A NAG test paper and preparation method thereof

Abstract

The present invention relates to a NAG detection test paper and its preparation method, comprising liquid A, liquid B, and liquid C. Liquid A is a substrate solution containing CNP-NAG, and the solvent is DMF and / or DMSO and purified water, and also contains TMN and / or ethylene glycol diethyl ether. Liquid B is a phosphate buffer solution. Liquid C is a fumaric acid-propylene sulfonic acid copolymer and / or one or more of nitrilotriacetic acid (NTA), sodium / potassium EDTA, diethylenetriaminepentaacetic acid (DTPA) and its salts, hydroxyethyldiphosphonic acid (HEDP), sodium ethylenediamine disuccinate (EDDS), and cyclohexanetriacyl ether (CDTA). The preparation process involves first spraying / soaking filter paper with liquid A and then drying it. Liquids B and C are then sprayed / soaked with the filter paper in separate steps, and then dried using hot air below 47°C. Finally, the paper is cut and sealed for storage to produce the NAG detection test paper. This method offers the advantages of simple operation, stable performance, and high sensitivity, while also improving the stability and shelf life of the test paper.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnostic reagents, and in particular to a NAG detection test paper and a preparation method thereof. Background Art

[0002] Acetylamino-β-D-glucosidase (NAG) is an important lysosomal hydrolase in the human body, widely present in various tissues, cells, and body fluids. It plays a role in regulating glucose metabolism, cell signaling, and clinically assessing cell damage. It is often used clinically to assist in the diagnosis of diseases such as liver and kidney damage and Trichomonas infection. Current methods for NAG detection include fluorescence analysis, spectrophotometry, and dry chemical enzymatic methods. Fluorescence analysis and spectrophotometry require expensive equipment and cumbersome procedures, and the substrate is often freeze-dried to maintain stability. Some dry chemical enzymatic methods use p-nitrobenzene-N-acetyl-β-D-glucosaminide (PNP-NAG, CAS: 3459-18-5) as a substrate for NAG detection strips. However, this requires the addition of a colorimetric solution or stop solution after incubation for alkalinization and color development, which is a cumbersome process. Other methods use 5-bromo-4-chloro-3-indolyl-N-β-D-glucosaminide (X-NAG, CAS: 4264-82-8), but this has low sensitivity. Furthermore, due to its highly degradable color development, CNP-NAG does not consistently produce a detectable color product in dry chemical enzymatic methods, limiting its application in this assay. Therefore, the development of a simple, stable, and highly sensitive NAG detection strip and its preparation method is crucial. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a NAG detection test paper and a preparation method thereof.

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

[0005] In one aspect, the present invention provides a NAG test strip comprising liquid A, liquid B, and liquid C;

[0006] The solution A is a substrate solution, including CNP-NAG, the solvent is DMF and / or DMSO and purified water, and also includes polyethylene glycol trimethyl nonyl ether (TMN) and / or ethylene glycol diethyl ether;

[0007] The B solution is a phosphate buffer solution;

[0008] The liquid C is one or more of fumaric acid-propylene sulfonic acid copolymer and / or aminotriacetic acid (NTA), EDTA sodium salt / potassium salt, diethylenetriaminepentaacetic acid (DTPA) and its salts, hydroxyethylenediphosphonic acid (HEDP), ethylenediamine disuccinate sodium (EDDS), and cyclohexanetriac acid (CDTA);

[0009] As an optimization, the concentration of CNP-NAG in the solution A is 0.2-5 g / L.

[0010] As an optimization, the concentration of CNP-NAG in the solution A is 1 g / L.

[0011] As an optimization, the concentration of TMN in the solution A does not exceed 1%.

[0012] As an optimization, the B solution is a phosphate buffer solution with a concentration of not less than 0.25 M / L and a pH of 5.0.

[0013] As an optimization, the solution B further includes one or more of TMN, NaCl, and EDTA-2Na electrolytes. Electrolytes with appropriate ion concentrations can stabilize the CNP-NAG on the filter paper.

[0014] As an optimization, the C liquid is 5% of fumaric acid-propylene sulfonic acid copolymer and / or one or more of aminotriacetic acid (NTA), EDTA sodium salt / potassium salt, diethylenetriaminepentaacetic acid (DTPA) and its salts, hydroxyethyldiphosphonic acid (HEDP), ethylenediamine disuccinate sodium (EDDS), and cyclohexanetricarboxylic acid (CDTA).

