Rapid colorimetric detection method and kit for crosslinking state of biological tissue

Abstract

The invention relates to a rapid colorimetric detection method and kit for the crosslinking state of biological tissues. The method comprises the following steps: preparing a color developing system solution containing ninhydrin, dipping a porous base material, and drying to obtain color developing test paper; the method comprises the following steps: providing standard biological tissue samples treated in different cross-linking states, calibrating the cross-linking states of the standard biological tissue samples by adopting thermal shrinkage temperature and / or mechanical property indexes, enabling a color result formed by the standard samples in the same color development mode as a to-be-detected sample to correspond to the cross-linking states, and establishing a standard colorimetric card with color gradation; the method comprises the following steps: soaking a to-be-detected biological tissue subjected to cross-linking treatment in a buffer solution to extract residual free amino groups, dropwise adding supernate onto color developing test paper, heating and developing, visually comparing with a standard colorimetric card under the same illumination condition, and outputting a cross-linking degree judgment result. The kit comprises color developing test paper, a standard colorimetric card and a detection interpretation instruction. The scheme is simple and convenient to operate, does not need complex instruments, and is suitable for on-site rapid grading and inter-batch consistency control.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomaterial detection and quality control in the production of medical devices, and particularly to a colorimetric detection method for rapidly determining the crosslinking degree of crosslinked biological tissues, and further to a kit for implementing the detection method. Background Art

[0002] In the manufacture of medical devices such as biological valves, glutaraldehyde crosslinking is a key process for treating biological tissues such as bovine pericardium to enhance their stability. The crosslinking degree directly determines the mechanical properties, anti-calcification ability, and long-term durability of the material. However, the current quality control detection methods for the crosslinking degree have significant limitations: traditional mechanical property tests and determination of thermal shrinkage temperature are both destructive and time-consuming off-line detection methods, and cannot rapidly and non-destructively screen intermediate products during the production process. This results in hysteresis and blind spots in the control of the crosslinking process, and there may be a quality risk that the performance of the entire batch of products does not meet the standards due to insufficient or excessive crosslinking, causing waste of resources and potential safety hazards.

[0003] Although there are some chemical detection methods (such as detecting free amino groups) in the prior art, their operations are cumbersome, require professional equipment and techniques, and are difficult to be rapidly applied on the production line site. Currently, there is a lack of a rapid detection scheme that can allow operators to instantaneously, intuitively, and semi-quantitatively judge whether the crosslinking degree is qualified, just like a pH test paper. Summary of the Invention

[0004] The present invention aims to solve the problems existing in the detection of the crosslinking degree of existing biological tissues, such as time-consuming detection processes, dependence on professional instruments, difficulty in rapidly releasing products on the production site, and non-intuitive judgment of batch-to-batch consistency, and provides a rapid colorimetric detection method for the crosslinking state of biological tissues. This method includes preparing a color-developing test paper containing ninhydrin, establishing a standard colorimetric card formed by standard biological tissue samples in different crosslinking states, and after immersing the crosslinked biological tissue to be detected in a buffer solution to extract residual free amino groups, taking the supernatant and dropping it onto the color-developing test paper for heating and color development, and visually comparing the color development result with the standard colorimetric card under the same light conditions, so as to output the determination result of the crosslinking degree of the biological tissue to be detected; at the same time, the present invention also provides a kit配套 with the method to meet the requirements of rapid, intuitive, and repeatable quality control of the crosslinking state on the production site.

[0005] According to the first aspect of the present invention, there is provided a rapid colorimetric detection method for the crosslinking state of biological tissues, including the following steps: Adding the biological tissue to be detected into a buffer solution for soaking to extract residual free amino groups, taking the supernatant and dropping it onto the color-developing test paper for color development, and obtaining the color development result of the sample to be detected; The color development results of the sample to be tested are visually compared with the standard colorimetric card, and the cross-linking degree of the biological tissue to be tested is determined. The standard colorimetric card is established based on a series of standard biological tissue samples with known cross-linking states. The standard colorimetric card contains color levels corresponding to different cross-linking states and indicates the degree of cross-linking determination results corresponding to each color level.

[0006] In some technical solutions, the colorimetric test paper includes a porous substrate and a colorimetric system loaded on the porous substrate, wherein the colorimetric system contains ninhydrin.

