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CN-122016774-A - Biological tissue crosslinking state rapid colorimetric detection method and kit

CN122016774ACN 122016774 ACN122016774 ACN 122016774ACN-122016774-A

Abstract

A method and a kit for detecting the cross-linking state of biological tissues in a rapid color matching way. The method comprises the steps of preparing a developing system solution containing ninhydrin, dipping and drying a porous substrate to obtain developing test paper, providing standard biological tissue samples treated in different crosslinking states, calibrating the crosslinking states by adopting heat shrinkage temperature and/or mechanical property indexes, corresponding color results formed by the standard samples in the same developing mode as that of the samples to be tested to the crosslinking states, establishing a standard color chart with color gradation, soaking the biological tissue to be tested subjected to crosslinking treatment in buffer solution to extract residual free amino, taking supernatant, dripping the supernatant onto the developing test paper, heating for developing, visually comparing the supernatant with the standard color chart under the same illumination condition, and outputting a crosslinking degree judging result. The kit comprises color development test paper, a standard color 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 batch-to-batch consistency control.

Inventors

  • GONG LINGYAN
  • SHANG DAPENG
  • WEN XIANTAO
  • YU QIFENG

Assignees

  • 成都纽脉生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260210

Claims (10)

  1. 1. A rapid colorimetric detection method for a biological tissue crosslinking state is characterized by comprising the following steps: Adding biological tissues to be detected into a buffer solution for soaking to extract residual free amino groups, taking supernatant, dripping the supernatant onto a chromogenic test paper and developing color to obtain a chromogenic result of a sample to be detected; visually comparing the color development result of the sample to be detected with a standard color card, and outputting a crosslinking degree judgment result of the biological tissue to be detected; the standard colorimetric card is established based on a series of standard biological tissue samples with known crosslinking states, comprises color levels corresponding to different crosslinking states, and marks the crosslinking degree judging results corresponding to the color levels.
  2. 2. The method of claim 1, wherein the chromogenic test strip comprises a porous substrate and a chromogenic system supported on the porous substrate, the chromogenic system comprising ninhydrin.
  3. 3. The method according to claim 2, wherein the color development system is a system formed by immersing a porous substrate in a color development system solution, taking out and drying the porous substrate, and the color development system solution comprises ninhydrin 5 g/L to 20g/L, cosolvent 100 g/L to 300 g/L, stabilizer 1 g/L to 5 g/L, and buffer 20g/L to 80 g/L.
  4. 4. The method of claim 3, wherein the step of, The cosolvent is at least one selected from ethylene glycol, propylene glycol and polyethylene glycol 400; the stabilizer is at least one selected from sodium carboxymethyl cellulose, polyvinylpyrrolidone and gum arabic; the buffer is selected from at least one of phosphate buffer pair and borate buffer pair.
  5. 5. The method of any one of claims 2 to 4, wherein the porous substrate is a chromatographic filter paper.
  6. 6. The method according to claim 1, wherein the means for extracting residual free amino groups comprises: the biological tissue to be detected is cut into test pieces, and the biological tissue to be detected is bovine pericardium tissue or other biological valve related tissues; The buffer solution is 1-3 mL of PBS buffer solution; the soaking and extracting time is 5-10 min.
  7. 7. The method according to claim 1, wherein the color development is performed by heating the supernatant liquid at 60-90 ℃ for 1-10 min after the supernatant liquid is dripped onto the color development test paper.
  8. 8. The method of claim 1, wherein the standard color chart is established by: providing standard biological tissue samples with different crosslinking states, and calibrating the crosslinking states of the standard biological tissue samples by using heat shrinkage temperature and/or mechanical property indexes; adding each standard biological tissue sample into a buffer solution for soaking to extract residual free amino, taking supernatant, and dripping the supernatant onto a chromogenic test paper for chromogenic; And (3) the color development results of the standard biological tissue samples are in one-to-one correspondence with the calibrated crosslinking states of the standard biological tissue samples to form standard color cards with color levels, and the crosslinking degree judgment results corresponding to the color levels are marked.
  9. 9. A kit for rapid colorimetric detection of a crosslinked state of biological tissue, comprising a chromogenic test paper and a standard colorimetric card, and comprising instructions for directing the detection and interpretation, wherein the instructions are for directing the execution of the method of any one of claims 1 to 8 and outputting a crosslinking degree determination result.
  10. 10. The kit according to claim 9, wherein the chromogenic test strip is a chromogenic test strip according to any one of claims 2 to 5, and/or, The standard color chart is obtained by the method of claim 8.