[0015] Another aspect of the present invention provides a method for preparing a NAG test strip, the specific preparation steps being:

[0016] S1. Prepare Solution A: Add TMN and / or ethylene glycol diethyl ether to DMF and / or DMSO to prepare a substrate solvent, and use this solvent to dissolve the substrate CNP-NAG to prepare a substrate mother liquor; then dilute the substrate mother liquor with purified water to the desired concentration to obtain Solution A; TMN and ethylene glycol diethyl ether can effectively slow down the spontaneous color development of CNP-NAG in the solvent and extend the shelf life of Solution A.

[0017] S2. Prepare Solution B: Use phosphate buffer to provide a buffer environment for the enzymatic reaction;

[0018] S3. Prepare Solution C: Use one or more of fumaric acid-propylene sulfonic acid copolymer and / or aminotriacetic acid (NTA), sodium / potassium EDTA, diethylenetriaminepentaacetic acid (DTPA) and its salts, hydroxyethyldiphosphonic acid (HEDP), sodium ethylenediamine disuccinate (EDDS), and cyclohexanetriac acid (CDTA). This helps stabilize the CNP-NAG on the filter paper and counteracts the spontaneous color development of the test paper, maintaining a negative colorless state.

[0019] S4. First, use the prepared solution A to spray / soak the filter paper so that the solution A evenly covers the filter paper, and then dry it for later use. The drying temperature must not exceed 47°C.

[0020] S5. Then, use the prepared solution B to spray / soak the filter paper obtained in S4, so that the solution B is evenly covered and dried;

[0021] S6. Next, use the prepared liquid C to spray / soak the filter paper obtained in S5, so that the liquid C evenly covers the filter paper and allows it to dry.

[0022] S7, cutting the filter paper obtained in S6, sealing and storing it, and finally preparing the NAG test paper;

[0023] In addition, the above S4-S6 must be prepared in steps, wherein S5 and S6 can be reversed in order.

[0024] The beneficial effects of the present invention are:

[0025] The present invention provides a NAG detection test paper, comprising liquid A, liquid B, and liquid C; the liquid A is a substrate solution, comprising CNP-NAG, and the solvent is DMF and / or DMSO and purified water, and also comprises TMN and / or ethylene glycol diethyl ether; the liquid B is a phosphate buffer; the liquid C is one or more of a fumaric acid-propylene sulfonic acid copolymer and / or nitrilotriacetic acid (NTA), sodium salt / potassium salt of EDTA, diethylenetriaminepentaacetic acid (DTPA) and its salts, hydroxyethyldiphosphonic acid (HEDP), sodium ethylenediamine disuccinate (EDDS), and cyclohexanetriac acid (CDTA); a method for preparing the NAG detection test paper comprises: S1, preparing liquid A: adding TMN and / or ethylene glycol diethyl ether to DMF and / or DMSO to obtain a substrate solvent, and dissolving the substrate CNP-NAG in the solvent to obtain a substrate mother liquor; then diluting the substrate mother liquor with purified water to a desired concentration to obtain liquid A; S2, preparing liquid B: using Phosphate buffer; S3, prepare liquid C: use one or more of fumaric acid-propylene sulfonic acid copolymer and / or aminotriacetic acid (NTA), EDTA sodium salt / potassium salt, diethylenetriaminepentaacetic acid (DTPA) and its salts, hydroxyethyldiphosphonic acid (HEDP), ethylenediamine disuccinate sodium (EDDS), and cyclohexanetriac acid (CDTA); S4, first, use the prepared liquid A to spray / soak the filter paper so that the liquid A evenly covers the filter paper, and then dry it for use. The drying temperature must not exceed 47°C; S5, then, use the prepared liquid B to spray / soak the filter paper obtained in S4 so that the liquid B evenly covers the filter paper and then dry it; S6, secondly, use the prepared liquid C to spray / soak the filter paper obtained in S5 so that the liquid C evenly covers the filter paper and then dry it; S7, cut the filter paper obtained in S6, seal it and store it, and finally prepare the NAG test paper; In addition, the above S4-S6 must be prepared in steps, and the order of S5 and S6 can be reversed. The present invention has the advantages of simple operation, stable performance and high sensitivity, and improves the stability and shelf life of the test paper. DETAILED DESCRIPTION