[0007] In some technical solutions, the color development system is a system formed on the porous substrate by immersing the porous substrate in the color development system solution, taking it out and drying it. The composition of the color development system solution is: ninhydrin 5 g / L to 20 g / L, cosolvent 100 g / L to 300 g / L, stabilizer 1 g / L to 5 g / L, and buffer 20 g / L to 80 g / L.

[0008] Ninhydrin: As a core colorimetric agent, it can react with the residual free amino groups extracted from the supernatant to form a colorimetric signal, thus converting the difference in free amino content into a visible difference in color intensity, thereby providing a direct reading basis for the grading of the degree of crosslinking.

[0009] Cosolvent: Used to improve the solubility of ninhydrin in aqueous colorimetric systems, so that ninhydrin can remain uniformly dispersed during preparation, impregnation and use, avoiding crystallization due to insufficient solubility, thereby ensuring the stability and effective colorimetric concentration of the colorimetric system.

[0010] Stabilizer: Used to improve the uniformity of the colorimetric system on porous substrates, so that the colorimetric reaction can spread more evenly on the surface and pores of the test paper after the addition of the supernatant, reducing spots and color differences caused by local enrichment, thereby improving the consistency and interpretability of the colorimetric results.

[0011] Buffer: Used to provide and maintain the pH environment required for the colorimetric reaction, offset the acid-base fluctuations caused by the sample extract and external factors, and ensure that the colorimetric reaction of ninhydrin and free amino groups is carried out under relatively constant conditions, thereby improving the stability of the correspondence between the color signal and the free amino content and enhancing the reliability of interpretation.

[0012] In some technical solutions, the co-solvent is selected from at least one of ethylene glycol, propylene glycol, and polyethylene glycol 400; the stabilizer is selected from at least one of sodium carboxymethyl cellulose, polyvinylpyrrolidone, and gum arabic; and the buffer is selected from at least one of phosphate buffer pairs and borate buffer pairs.

[0013] In some technical solutions, the porous substrate is chromatography filter paper.

[0014] However, the present invention is not limited thereto. The porous substrate may also be other porous materials with liquid absorption and transport capabilities and capable of supporting the color development system, as long as they can achieve stable wetting and color development reaction and output interpretable color results after the addition of clear liquid. Examples include filter paper, cellulose-based porous membrane, non-woven fabric, porous polymer membrane or combinations thereof. The specific selection of the substrate may be adjusted according to the properties of the extract of the target sample, the requirements for color uniformity, and the preparation and storage conditions, so as to obtain color development effect and repeatability that meet the requirements of rapid on-site colorimetric determination.

[0015] In some technical solutions, the biological tissue to be tested is cut into test pieces of a specified size and the surface is kept clean and undamaged to ensure uniform sampling volume and effective extraction area, and to avoid extraction deviations caused by contamination, damage or edge defects.

[0016] In some technical solutions, the test subject is preferably bovine pericardial tissue or other bioprosthetic valve-related tissue, in order to match the actual quality control scenario and ensure compatibility with the standard colorimetric card.

[0017] In some technical solutions, the specific method for extracting residual free amino groups is as follows: using 1-3 mL of PBS buffer and controlling the soaking and extraction time to 5-10 min, so that the extraction process can fully release the residual free amino groups that reflect the degree of cross-linking within a controlled volume and time window, while avoiding excessive extraction that could lead to increased background or amplified batch-to-batch fluctuations.

[0018] In some technical solutions, the color development method is as follows: after adding the supernatant droplet to the color development test paper, it is heated at 60℃~90℃ for 1~10 min. This is to enable the color development reaction to be completed quickly and the color to be stably formed under controllable thermal activation conditions, thereby improving the color development uniformity, interpretation consistency and on-site detection efficiency.

[0019] In some technical solutions, the standard colorimetric card is established through the following steps: Standard biological tissue samples with different cross-linking states are provided, and the cross-linking state of each standard biological tissue sample is calibrated using heat shrinkage temperature and / or mechanical property indicators; Each standard biological tissue sample was soaked in buffer solution to extract residual free amino groups. The supernatant was then dropped onto colorimetric paper and colorimetric analysis was performed. The color development results of each standard biological tissue sample are matched one-to-one with their calibrated cross-linking states to form a standard colorimetric card with color levels, and the cross-linking degree judgment results corresponding to each color level are marked.

