A rapid detection gel for detecting pepsin in saliva and a preparation method thereof

Abstract

The application discloses a rapid detection gel for detecting pepsin in saliva and a preparation method of the rapid detection gel. The structure of the rapid detection gel comprises casein gel, carbon ink blue particles which are uniformly distributed in the casein gel, are insoluble in water and have a particle size greater than a pore size of the gel. The detection is "enzyme activity" instead of "protein presence". False positive caused by inactivated pepsin is avoided, and the rapid detection gel is more in line with clinical physiological significance. The material is cheap, does not need antibody and has simple process. The preparation can be completed by using casein and dye particles, and the rapid detection gel is suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of detection reagent technology, and in particular to a rapid detection gel for salivary pepsin detection and its preparation method. Background Technology

[0002] Pepsin is an aspartic protease that primarily functions in the stomach, exhibiting strong proteolytic activity and an optimal pH of 1.5–2.0. During physiological or pathological reflux of gastric contents (such as GERD and LPR), pepsin is often carried to the esophagus and even the pharynx, thus becoming a characteristic biomarker detectable in saliva. In recent years, numerous clinical studies have validated that salivary pepsin can be used for the rapid auxiliary diagnosis of gastroesophageal reflux disease.

[0003] In existing IVD technology systems, the detection of Pepsin in saliva mainly relies on the following methods: (1) Immunochromatography (e.g., Peptest): relies on antibody recognition of the pepsin molecule itself; (2) ELISA: Specific antibody capture detection; (3) Protein spectrum detection: Used for scientific research, not suitable for on-site detection; (4) pH measurement: non-specific and cannot directly prove Pepsin activity.

[0004] However, these methods have obvious limitations: 1) Antibody methods detect the "presence" of protein, not its "activity," and therefore cannot distinguish between active and inactive pepsin. Pepsin is rapidly inactivated in a neutral environment, but its protein structure can still be recognized by antibodies. Therefore: ① Positive result ≠ active; ② Cannot be used for functional diagnosis; ③ False positives are frequent in clinical practice. (2) Antibodies are expensive and require strict storage conditions, while test strips have strict requirements for production and transportation. ① Antibodies are sensitive to temperature and humidity; ② There are large batch-to-batch variations; ③ Production costs are high and shelf life is limited; ④ This makes IVDR registration and mass production difficult.

[0005] (3) Currently, there is a lack of enzyme activity detection systems that can provide rapid on-site readings. Traditional enzyme activity assay systems (such as those based on quantitative fluorescent substrates) require: ① Precise pH control; ② Optical instruments; ③ Temperature control conditions.

[0006] This results in the vast majority of existing products being unsuitable for home or point-of-care testing (POCT).

[0007] (4) Existing chromogenic substrates (such as BSA-FITC and proteolytic fluorescent substrates) are expensive and difficult to solidify onto visualization gel platforms. They are difficult to use for low-cost rapid detection. Therefore, there is an urgent need for a rapid detection technology that does not require antibodies, can be stored at room temperature, can be quantitative or semi-quantitative, can detect pepsin activity, and can be applied in saliva scenarios. Summary of the Invention

[0008] Therefore, to address the aforementioned problems, this invention, based on the controllable enzymatic degradation characteristics of casein hydrogels and combined with the dye particle release mechanism, constructs a rapid colorimetric detection system suitable for saliva Pepsin, overcoming several limitations of existing antibody detection methods.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: a rapid detection gel for salivary pepsin detection and its preparation method, the structure of which includes a casein gel and carbon black blue particles uniformly distributed therein, which are insoluble in water and have a particle size larger than the gel pore size.

[0010] Furthermore, the steps include: 1) Prepare the casein solution: Add casein powder to deionized water, heat to 60–80℃, stir until completely dissolved, and adjust the pH to 7.0–7.5. 2) Formation of a gel network, acid-induced method: Slowly add lactic acid / acetic acid to the casein solution until the pH reaches 4.3–4.6, causing casein micelles to aggregate and form a flexible hydrogel. 3) Visualization of microparticle encapsulation methods, taking carbon black blue microparticles as an example: Carbon blue microparticles with a particle size of approximately 300 nm were uniformly dispersed in a casein solution. Acid-induced gelation simultaneously achieved "physical embedding," resulting in gel columns or sheets with uniform appearance, deep color, and no leakage. Encapsulation principle: The microparticles are stable and do not leak in the gel. After Pepsin digestion, the pore size increases, leading to rapid release.