[0026] The following specific embodiments further describe the present application in detail. It is particularly noted that the following embodiments are only used to illustrate the present application and do not limit the scope of the present application. Similarly, the following embodiments are only some of the embodiments of the present application and not all of them. All other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0027] Example 1

[0028] A NAG detection test strip comprises liquid A, liquid B and liquid C; the liquid A is a substrate solution comprising CNP-NAG, the solvent being DMF and / or DMSO and purified water, and also comprising TMN and / or ethylene glycol diethyl ether; the final concentration of CNP-NAG in the liquid A is 1 g / L, and the concentration of TMN in the liquid A is 0.5%; the liquid B is a 0.5 M / L, pH 5.0 phosphate buffer solution, further comprising 10 mM / L each of NaCl and EDTA-2Na; and the liquid C comprises 5% fumaric acid-propylene sulfonic acid copolymer and EDTA sodium salt.

[0029] A method for preparing a NAG test strip comprises the following steps:

[0030] S1. Prepare Solution A: Add TMN and / or ethylene glycol diethyl ether to DMF and / or DMSO to prepare a substrate solvent, and dissolve the substrate CNP-NAG in the solvent to prepare a 10 g / L substrate mother solution, wherein the volume ratio of TMN and / or ethylene glycol diethyl ether is 0.5%; then dilute the substrate mother solution to 1 g / L with purified water;

[0031] S2. Prepare Solution B: Use 0.5 M / L, pH 5.0 phosphate buffer, and include 10 mM / L each of NaCl and EDTA-2Na;

[0032] S3. Prepare Solution C: Use 5% fumaric acid-propylene sulfonic acid copolymer and EDTA sodium salt;

[0033] S4. First, use the prepared solution A to spray / soak the filter paper so that the solution A evenly covers the filter paper, and then dry it for later use. The drying temperature must not exceed 47°C.

[0034] S5. Then, use the prepared liquid B to spray / soak the filter paper obtained in S4, so that the liquid B is evenly covered and dried; S6. Secondly, use the prepared liquid C to spray / soak the filter paper obtained in S5, so that the liquid C is evenly covered and dried; S7. Cut the filter paper obtained in S6, seal it for storage, and finally prepare the NAG detection test paper.

[0035] Example 2

[0036] In Example 1, the order of S5 and S6 is reversed, and the rest is the same as in Example 1.

[0037] Comparative Example 1

[0038] There is no C solution in the NAG test paper, and the preparation method of the NAG test paper does not contain steps related to C solution. Other aspects are the same as those in Example 1.

[0039] Comparative Example 2

[0040] There is no B solution in the NAG test paper, and the preparation method of the NAG test paper does not contain steps related to B solution. Other steps are the same as those in Example 1.

[0041] Comparative Example 3

[0042] In the preparation method of the NAG test strip, solution A and solution B are mixed together, and solution C is prepared separately. Other aspects are the same as in Example 1.

[0043] Comparative Example 4

[0044] In the preparation method of the NAG test strip, liquid A and liquid C are mixed together, and liquid B is prepared separately. Other aspects are the same as in Example 1.

[0045] Comparative Example 5

[0046] In the preparation method of the NAG test strip, solution B and solution C are mixed together, and the rest is the same as in Example 1.

[0047] Comparative Example 6

[0048] In the preparation method of the NAG test strip, liquid A, liquid B and liquid C are mixed together, and the rest is the same as in Example 1.

[0049] Thermal acceleration comparison results

[0050] Examples 1 and 2 of the present invention and Comparative Examples 1-6 were simultaneously subjected to a 47°C thermal acceleration test. The base color (visual inspection) and the color development of the negative and positive quality controls were respectively tested at 0, 1, 3, 5, and 7 days. The results showed that the base color of Comparative Examples 1, 2, and 6 turned yellow after 1 day. The negative control immediately turned yellow after addition and was indistinguishable from the positive control after 15 minutes. Comparative Examples 2, 3, and 4 had a white base color, but the negative control also immediately turned yellow after addition and was indistinguishable from the positive control after 15 minutes. The thermal acceleration method of the present invention remained effective even after 7 days. Specific comparative results are shown in Table 1 below.

