzenesulfonate, sodium o-hydroxybenzenesulfonate, sodium methylhydroxybenzenesulfonate, sodium ethylhydroxybenzenesulfonate, more preferably sodium 3,5-dichloro-2-hydroxybenzenesulfonate or sodium 3-amino-4-hydroxybenzenesulfonate, and most preferably sodium 3,5-dichloro-2-hydroxybenzenesulfonate.

4. The salivary glucose test strip according to any one of claims 1 to 3, characterized in that, The salivary glucose test strip with extended room temperature storage time also includes: preservatives, gelatin, and desiccant; The preservative is selected from one or more of the following: Proclin 300, Proclin 950, KroVin 300, KroVin 950, Rozone S16, carbamate, dimethyl sulfide, Na2EDTA+NaF, sodium thiosulfate, preferably selected from one or more of the following: Proclin 300, Proclin 950, KroVin 300, KroVin 950, Rozone S16, more preferably selected from one or more of the following: Proclin 300, Proclin 950, KroVin 300, KroVin 950; and / or The desiccant is selected from one or more of the following: silica gel, activated carbon, molecular sieve, WiseFlex+ composite drying filter membrane, WiseSheet polymer drying sheet, preferably WiseFlex+ composite drying filter membrane and / or WiseSheet polymer drying sheet, more preferably WiseSheet polymer drying sheet.

5. A method for preparing a salivary glucose test strip according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: (1) Prepare an impregnation solution containing glucose oxidase, horseradish peroxidase, vitamin C oxidase solution, color reagent, enzyme / protein protectant and enzyme stabilizer and cool it for later use. (2) Immerse the test paper in the impregnation solution prepared in step (1), dry it and coat it with a protective film.

6. The method according to claim 5, characterized in that, In step (1), preparing the impregnation solution includes the following steps: a) Pre-prepared mixed solution; b) Add glucose oxidase, horseradish peroxidase, vitamin C oxidase solution, bovine serum albumin, polyethylene glycol, enzyme / protein protectant, enzyme stabilizer, preservative, and chromogenic agent to a petri dish, disperse by sonication, then add citral and the pre-prepared mixed solution from step a), allow to stand, and filter; and c) Add the gelatin solution and sodium alginate solution cooled to room temperature, stir well, and the impregnation solution is obtained.

7. The method according to claim 6, characterized in that: In step a), the mixed solution includes: a mixed solution of acetic acid and chitosan and a non-enzyme activator solution; the mass ratio of chitosan to acetic acid is preferably 1:50-500, more preferably 1:100-300, and most preferably 1:

160. In steps b) and c), the vitamin C oxidase solution and the gelatin solution are prepared 24–36 hours in advance, preferably 24 hours in advance; and / or In step c), the amount of sodium alginate solution used is 0-5 mL, preferably 0-3 mL, more preferably 0-2 mL, and most preferably 1 mL.