Description

Biological tissue crosslinking state rapid colorimetric detection method and kit Technical Field The invention relates to the technical field of biological material detection and medical instrument production quality control, in particular to a colorimetric detection method for quickly judging the crosslinking degree of crosslinked biological tissues, and further relates to a kit for implementing the detection method. Background Glutaraldehyde crosslinking is a key process for treating biological tissues such as bovine pericardium to enhance stability in the manufacture of medical devices such as biological valves. The degree of crosslinking directly determines the mechanical properties, calcification resistance and long-term durability of the material. However, the quality control detection method for the crosslinking degree has the remarkable limitation that the traditional mechanical property test and the thermal shrinkage temperature measurement are destructive and time-consuming off-line detection methods, and the intermediate products in the production process cannot be rapidly and nondestructively screened. This results in hysteresis and dead zones in the control of the crosslinking process, and there may be quality risks that the performance of the whole batch of products is not up to standard due to insufficient or excessive crosslinking, resulting in resource waste and potential safety hazards. In the prior art, although some chemical detection methods (such as free amino detection) exist, the operation is complicated, professional equipment and technology are required, and the method is difficult to rapidly apply to the production line on site. At present, a rapid detection scheme which can instantly, intuitively and semi-quantitatively judge whether the crosslinking degree is qualified like a pH test paper is lacking. Disclosure of Invention The invention aims to solve the problems that the detection flow is time-consuming, a professional instrument is relied on, quick release is difficult in a production site, consistency judgment among batches is not visual and the like in the conventional biological tissue crosslinking degree detection, and provides a quick colorimetric detection method for the biological tissue crosslinking state. The method comprises the steps of preparing a color development test paper containing ninhydrin, establishing a standard colorimetric card formed by standard biological tissue samples in different crosslinking states, soaking the crosslinked biological tissue to be tested in a buffer solution to extract residual free amino, taking supernatant, dripping the supernatant onto the color development test paper to heat and develop color, visually comparing the color development result with the standard colorimetric card under the same illumination condition, and outputting a crosslinking degree judgment result of the biological tissue to be tested. According to a first aspect of the present invention, there is provided a rapid colorimetric detection method of a crosslinked state of biological tissue, comprising the steps of: Adding biological tissues to be detected into a buffer solution for soaking to extract residual free amino groups, taking supernatant, dripping the supernatant onto a chromogenic test paper and developing color to obtain a chromogenic result of a sample to be detected; visually comparing the color development result of the sample to be detected with a standard color card, and outputting a crosslinking degree judgment result of the biological tissue to be detected; the standard colorimetric card is established based on a series of standard biological tissue samples with known crosslinking states, comprises color levels corresponding to different crosslinking states, and marks the crosslinking degree judging results corresponding to the color levels. In some embodiments, the chromogenic test strip comprises a porous substrate and a chromogenic system supported on the porous substrate, the chromogenic system comprising ninhydrin. In some technical schemes, the color development system is a system formed by immersing a porous substrate in a color development system solution, taking out and drying the porous substrate, wherein the color development system solution comprises ninhydrin 5 g/L-20 g/L, cosolvent 100 g/L-300 g/L, stabilizer 1 g/L-5 g/L and buffer 20 g/L-80 g/L. Ninhydrin as core color developing agent can react with residual free amino extracted from supernatant to form characteristic color signal, and the free amino content difference is converted into visible color strength difference, so as to provide direct reading basis for grading judgment of crosslinking degree. The cosolvent is used for improving the solubility of the ninhydrin in the aqueous color development system, so that the ninhydrin can be uniformly dispersed in the preparation, impregnation and use processes, and crystallization precipitation caused by insufficient solubility is