Rapid qualitative and quantitative detection kit and detection method for collagen triple-helix structure

Abstract

The invention belongs to the technical field of biological material structure and performance analysis, and discloses a rapid qualitative and quantitative detection kit for a collagen triple-helix structure and a detection method. The detection kit comprises an enzymolysis system solution A of a non-triple helix structure of specific enzymolysis collagen; an ultrafiltration centrifugal tube; the enzymolysis system liquid B is used for performing non-specific enzymolysis on small-molecule polypeptide; colloidal gold test paper; the colloidal gold test paper is sequentially provided with a sample pad, a combination pad, an NC membrane and a water absorption pad from left to right; the NC membrane is sequentially coated with a T line and a C line from left to right; the marker on the combination pad is a gold-labeled antibody prepared from a hydroxyproline monoclonal antibody and colloidal gold particles through electrostatic coupling; and a coating substance on the T line is a hydroxyproline-carrier protein conjugate. The detection kit provided by the invention realizes a rapid, simple and good-specificity qualitative and quantitative detection method for the collagen triple-helix structure.

Description

Technical Field

[0001] This invention belongs to the field of biomaterial structure and performance analysis technology, and more specifically, relates to a rapid qualitative and quantitative detection kit and detection method for collagen triple helix structure. Background Technology

[0002] Collagen is the most abundant protein in animals, and its unique triple helix structure is fundamental to maintaining its biological functions (such as promoting cell proliferation and tissue repair) and mechanical properties. Therefore, accurate and rapid qualitative and even quantitative detection of the collagen triple helix structure is of vital practical significance in fields such as biomaterial preparation, tissue engineering, cosmetics, and food quality control.

[0003] Currently, methods for detecting the triple helix structure of collagen can be mainly divided into three categories: The first category is analytical methods based on large-scale instruments, such as circular dichroism chromatography, Fourier transform infrared spectroscopy, X-ray diffraction, and differential scanning calorimetry. These methods can provide detailed information on the secondary structure of collagen from different perspectives, but they generally have inherent drawbacks such as being cumbersome to operate, time-consuming, dependent on expensive specialized equipment, and requiring professional personnel to operate, making them difficult to apply to rapid screening and real-time monitoring in production sites, quality control processes, or clinical institutions.

[0004] The second category is rapid biochemical detection methods based on specific binding (rapid dye methods), aiming to address the pain points of the first category mentioned above. Among these, the reaction of dyes such as Sirius Red specifically binding to collagen under acidic conditions to form a precipitate has attracted much attention due to its speed and simplicity, leading to commercial kits such as Sircol and related improvement patents (e.g., CN110095421B). The core process of this type of method involves collecting the dye-collagen complex precipitate by centrifugation, washing, alkali resolution, and then performing spectrophotometric quantification. However, the centrifugation separation step has inherent limitations; it cannot effectively distinguish between natural active collagen with a complete triple helix structure and its denatured or hydrolyzed products (such as gelatin and hydrolyzed collagen). These collagen analogues (denatured or hydrolyzed products) and the tiny precipitates bound to the dye are difficult to completely remove during centrifugation and washing, resulting in severe interference with the quantitative results and preventing the specific detection of the collagen triple helix structure.

[0005] The third category is analytical methods based on enzymatic hydrolysis characteristics (existing enzymatic hydrolysis methods). These methods utilize the difference in resistance of collagen's triple-helix structure to specific proteases (such as trypsin) compared to the ease with which non-triple-helix structures are hydrolyzed to achieve detection (e.g., disclosed in CN108659117B and CN10988415B). The basic procedure is as follows: the sample to be tested is divided into two parts; one part is fully degraded with protease, and the other serves as a control. Subsequently, the content of collagen's triple-helix structure is calculated by measuring the change in the content of characteristic collagen amino acids (such as hydroxyproline) before and after enzymatic hydrolysis, or by analyzing the hydrolyzed fragments via electrophoresis. This method has high specificity and can effectively distinguish between triple-helix and non-triple-helix structures. However, its operation is extremely cumbersome, involving multiple steps such as enzymatic reaction, hydrolysis, derivatization, or electrophoresis. The detection time can range from several hours to tens of hours, and it also requires specialized laboratory equipment and skills, failing to meet the needs for rapid, on-site detection.