[0020] In some technical solutions, the standard colorimetric card includes one or more of the following: (1) The cross-linking time of the standard biological tissue sample shall include at least two or more of the following: 0 h, 24 h, 72 h, and 168 h; (2) Mark the degree of crosslinking on the standard colorimetric card, wherein the degree of crosslinking includes one or more of the following: "not crosslinked", "insufficient crosslinking", "qualified crosslinking" and "excessive crosslinking".

[0021] The selection of crosslinking time can be understood as follows: under the same crosslinking system, the same tissue source, and the same treatment conditions, by setting at least two different crosslinking time points, the standard sample covers different stages of the crosslinking reaction from the initial stage to a more complete stage, thereby forming a color reference range that can distinguish the differences in the degree of crosslinking of the test sample. On this basis, multiple crosslinking time points can be further selected to improve the color gradation resolution and interpretation stability. The crosslinking time can include at least two or more of 0h, 24h, 72h, and 168h, where 0h corresponds to the uncrosslinked baseline state, 24h and 72h are used to cover the typical range of transition from under-crosslinking to qualified crosslinking, and 168h is used to cover the state of further deepening crosslinking.

[0022] For the setting of crosslinking status grading, the above color levels can be divided and labeled as one or more of "not crosslinked," "insufficient crosslinking," "qualified crosslinking," and "excessive crosslinking" according to the production quality control objectives. Specifically, preferably, a four-level grading is adopted, with 0 h, 24 h, 72 h, and 168 h as the corresponding representative time points, so that the boundaries of each level are clear and the operation is strong. In scenarios where cost or management needs are relatively simplified, it can also be combined into two or three levels. For example, "not crosslinked" and "insufficient crosslinking" can be combined into the unqualified area, "qualified crosslinking" can be used as the qualified area, and "excessive crosslinking" can be used as the over-crosslinking indication area. The above crosslinking time points and the number of grades can be adjusted according to the actual tissue type, crosslinking agent concentration, temperature, and process window. The selection logic is to ensure that the color level range of the standard colorimetric card can cover the crosslinking fluctuation range of the actual production sample, and that there are distinguishable color differences between each grade, so as to achieve stable, intuitive, and repeatable on-site interpretation.

[0023] According to a second aspect of the present invention, a kit for rapid colorimetric detection of cross-linking status of biological tissues is further provided, comprising the above-described colorimetric test strip and standard colorimetric card, and including instructions for guiding detection and interpretation, wherein the instructions are used to guide the execution of the above-described method and output the cross-linking degree determination result.

[0024] The present invention, by employing the above technical solution, has at least the following beneficial effects: 1. Achieve rapid and intuitive determination of cross-linking state. This invention transforms the differences in residual free amino groups in cross-linked biological tissues into directly observable color differences, thereby enabling rapid determination of the degree of cross-linking in a short time, meeting the needs of rapid release and process monitoring in production sites.

[0025] 2. Lowering the testing threshold and improving on-site usability. The interpretation method of this invention is visual comparison with the standard colorimetric card, which does not rely on complex spectrometers or colorimeters. The operation steps are simple and easy to promote and apply in production workshops or quality control links, which can effectively reduce testing costs and operator training costs.

[0026] 3. Improve the consistency and traceability of judgments. This invention establishes a standard colorimetric card using standard biological tissue samples treated with different cross-linking states, and calibrates the cross-linking state of the standard samples using heat shrinkage temperature and / or mechanical property indicators. This establishes a clear correspondence between color grading and cross-linking state, thereby reducing subjective judgment fluctuations, improving the consistency of interpretation between different batches and different personnel, and enhancing the traceability of test results.

[0027] 4. High applicability and easy product packaging. This invention further provides a matching solution for colorimetric test strips, standard colorimetric cards and reagent kits, which standardizes and modularizes the detection elements, facilitates batch preparation, transportation and storage, and is suitable for rapid detection of cross-linking status in biological valve-related tissues such as bovine pericardium. It can also be extended to quality control scenarios for other cross-linked biological tissues. Detailed Implementation

[0028] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0029] Example 1: Preparation of colorimetric test paper Prepare a mixed colorimetric solution containing 40 g / L phosphate buffer, 200 g / L ethylene glycol, 12 g / L ninhydrin, and 3 g / L sodium carboxymethyl cellulose using 100 mL of pure water. Completely immerse a 10 mm wide chromatography filter paper in the solution for 15 minutes, then remove and dry in a 60°C oven in the dark for 30 minutes until completely dry. This yields colorimetric test strip #1.