[0011] Furthermore, the steps are as follows: Enzyme digestion reaction system: 1) Acidify to activate saliva Pepsin. Saliva pH is usually 6.5–7.2 and needs to be acidified to pH 1.5–2.0. Use pre-prepared acidification buffer and ensure the final pH is stable by using test strips or a fixed ratio.

[0012] 2) Add gel particles or gel columns, 5–10 mg / gel block, mix with acidified saliva, and incubate at room temperature for 15 minutes.

[0013] Compared with traditional antibody-based pepsin detection methods, this invention has the following significant advantages: The test measures "enzyme activity" rather than "protein presence." This avoids false positives caused by pepsin inactivation and is more consistent with clinical physiological significance.

[0014] The materials are inexpensive, require no antibodies, and the process is simple. It can be prepared using casein and dye microparticles, making it suitable for large-scale production.

[0015] The pH activation + enzyme cleavage release mechanism is stable and reliable, provides rapid color development, strong signal, and does not rely on precision instruments.

[0016] It can be stored at room temperature and has high stability. Casein gel can be stable for more than six months under slightly dry or sealed conditions.

[0017] Suitable for home self-testing, primary care hospitals, and POCT scenarios, it can be operated without professional personnel.

[0018] It can be extended to the detection of other protease activities, such as pepsinogen, trypsin, and MMPs. Attached Figure Description

[0019] Figure 1 This is a simplified reaction procedure for this scheme. Detailed Implementation

[0020] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments.

[0021] This embodiment provides a rapid detection gel for salivary pepsin detection and its preparation method. The structure includes a casein gel and carbon black blue particles uniformly distributed therein, which are insoluble in water and have a particle size larger than the gel pore size.

[0022] Furthermore, the steps include: 1) Prepare the casein solution: Add casein powder to deionized water, heat to 60–80℃, stir until completely dissolved, and adjust the pH to 7.0–7.5. 2) Formation of a gel network, acid-induced method: Slowly add lactic acid / acetic acid to the casein solution until the pH reaches 4.3–4.6, causing casein micelles to aggregate and form a flexible hydrogel. 3) Visualization of microparticle encapsulation methods, taking carbon black blue microparticles as an example: Carbon blue microparticles with a particle size of approximately 300 nm were uniformly dispersed in a casein solution. Acid-induced gelation simultaneously achieved "physical embedding," resulting in gel columns or sheets with uniform appearance, deep color, and no leakage. Encapsulation principle: The microparticles are stable and do not leak in the gel. After Pepsin enzyme digestion, the pore size increases, leading to rapid release.

[0023] Enzyme digestion reaction system: 1) Acidify to activate saliva Pepsin. Saliva pH is usually 6.5–7.2 and needs to be acidified to pH 1.5–2.0. Use pre-prepared acidification buffer and ensure the final pH is stable by using test strips or a fixed ratio.

[0024] 2) Add gel particles or gel columns, 5–10 mg / gel block, mix with acidified saliva, and incubate at room temperature for 15 minutes.

[0025] Result determination Negative (inactive pepsin): The gel remains intact, and the supernatant shows no obvious color; In the absence of Pepsin or under non-acidic conditions, the gel remains intact with a distinct interface; Saliva itself has a neutral pH (6.5–7.2), under which Pepsin is almost inactive and therefore does not attack the casein gel structure.

[0026] In negative cases, the gel block will deposit at the bottom of the test tube or be suspended in the liquid, maintaining a "solid-liquid interface" with the liquid above.

[0027] Meanwhile, because the carbon blue particles cannot escape, the solution remains completely clear, without dispersion or false positive color development.