[0006] Therefore, developing a method for detecting the triple helix content of collagen in various physical states (such as solutions, gels, and membranes) that is easy to operate, has a rapid response, can specifically distinguish between triple helix and non-triple helix structures, and is applicable to various physical states of collagen (such as solutions, gels, and membranes) has become an urgent technical problem to be solved in this field. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing a rapid qualitative and quantitative detection kit and method for the triple helix structure of collagen. The detection kit of this invention enables a rapid, simple, and highly specific method for the qualitative and quantitative detection of the triple helix structure of collagen.

[0008] To achieve the above objectives, the present invention provides a rapid qualitative and quantitative detection kit for the triple helix structure of collagen, the detection kit comprising: Enzymatic hydrolysis system A for specific enzymatic hydrolysis of collagen with non-triple helix structure; Ultrafiltration centrifuge tubes; Enzymatic hydrolysis system B for non-specific enzymatic hydrolysis of small molecule peptides; Colloidal gold test paper; the colloidal gold test paper is provided with a sample pad, a conjugate pad, an NC membrane and an absorbent pad from left to right; the NC membrane is coated with a T line and a C line from left to right; The marker on the binding pad is a gold-labeled antibody prepared by electrostatic coupling of hydroxyproline monoclonal antibody and colloidal gold particles; The coating on the T-line is a hydroxyproline-carrier protein conjugate.

[0009] According to the present invention, preferably, the enzymatic hydrolysis system A for specifically hydrolyzing collagen non-triple helix structures is obtained by mixing an enzyme for specifically hydrolyzing collagen non-triple helix structures with a first buffer solution.

[0010] According to the present invention, preferably, the enzyme that specifically hydrolyzes the non-triple helix structure of collagen is at least one of pepsin, trypsin, figase and papain.

[0011] According to the present invention, preferably, the first buffer solution is at least one selected from glycine-hydrochloric acid, citrate-sodium citrate, acetic acid-sodium acetate, Tris-HCl, sodium dihydrogen phosphate-disodium hydrogen phosphate, 2-morpholine ethanesulfonic acid, 3-morpholine propanesulfonic acid, piperazine-N,N'-bis(2-ethanesulfonic acid), N-(2-acetamido)-2-aminoethanesulfonic acid, and triethanolamine-hydrochloric acid.

[0012] According to the present invention, preferably, the pH of the enzymatic hydrolysis system A for the specific enzymatic hydrolysis of collagen non-triple helix structure is 1-9.

[0013] According to the present invention, preferably, the enzymatic hydrolysis system B for non-specific enzymatic hydrolysis of small molecule polypeptides is obtained by mixing an enzyme for non-specific enzymatic hydrolysis of small molecule polypeptides and a second buffer solution.

[0014] According to the present invention, preferably, the enzyme for non-specific enzymatic hydrolysis of small molecule polypeptides is at least one selected from collagenase, pepsin, trypsin, figase, papain, proline endopeptidase and matrix metalloproteinase.

[0015] According to the present invention, preferably, the second buffer solution is at least one selected from glycine-hydrochloric acid, citrate-sodium citrate, acetic acid-sodium acetate, Tris-HCl, sodium dihydrogen phosphate-disodium hydrogen phosphate, 2-morpholine ethanesulfonic acid, 3-morpholine propanesulfonic acid, piperazine-N,N'-bis(2-ethanesulfonic acid), N-(2-acetamido)-2-aminoethanesulfonic acid, and triethanolamine-hydrochloric acid.

[0016] According to the present invention, preferably, the pH of the enzymatic hydrolysis system B for non-specific enzymatic hydrolysis of small molecule polypeptides is 1-9.

[0017] According to the present invention, preferably, the molecular weight cutoff of the ultrafiltration centrifuge tube is 3-100 kDa.

[0018] According to the present invention, preferably, the coating on the C line is at least one of goat anti-mouse IgG, rabbit anti-mouse IgG and staphylococcal A protein.