[0051] Table 1 Thermal acceleration comparison results

[0052]

[0053] As shown in Table 1, Comparative Examples 1-6 all spontaneously developed color within 24 hours of thermal acceleration at 47°C, resulting in failure. However, the present invention remained effective even after 7 days of thermal acceleration. This indicates that the present invention, by first spraying / soaking the filter paper with Liquid A and then drying it, then sequentially spraying / soaking the filter paper with Liquid B and Liquid C, or Liquid C and Liquid B, followed by drying with hot air below 47°C, and finally cutting and sealing the paper for storage, produces NAG test strips with superior stability and a longer shelf life.

[0054] Unexpected technical effects are achieved by adopting the formulation and preparation method of the present invention.

[0055] Comparative Example 7

[0056] We purchased NAG detection kits from 8 companies. Among them, the name of the commercially available product No. 1 is "Vaginitis Combined Detection Kit (Dry Chemical Enzyme Method)", the name of the commercially available product No. 2 is "Vaginitis Combined Detection Kit (Enzyme Chemical Reaction Method)", and the name of the commercially available product No. 3 is "Vaginal Microecological Detection Kit (Enzyme Chemical Reaction Method)", and the substrates used are all p-nitrobenzene-N-acetyl-β-D-glucosamine (PNP-NAG, CAS: 3459-18--5); the names of the commercially available products No. 4, No. 5 and No. 6 are "Vaginitis Seven-Combination Detection Kit (Dry Chemical Method)", and the substrates are Both use 5-bromo-4-chloro-3-indolyl-N-β-D-glucosaminide (CAS: 4264-82-8). Commercially available product No. 7, called the "Mixed Vaginal Infection Detection Kit (Dry Chemical Enzymatic Method)," and product No. 8, called the "Vaginitis Five-Test Kit II (Dry Chemical Enzymatic Method)," both utilize acetylglucosaminide salts in combination with diazonium salts. Product No. 7 utilizes a layered filter paper structure, immobilizing the diazonium salt on a single layer of filter paper, while product No. 8 utilizes a separate liquid vial containing the diazonium salt. Currently, there are no commercially available dry chemical enzymatic kits for detecting NAG using CNP-NAG. Performance comparison results are shown in Table 2 below.

[0057] Table 2 Performance comparison results

[0058]

[0059] As shown in Table 2, the kits of commercial products No. 1, No. 2, and No. 3 using p-nitrobenzene-N-acetyl-β-D-glucosaminide (PNP-NAG, CAS: 3459-18-5) as substrate are cumbersome and require additional addition of colorimetric solution or stop solution after incubation for alkalinization and color development. In addition, the sensitivity of commercial product No. 2 is average. The kits of commercial product No. 3 using 5-bromo-4-chloro-3-indolyl-N-β-D-glucosaminide (CAS: 4264-82-8) as substrate are cumbersome and require additional addition of colorimetric solution or stop solution after incubation for alkalinization and color development. In addition, the sensitivity of commercial product No. 2 is average. Although the commercially available test kits 4, 5, and 6 are easy to use and do not require the addition of a developer or stop solution, they all have low sensitivity. Commercially available test kit 7, which uses an acetylglucosamine salt in combination with a diazonium salt, is easy to use and does not require the addition of a developer, but its sensitivity and stability are average. Commercially available test kit 8, which uses an acetylglucosamine salt in combination with a diazonium salt, is complex, requires the addition of a developer (diazonium salt), and exhibits average sensitivity. The present invention, however, utilizes solutions A, B, and C to produce NAG test strips in separate steps. These test strips are easy to use, do not require the addition of a developer or stop solution, and exhibit high sensitivity, good stability, and a long shelf life. Different from the prior art, TMN and ethylene glycol diethyl ether in the liquid A of the present invention can effectively slow down the spontaneous color development of CNP-NAG in the solvent and extend the shelf life of liquid A; liquid B provides a buffer environment for the enzymatic reaction, in which the electrolyte with an appropriate ion concentration can stabilize the CNP-NAG on the filter paper; liquid C uses one or more of fumaric acid-propylene sulfonic acid copolymer and / or aminotriacetic acid (NTA), EDTA sodium salt / potassium salt, diethylenetriaminepentaacetic acid (DTPA) and its salts, hydroxyethyldiphosphonic acid (HEDP), sodium ethylenediamine disuccinate (EDDS), and cyclohexanetriac acid (CDTA), which, on the one hand, help stabilize the CNP-NAG on the filter paper, and on the other hand, offset the spontaneous color development of the test paper to maintain a negative colorless state.