[0030] Comparative Example 1: Preparation of Colorimetric Test Paper Prepare a mixed colorimetric solution containing 40 g / L phosphate buffer, 12 g / L ninhydrin, and 3 g / L sodium carboxymethyl cellulose using 100 mL of pure water. Completely immerse a 10 mm wide chromatography filter paper in the solution for 15 minutes, then remove and dry in a 60°C oven in the dark for 30 minutes until completely dry. This yields colorimetric test strip #2.

[0031] Comparative Example 2: Preparation of Colorimetric Test Paper Prepare a mixed colorimetric solution containing 40 g / L phosphate buffer, 12 g / L ninhydrin, and 200 g / L ethylene glycol using 100 mL of pure water. Completely immerse a 10 mm wide chromatography filter paper in the solution for 15 minutes, then remove and dry in a 60°C oven in the dark for 30 minutes until completely dry. This yields colorimetric test strip #3.

[0032] Comparative Example 3: Preparation of Colorimetric Test Paper Prepare a mixed colorimetric solution containing 12 g / L ninhydrin, 200 g / L ethylene glycol, and 3 g / L sodium carboxymethyl cellulose using 100 mL of pure water. Completely immerse a 10 mm wide chromatography filter paper in the solution for 15 minutes, then remove and dry in a 60°C oven in the dark for 30 minutes until completely dry. This yields colorimetric test strip #4.

[0033] Comparing the color development results of colorimetric test strips #1 to #4, we can see that: Colorimetric test strip #1 shows uniform color development, a clear pale yellow, and is easily readable; colorimetric test strip #2 shows a large number of crystalline spots, resulting in uneven color development and making it unreadable; colorimetric test strip #3 forms halos of varying shades with blurred boundaries, resulting in poor consistency in interpretation; colorimetric test strip #4 shows unstable color, with significant differences between different batches of test strips.

[0034] Therefore, the readability and batch-to-batch consistency of colorimetric test strips depend on the overall compatibility of the colorimetric system: when the colorimetric system contains ninhydrin, solubilizer, stabilizer and buffer, it can inhibit crystal precipitation, reduce spots and halos and stabilize reaction conditions, thereby ensuring the stability and effectiveness of the colorimetric system and obtaining uniform, stable and repeatable colorimetric results.

[0035] Test Example 1: Cut small test pieces (5 mm × 5 mm) from bovine pericardium that has passed cross-linking (cross-linking for 72 hours). Place the test pieces in centrifuge tubes, add 1.0 mL of PBS buffer, and extract by shaking in a shaker for 10 min. Add the extract dropwise onto the test paper prepared in Example 1 and heat in a 70°C oven for 2 minutes.

[0036] Test Example 2: Cut small test pieces (5 mm × 5 mm) from bovine pericardium that has passed cross-linking (cross-linking for 72 hours). Place the test pieces in centrifuge tubes, add 3.0 mL of PBS buffer, and extract by shaking in a shaker for 10 min. Add the extract dropwise onto the test paper prepared in Example 1 and heat in a 70°C oven for 2 minutes.

[0037] Test Example 3: Cut small test pieces (5 mm × 5 mm) from bovine pericardium that has passed cross-linking (cross-linking for 72 hours). Place the test pieces in centrifuge tubes, add 1.0 mL of PBS buffer, and extract by shaking in a shaker for 10 min. Add the extract dropwise onto the test paper prepared in Example 1 and heat in a 70°C oven for 5 minutes.

[0038] Test Comparison Example 1: Cut small test pieces (5 mm × 5 mm) from bovine pericardium that has passed cross-linking (cross-linking for 72 hours). Place the test pieces in centrifuge tubes, add 5.0 mL of PBS buffer, and extract by shaking in a shaker for 10 min. Add the extract dropwise to the test paper prepared in Example 1 and heat in a 70°C oven for 2 minutes.

[0039] Test Comparison Example 2: Cut small test pieces (5 mm × 5 mm) from bovine pericardium that has passed cross-linking (cross-linking for 72 hours). Place the test pieces in centrifuge tubes, add 1.0 mL of PBS buffer, and extract by shaking in a shaker for 3 min. Add the extract dropwise onto the test paper prepared in Example 1 and heat in a 70°C oven for 2 minutes.