[0019] According to the present invention, preferably, the method for electrostatic coupling of the hydroxyproline monoclonal antibody with colloidal gold particles includes: incubating the hydroxyproline monoclonal antibody with colloidal gold particles to obtain a blend; mixing the blend with bovine serum albumin and centrifuging to collect the precipitate; resuspending the precipitate in a stabilizing solution to obtain the gold-labeled antibody; as a preferred embodiment, the method for electrostatic coupling of the hydroxyproline monoclonal antibody with colloidal gold particles includes: incubating the hydroxyproline monoclonal antibody with colloidal gold particles for 60 minutes to obtain a blend; mixing the blend with bovine serum albumin (final concentration 1%), using the bovine serum albumin to block the remaining sites on the surface of the colloidal gold particles, and then purifying by high-speed centrifugation to remove unbound antibodies and impurities, collecting the precipitate; resuspending the precipitate in a stabilizing solution to obtain the gold-labeled antibody, wherein the stabilizing solution is a borate buffer containing 1% bovine serum albumin, 0.05% NaN3, and 2% sucrose.

[0020] According to the present invention, preferably, the particle size of the colloidal gold particles is 15-80 nm.

[0021] According to the present invention, preferably, the coupling ratio of the hydroxyproline-carrier protein conjugate is (5-10):1.

[0022] According to the present invention, preferably, the carrier protein is at least one of bovine serum albumin, ovalbumin, and hemocyanin.

[0023] According to the present invention, preferably, the preparation method of the hydroxyproline-carrier protein conjugate includes the following steps: the preparation method of the hydroxyproline-carrier protein conjugate includes the following steps: mixing and stirring hydroxyproline, PBS buffer, EDC and NHS, and obtaining an activated hydroxyproline solution through a first reaction; adding the activated hydroxyproline solution dropwise to a PBS buffer containing a carrier protein, stirring, obtaining a product mixture system through a second reaction, and obtaining the hydroxyproline-carrier protein conjugate by dialysis; Preferably, the temperature of the first reaction is 25°C and the time is 15-30 min; Preferably, the temperature of the second reaction is 10°C and the time is 2-4 hours.

[0024] According to the present invention, preferably, the storage temperature of the test kit is ≤10℃.

[0025] According to the present invention, preferably, the test sample to be detected by the test kit is at least one of collagen solution, collagen gel, collagen sponge and collagen dressing.

[0026] Another aspect of the present invention provides a rapid qualitative and quantitative detection method for the triple helix structure of collagen. The detection method uses the aforementioned detection kit and includes the following steps: S1: Mix the sample to be tested with the specific enzymatic hydrolysis system A containing non-triple helix collagen by shaking, and perform the first enzymatic hydrolysis step to obtain a hydrolysate containing small molecule polypeptides. S2: The enzymatic hydrolysate containing small molecule polypeptides is subjected to ultrafiltration and centrifugation to obtain the filtrate; S3: The filtrate is mixed with the enzymatic hydrolysis system B of the non-specific enzymatic hydrolysis of small molecule peptides by shaking, and the second enzymatic hydrolysis is carried out to obtain the enzymatic hydrolysate containing free hydroxyproline; S4: Add the free hydroxyproline enzymatic hydrolysate to the sample pad of the colloidal gold test paper. After the colloidal gold test paper has developed color, perform qualitative analysis by visual inspection and / or semi-quantitative analysis using a colloidal gold reader.

[0027] In this invention: Step 1 Enzymatic hydrolysis: The sample to be tested is mixed with enzyme hydrolysis solution A and reacted. The part with the triple helix structure can resist enzymatic hydrolysis, while the part without the triple helix structure is hydrolyzed into small molecule peptides. Ultrafiltration separation: The enzymatic hydrolysate (containing small molecule polypeptide hydrolysate) from the first enzymatic hydrolysis step is transferred into an ultrafiltration centrifuge tube for ultrafiltration centrifugation. The part with the triple helix structure is retained and discarded, while the small molecule polypeptides produced in the first enzymatic hydrolysis step enter the filtrate and are collected, thus achieving effective separation of the target analyte (non-triple helix structure part) and the interfering analyte (triple helix structure part) detected by colloidal gold test strips. The second step is enzymatic hydrolysis: Enzymatic hydrolysis system B is added to the filtrate to further enzymatically hydrolyze the small molecule peptides in the filtrate, releasing a unified detection signal molecule—free hydroxyproline (from a non-triple helix structure). Colloidal gold test strip detection (using competitive inhibition method): A self-made colloidal gold test strip is used to perform qualitative and / or semi-quantitative analysis of hydroxyproline (from non-triple helix structures) in the enzymatic hydrolysate (containing free hydroxyproline hydrolysate) from the second enzymatic hydrolysis step. The semi-quantitative analysis includes: reading the T / C ratio using a portable colloidal gold reader, substituting it into a pre-established linear standard curve of triple helix structure content versus T / C ratio, to obtain the relative content of triple helix structures in the sample (the self-made colloidal gold test strip of this invention measures the specific free hydroxyproline content from non-triple helix structures).