8. The method according to claim 6 or 7, characterized in that, In step b): The amount of glucose oxidase added is 5-50 mg, preferably 5-30 mg, more preferably 5-20 mg, and even more preferably 5-10 mg; The amount of horseradish peroxidase added is 2.5-30 mg, preferably 2.5-20 mg, more preferably 2.5-10 mg, and even more preferably 5.0-10 mg; The amount of vitamin C oxidase solution added is 5-50 μL, preferably 10-50 μL, more preferably 10-40 μL, and even more preferably 10-30 μL. The amount of bovine serum albumin added is 30-300 mg, preferably 50-250 mg, more preferably 50-200 mg, and even more preferably 50-100 mg; The amount of polyethylene glycol added is 0.25-3 mL, preferably 0.25-2 mL, more preferably 0.25-1 mL, and even more preferably 0.5-1 mL; The amount of the enzyme / protein protectant added is 0.1% to 5%, preferably 0.3% to 3%, more preferably 0.5% to 2%, and even more preferably 1% to 2%. The amount of enzyme stabilizer added is 0.5% to 5%, preferably 0.5% to 3%, more preferably 0.8% to 2%, and even more preferably 1% to 1.5%. The amount of the preservative added is 0.1% to 3%, preferably 0.3% to 2.5%, more preferably 0.5% to 2%, and even more preferably 1% to 1.5%. The amount of 2,4,6-tribromo-3-hydroxybenzoic acid added in the colorimetric reagent is 10-60 mg, preferably 20-60 mg, more preferably 30-60 mg, further preferably 40-60 mg, and most preferably 50 mg; The amount of 4-aminoantipyrine added in the colorimetric reagent is 100-400 mg, preferably 100-300 mg, more preferably 150-250 mg, further preferably 150-200 mg, and most preferably 150 mg; The amount of citral added is 3-12 μL, preferably 5-10 μL, more preferably 5-8 μL, and even more preferably 5-7 μL; The amount of the acetic acid and chitosan mixed solution added is 0.3-3 mL, preferably 0.3-2 mL, more preferably 0.3-1 mL, further preferably 0.5-0.8 mL, and most preferably 0.5 mL; The amount of the non-enzyme activator solution added is 50–500 μL, preferably 50–400 μL, and more preferably 50–300 μL. Further preferred is 50–200 μL, most preferably 100 μL; The ultrasonic dispersion time is 2–10 min, preferably 3–6 min, more preferably 3–5 min; and / or The settling time is 2 to 20 minutes, preferably 5 to 15 minutes, and more preferably 5 to 10 minutes.

9. The method according to any one of claims 5 to 8, characterized in that, Step (2) includes the following steps: placing the rough side of the filter paper down, immersing it in the impregnation solution in an oven and drying it, then immersing the rough side of the filter paper down in a protective film solution, and drying it to obtain the saliva glucose test strip; wherein, The temperature of the oven is 20-50℃, preferably 20-40℃, and most preferably 30℃; The soaking time in the impregnation solution is 20-36 hours, more preferably 24-36 hours, and most preferably 24 hours; The immersion time in the protective film solution is 1.5 to 5 minutes, more preferably 1.5 to 3 minutes, and most preferably 2 minutes; and / or The protective film solution must completely cover the test paper.

10. The method according to any one of claims 5 to 9, characterized in that, The method further includes: (3) Cut the test strip prepared in step (2), store it at room temperature and in a vacuum seal to obtain the salivary glucose test strip with extended room temperature storage time.

11. The use of the salivary glucose test strip with extended room temperature storage time according to any one of claims 1 to 4 in the preparation of products for diagnosing blood glucose disorders or oral diseases; wherein, The room temperature storage time is 0 to 12 months, preferably 1 to 10 months, more preferably 2 to 8 months, and even more preferably 6 months; Preferably, the blood glucose disorder is selected from one or more of the following: diabetes mellitus, hypoglycemia, prediabetes, insulin resistance, obesity, insulinoma, thyroid dysfunction, Cushing's syndrome; more preferably, it is selected from one or more of the following: diabetes mellitus, hypoglycemia, prediabetes; and most preferably, diabetes mellitus; and / or Preferably, the oral disease is selected from one or more of the following: periodontitis, dental caries, gingivitis, dental plaque, thrush; more preferably, it is selected from one or more of the following: periodontitis, dental caries, gingivitis, dental plaque; and even more preferably, dental caries or dental plaque.

12. A method for diagnosing blood glucose disorders, characterized in that, The method includes diagnosis using a salivary glucose test strip with an extended room temperature storage time as described in any one of claims 1 to 4.

13. A method for diagnosing oral diseases, characterized in that, The method includes diagnosis using a salivary glucose test strip with an extended room temperature storage time as described in any one of claims 1 to 4.

14. A method for detecting salivary glucose, characterized in that, The method includes testing a saliva sample or a subject in need using a saliva glucose test strip as described in any one of claims 1 to 4.

15. A product or device for detecting salivary glucose, characterized in that, The detection product or device comprises the salivary glucose test strip with extended room temperature storage time as described in any one of claims 1 to 4; Preferably, the detection product is a detection kit.

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