[0040] Test Comparison Example 3: Cut small test pieces (5 mm × 5 mm) from bovine pericardium that has passed cross-linking (cross-linking for 72 hours). Place the test pieces in centrifuge tubes, add 1.0 mL of PBS buffer, and extract by shaking on a shaker for 15 min. Add the extract dropwise to the test paper prepared in Example 1 and heat in a 70°C oven for 2 minutes.

[0041] Test Comparison Example 4: Cut small test pieces (5 mm × 5 mm) from bovine pericardium that has passed cross-linking (cross-linking for 72 hours). Place the test pieces in centrifuge tubes, add 1.0 mL of PBS buffer, and extract by shaking in a shaker for 10 min. Add the extract dropwise onto the test paper prepared in Example 1 and heat in a 50°C oven for 2 minutes.

[0042] Test Comparison Example 5: Cut small test pieces (5 mm × 5 mm) from bovine pericardium that has passed cross-linking (cross-linking for 72 hours). Place the test pieces in centrifuge tubes, add 1.0 mL of PBS buffer, and extract by shaking in a shaker for 10 min. Add the extract dropwise to the test paper prepared in Example 1 and heat in a 100°C oven for 2 minutes.

[0043] Test Comparison Example 6: Cut small test pieces (5 mm × 5 mm) from bovine pericardium that has passed cross-linking (cross-linking for 72 hours). Place the test pieces in centrifuge tubes, add 1.0 mL of PBS buffer, and extract by shaking in a shaker for 10 min. Add the extract dropwise onto the test paper prepared in Example 1 and heat in a 70°C oven for 15 minutes.

[0044] The color development results based on test examples 1-3 and test comparative examples 2-6 are shown in Table 1:

[0045] In summary, the rapid colorimetric detection in this case demonstrates a clear process window for "extraction volume / time" and "heating temperature / time": under conditions of appropriate extraction volume, sufficient extraction, and suitable color development conditions, the test paper displays a light blue-purple color and can be reliably determined as indicating successful cross-linking (as in test examples 1-3). Conversely, when excessive extraction dilutes the free amino groups, or insufficient extraction leads to inadequate dissolution of the free amino groups, the concentration of effective reactants added to the test paper decreases, and the color development tends to be nearly colorless, easily resulting in false negatives that misjudge the sample as over-cross-linked (as in test comparative examples 1 and 2). This indicates the need for strict control of the cross-linking process. While controlling the extraction volume and time to ensure sufficient extraction, it's crucial to pay attention to extraction time control. After a certain point, increasing the extraction time does not improve the extraction effect (as in Comparative Example 3). During color development, heating conditions significantly influence the success or failure. Insufficient temperature leads to incomplete color development, resulting in a pale yellow color that is difficult to determine (as in Comparative Example 4). Excessive temperature or prolonged heating can damage the system's stability or cause abnormal colors such as burnt yellow / grayish brown, making the colorimetric analysis impossible (as in Comparative Examples 5 and 6). Heating within a suitable window ensures a complete reaction and stable results. Overall, these results demonstrate that this study, through parameter window control, can achieve rapid, stable, and consistent on-site interpretation of the crosslinking state with objective performance indicators.

[0046] Example 2: Preparation of Standard Colorimetric Card Fresh bovine pericardium tissue was taken, cleaned, and cut into uniform sizes (2.0cm × 2.0cm). These were divided into four groups and immersed in 0.5% (w / v) glutaraldehyde solution, respectively, for cross-linking treatment at 4°C for 0 hours, 24 hours, 72 hours, and 168 hours. After cross-linking, the samples were rinsed thoroughly with deionized water to terminate the reaction and remove free glutaraldehyde. Small test pieces (5mm × 5mm) were cut from the four standard samples. The test pieces were placed in centrifuge tubes, 1.0 mL of PBS buffer was added, and the samples were extracted by shaking for 10 minutes. The extract was dropped onto the test paper prepared in Example 1 and heated in a 70°C oven for 2 minutes. The color of the test paper was recorded according to the determined cross-linking state, resulting in a standard colorimetric card. The correspondence between cross-linking state and color is shown in Table 2.