[0028] According to the present invention, preferably, the temperature of the first enzymatic hydrolysis step is 10-30°C; the temperature of the second enzymatic hydrolysis step is 10-50°C; and the time for the first enzymatic hydrolysis step and the second enzymatic hydrolysis step are each independently 10-60 min.

[0029] In this invention, as a preferred embodiment, the oscillation mixing method for the first and second enzymatic hydrolysis steps can be manual oscillation, or oscillation and mixing can be assisted by mixing equipment such as a water bath shaker, a constant temperature incubator shaker, or a vortex shaker.

[0030] According to the present invention, preferably, the centrifugal force of the ultrafiltration centrifugation is 1000-8000g, the centrifugation temperature is 3-30℃, and the centrifugation time is 8-30min.

[0031] According to the present invention, preferably, the colloidal gold test strip employs a competitive inhibition method; the detection environment temperature in step S4 is 4-40℃; and the time taken from adding the free hydroxyproline enzyme hydrolysate to the sample pad of the colloidal gold test strip to the completion of color development of the colloidal gold test strip is 5-20 minutes.

[0032] The qualitative analysis principle of the self-made colloidal gold test strip of the present invention is as follows: Positive result (non-triple helix structure in the sample): The test solution (enzymatic digest from the second step) contains a large amount of hydroxyproline, and all the gold-labeled antibodies in the conjugate pad are bound. When passing through the T line, there are no remaining gold-labeled antibodies that can bind to the hydroxyproline-carrier protein conjugate, so the T line does not show color; the gold-labeled antibodies that bind to hydroxyproline are captured by goat anti-mouse IgG / rabbit anti-mouse IgG at the C line, so the C line shows normal color. Weak positive result (non-triple helix structure in the sample): The test solution contains a certain amount of hydroxyproline. Some of the gold-labeled antibody in the conjugate pad is partially bound. When passing through the T line, the remaining gold-labeled antibody can bind to the hydroxyproline-carrier protein conjugate, so the T line appears light-colored. Both the gold-labeled antibody bound to hydroxyproline and the gold-labeled antibody not bound to hydroxyproline can be captured by the goat anti-mouse IgG / rabbit anti-mouse IgG of the C line, so the C line shows normal color. Negative result (no non-triple helix structure in the sample): The test solution does not contain hydroxyproline, and the gold-labeled antibodies in the conjugate pad are not bound. When passing through the T line, a large number of gold-labeled antibodies bind to the hydroxyproline-carrier protein conjugate, so the T line appears dark. The gold-labeled antibodies that do not bind to hydroxyproline can also be captured by goat anti-mouse IgG / rabbit anti-mouse IgG at the C line, so the C line appears normally.

[0033] The process of establishing the linear standard curve of the triple helix structure content and the T / C ratio is as follows: A bovine Achilles tendon type I collagen solution (concentration 2.0 mg / mL) with a complete triple helix structure was used as a standard with 100% triple helix structure content. A sample with the same concentration but completely destroyed by heat treatment at 80°C for 20 minutes was used as a standard with 0% triple helix structure content. The two standards (100% and 0%) were mixed in volume ratio to prepare a series of standards with triple helix structure contents of 0%, 10%, 20%, 50%, 80%, and 100% (the total collagen concentration of all samples was kept constant at 2.0 mg / mL). Using the detection kit and method of Example 1 of this invention, the corresponding T / C ratio was obtained using a colloidal gold reader. A linear fit was performed between the triple helix structure content (y, %) and the T / C ratio (x) (which showed a significant positive correlation). The linear standard curve equation for the triple helix structure content and the T / C ratio was y = 127.18x - 19.87, with a correlation coefficient R0. 2 =0.993. More preferably, since theoretically, the T / C ratio (x) and the triple helix structure content (y) have an exponential function relationship, in order to obtain more accurate quantitative results, a four-parameter Sigmoid model can be used to fit the triple helix structure content (y, %) and the T / C ratio (x) to obtain a nonlinear standard curve.