[0060] The above description shows the main features, basic principles, and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments or examples, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, the above embodiments or examples should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any simple modification, equivalent change and modification of the above embodiments based on the technical essence of the present invention, without departing from the technical principle of the present invention, should fall within the scope of patent protection of the present invention.

Claims

1. A NAG test strip, characterized by: Including liquid A, liquid B and liquid C; The solution A is a substrate solution, including CNP-NAG, the solvent is DMF and / or DMSO and purified water, and also includes polyethylene glycol trimethyl nonyl ether (TMN) and / or ethylene glycol diethyl ether; The B solution is a phosphate buffer solution; The C liquid is one or more of fumaric acid-propylene sulfonic acid copolymer and / or aminotriacetic acid (NTA), EDTA sodium salt / potassium salt, diethylenetriaminepentaacetic acid (DTPA) and its salts, hydroxyethylenediphosphonic acid (HEDP), ethylenediamine disuccinate sodium (EDDS), and cyclohexanetriac acid (CDTA).

2. The NAG test paper according to claim 1, wherein: The concentration of CNP-NAG in the solution A is 0.2-5 g / L.

3. The NAG test strip according to claim 2, wherein: The concentration of CNP-NAG in the solution A is 1 g / L.

4. The NAG test paper according to claim 2, wherein: The concentration of polyethylene glycol trimethyl nonyl ether (TMN) in the liquid A does not exceed 1%.

5. The NAG test paper according to claim 1, wherein: The solution B is a phosphate buffer solution with a pH of not less than 0.25 M / L and a pH of 5.

0.

6. The NAG test paper according to claim 5, wherein: The B solution further includes one or more of polyethylene glycol trimethyl nonyl ether (TMN), NaCl and EDTA-2Na electrolytes.

7. The NAG test strip according to claim 1, wherein: The C liquid is one or more of fumaric acid-propylene sulfonic acid copolymer and / or aminotriacetic acid (NTA), EDTA sodium salt / potassium salt, diethylenetriaminepentaacetic acid (DTPA) and its salts, hydroxyethylenediphosphonic acid (HEDP), ethylenediamine disuccinate sodium (EDDS), and cyclohexanetriac acid (CDTA), with a total concentration of 5%.

8. A method for preparing a NAG test paper, characterized in that: The NAG test paper according to any one of claims 1 to 7 is prepared in the following steps: S1. Prepare Solution A: Add polyethylene glycol trimethyl nonyl ether (TMN) and / or ethylene glycol diethyl ether to DMF and / or DMSO to prepare a substrate solvent, and dissolve the substrate CNP-NAG in the solvent to prepare a substrate mother liquor; then dilute the substrate mother liquor with purified water to the desired concentration to obtain Solution A; S2. Prepare Solution B: Solution B uses phosphate buffer; S3. Prepare Liquid C: Liquid C uses one or more of fumaric acid-propylene sulfonic acid copolymer and / or aminotriacetic acid (NTA), EDTA sodium salt / potassium salt, diethylenetriaminepentaacetic acid (DTPA) and its salts, hydroxyethylenediphosphonic acid (HEDP), ethylenediamine disuccinate sodium (EDDS), and cyclohexanetriac acid (CDTA); S4. First, use the prepared solution A to spray / soak the filter paper so that the solution A evenly covers the filter paper, and then dry it for later use. The drying temperature must not exceed 47°C. S5. Then, use the prepared solution B to spray / soak the filter paper obtained in S4, so that the solution B is evenly covered and dried; S6. Next, use the prepared liquid C to spray / soak the filter paper obtained in S5, so that the liquid C evenly covers the filter paper and allows it to dry. S7, cutting the filter paper obtained in S6, sealing and storing it, and finally preparing the NAG test paper; In addition, the above S4-S6 must be prepared in steps, wherein S5 and S6 can be reversed in order.

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