[0047] Ten bovine pericardium samples with different degrees of cross-linking were randomly selected and tested using the test strip from Example 1. The results were compared with the aforementioned standard colorimetric card. Samples deemed "cross-linking qualified" by the test strip all had a tensile strength (Ts) between 82 and 86°C and a tensile strength > 12 MPa. Samples deemed "insufficiently cross-linked" all had a Ts < 75°C and a tensile strength < 10 MPa, meeting the company's internal control standards. Overall, the results were consistent with the standard. The same sample was tested three times using the same method, and the results remained consistent throughout.

[0048] Test Example 1 (Test Sample) - To verify the accuracy of the established system and colorimetric card, the test sample on the production line was tested: Take a small piece (5mm × 5mm) of glutaraldehyde-crosslinked bovine pericardium tissue (e.g., an intermediate product from a production line) and blot dry with filter paper. Place it in a centrifuge tube, add 1.0 mL of PBS buffer, and vortex for 10 min. Drop the extract onto the test paper and heat in a 70°C oven for 2 minutes.

[0049] Test results:

[0050] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A rapid colorimetric detection method for the cross-linking state of biological tissues, characterized in that, Includes the following steps: The biological tissue to be tested is immersed in buffer solution to extract residual free amino groups. The supernatant is then dropped onto the colorimetric test paper and colorimetric analysis is performed to obtain the colorimetric result of the sample to be tested. The color development results of the sample to be tested are visually compared with the standard colorimetric card, and the cross-linking degree of the biological tissue to be tested is determined. The standard colorimetric card is established based on a series of standard biological tissue samples with known cross-linking states. The standard colorimetric card contains color levels corresponding to different cross-linking states and indicates the degree of cross-linking determination results corresponding to each color level.

2. The method according to claim 1, characterized in that, The colorimetric test paper includes a porous substrate and a colorimetric system loaded on the porous substrate, wherein the colorimetric system contains ninhydrin.

3. The method according to claim 2, characterized in that, The color development system is a system formed on the porous substrate by immersing the porous substrate in the color development system solution, taking it out and drying it. The composition of the color development system solution is: ninhydrin 5 g / L to 20 g / L, cosolvent 100 g / L to 300 g / L, stabilizer 1 g / L to 5 g / L, and buffer 20 g / L to 80 g / L.

4. The method according to claim 3, characterized in that, The co-solvent is selected from at least one of ethylene glycol, propylene glycol, and polyethylene glycol 400; The stabilizer is selected from at least one of sodium carboxymethyl cellulose, polyvinylpyrrolidone, and gum arabic. The buffer is selected from at least one of phosphate buffer pairs and borate buffer pairs.

5. The method according to any one of claims 2 to 4, characterized in that, The porous substrate is chromatography filter paper.

6. The method according to claim 1, characterized in that, The method for extracting residual free amino groups includes: The biological tissue to be tested was cut into a test piece; the biological tissue to be tested was bovine pericardial tissue or other bio-valve-related tissue. The buffer solution is 1-3 mL of PBS buffer; The soaking and extraction time is 5 to 10 minutes.

7. The method according to claim 1, characterized in that, The color development method is as follows: after adding the supernatant to the color development test paper, heat it at 60℃~90℃ for 1~10 min to complete the color development.

8. The method according to claim 1, characterized in that, The standard colorimetric card is established through the following steps: Standard biological tissue samples with different cross-linking states are provided, and the cross-linking state of each standard biological tissue sample is calibrated using heat shrinkage temperature and / or mechanical property indicators; Each standard biological tissue sample was soaked in buffer solution to extract residual free amino groups. The supernatant was then dropped onto colorimetric paper and colorimetric analysis was performed. The color development results of each standard biological tissue sample are matched one-to-one with their calibrated cross-linking states to form a standard colorimetric card with color levels, and the cross-linking degree judgment results corresponding to each color level are marked.

9. A kit for rapid colorimetric detection of cross-linking states in biological tissues, characterized in that, It includes colorimetric test strips and a standard colorimetric card, and contains instructions for guiding detection and interpretation, wherein the instructions are used to guide the execution of the method according to any one of claims 1 to 8 and output the crosslinking degree determination result.

10. The reagent kit according to claim 9, characterized in that, The colorimetric test paper is the colorimetric test paper according to any one of claims 2 to 5, and / or, The standard colorimetric card is a standard colorimetric card obtained by the method described in claim 8.

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