[0028] Positive (active pepsin): The gel is partially or completely hydrolyzed, releasing carbon blue microparticles → the supernatant turns blue rapidly; In acidified saliva containing active Pepsin, the gel degrades and releases carbon blue particles; After saliva is acidified to pH 1.5–2.0, Pepsin regains its activity and begins to rapidly cleave peptide bonds in casein gels.

[0029] As the gel structure is destroyed, its internal pore size increases, micelles disperse, and eventually the gel block gradually dissolves and disappears.

[0030] Carbon blue particles encapsulated in the gel are released into the solution as the structure breaks down, causing the solution to quickly turn blue / dark, forming a visible positive signal.

[0031] It can achieve: ① Qualitative analysis by naked eye; ② Quantitative analysis by smartphone images; ③ Semi-quantitative analysis by optical density (600–700 nm).

[0032] Example: Detection of the activity of standard Pepsin Prepare gels with three different casein contents (5%, 8%, and 10%). Samples with added 0, 10, 50, 100, and 500 ng / mL Pepsin Acidify to pH 1.8 The colorimetric readings after 10 minutes are as follows:

[0033] Compared with traditional antibody-based pepsin detection methods, this invention has the following significant advantages: The test measures "enzyme activity" rather than "protein presence." This avoids false positives caused by pepsin inactivation and is more consistent with clinical physiological significance.

[0034] The materials are inexpensive, require no antibodies, and the process is simple. It can be prepared using casein and dye microparticles, making it suitable for large-scale production.

[0035] The pH activation + enzyme cleavage release mechanism is stable and reliable, provides rapid color development, strong signal, and does not rely on sophisticated instruments.

[0036] It can be stored at room temperature and has high stability. Casein gel can be stable for more than six months under slightly dry or sealed conditions.

[0037] Suitable for home self-testing, primary care hospitals, and POCT scenarios, it can be operated without professional personnel.

[0038] It can be extended to the detection of other protease activities, such as pepsinogen, trypsin, MMPs, etc.

[0039] The enzyme digestion and colorimetric platform of this invention can be extended to: Rapid detection of other digestive enzyme activities, such as trypsin, chymotrypsin, and pepsinogen.

[0040] Rapid evaluation of food digestion simulation: such as solid protein digestibility analysis.

[0041] Drug release system research: using enzyme-cleaved hydrogels to control drug release.

[0042] Construction of biodegradable hydrogel sensors: with application potential in medical implantation and biosensors.

[0043] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0044] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A rapid detection gel for salivary pepsin detection and its preparation method, characterized in that: Its structure includes a casein gel and carbon blue particles that are uniformly distributed therein, are insoluble in water, and have a particle size larger than the gel pore size.

2. The method for preparing a rapid detection gel for salivary pepsin detection according to claim 1, characterized in that: The steps include: 1) Prepare casein solution; Add casein powder to deionized water, heat to 60–80°C, stir until completely dissolved, and adjust the pH to 7.0–7.5 (for easier subsequent operations). 2) Formation of a gel network; Acid-induced method: Lactic acid / acetic acid is slowly added dropwise to the casein solution until the pH reaches 4.3–4.6, causing casein micelles to aggregate and form a flexible hydrogel. 3) Visualization of microparticle encapsulation methods; Take carbon black blue microparticles as an example: Carbon blue microparticles with a particle size of approximately 300 nm were uniformly dispersed in a casein solution. Acid-induced gelation simultaneously achieved "physical embedding," resulting in gel columns or sheets with uniform appearance, deep color, and no leakage. Encapsulation principle: The microparticles are stable and do not leak in the gel. After Pepsin digestion, the pore size increases, leading to rapid release.

3. The rapid detection gel for salivary pepsin detection and its preparation method according to claim 1, characterized in that: The steps are as follows: Enzyme digestion reaction system: 1) Acidify to activate saliva Pepsin. Saliva pH is usually 6.5–7.2 and needs to be acidified to pH 1.5–2.

0. Use pre-prepared acidification buffer (diluted HCl solution) and ensure the final pH is stable by using test strips or a fixed ratio. 2) Add gel particles or gel columns, 5–10 mg / gel block, mix with acidified saliva, and incubate at room temperature for 15 minutes.

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