[0034] The beneficial effects of the technical solution of this invention are as follows: The detection kit of this invention provides a rapid, simple, and highly specific method for the qualitative and quantitative detection of the triple helix structure of collagen. The detection kit of this invention overcomes the technical shortcomings of existing enzymatic digestion methods, which are cumbersome and time-consuming, and rapid dye methods, which are easily interfered with by collagen analogs (denaturing or hydrolyzing products) and have difficulty specifically reflecting the triple helix structure content. Specifically: First, this invention significantly accelerates the separation and acquisition of specific free hydroxyproline from non-triple helical structures by coupling ultrafiltration centrifugation technology with a stepwise enzymatic hydrolysis strategy, greatly shortening the traditional process that takes several hours to tens of hours. Secondly, this invention utilizes self-made colloidal gold test paper for rapid and highly sensitive visual detection of free hydroxyproline, replacing traditional hydroxyproline quantification methods (such as chromatography and colorimetry) that require large instruments or long-term derivatization, thus enabling rapid reading and semi-quantitative analysis of the detection signal. In summary, this invention organically combines the aforementioned "ultrafiltration centrifugation technology coupled with stepwise enzymatic hydrolysis and self-made colloidal gold test strips" to establish a new method integrating rapid sample pretreatment and rapid detection. This method ensures a clear distinction between non-triple-helix and triple-helix structures (i.e., this invention detects collagen based on the biological nature of the triple-helix structure (protease resistance), effectively distinguishing active collagen from collagen analogs (denatured or hydrolyzed products)). It achieves rapid qualitative assessment of the integrity of the collagen triple-helix structure (typically completed within 60 minutes, significantly shortening the detection time) and on-site, rapid, and semi-quantitative evaluation of the collagen triple-helix structure content. The results are accurate and reliable, and the operation is simple, requiring no large instruments. It is particularly suitable for rapid on-site screening and quality control, meeting the urgent need for rapid determination of collagen bioactivity in production quality control, raw material acceptance, and other scenarios.

[0035] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0036] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0037] Figure 1 The diagram shows a flowchart of a rapid qualitative and quantitative detection method for the triple helix structure of collagen provided in Embodiment 1 of the present invention.

[0038] Figure 2 The diagram shows a schematic diagram of the colloidal gold test strip and a schematic diagram of the qualitative analysis principle of a rapid qualitative and quantitative detection kit for the triple helix structure of collagen provided by the present invention.

[0039] Figure 3 The linear standard curve of the triple helix structure content and T / C ratio established in this invention is shown.

[0040] Figure 4 The circular dichroism (CD) curves (“CD(mdeg), milliseconds”) of the test samples with different triple helix structure contents obtained by circular dichroism chromatography in Comparative Example 1 of this invention are shown; from Figure 4 As the content of the triple helix structure in the sample decreases, the signal value of the circular dichroism chromatogram at 220 nm gradually decreases.

[0041] Figure 5 The results of linear regression analysis of the T / C ratio (x) of Example 17 of the present invention and the signal value (y) of the circular dichroism chromatogram of Comparative Example 1 at 220 nm are shown.

[0042] Figure 6 The standard curve of absorbance at 558 nm wavelength versus hydroxyproline content for Comparative Example 3 of the present invention is shown. Detailed Implementation

[0043] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0044] In the following embodiments and comparative examples: The hydroxyproline monoclonal antibody was purchased from Creative Biolabs (CBP4797). Colloidal gold particles, prepared by reducing chloroauric acid with trisodium citrate, have a particle size of 15-80 nm. Hydroxyproline was purchased from Aladdin and was ≥99%; Type I collagen from bovine Achilles tendon is obtained by acid-enzyme extraction and freeze-drying from bovine Achilles tendon. Fish scale type I collagen is obtained by extracting grass carp scales using an acid-enzyme method and then freeze-drying. Type I collagen from pig skin is obtained by extracting pig skin using an acid-enzyme method and then freeze-drying it. Human placental type IV collagen, purchased from Sigma-Aldrich (C8374). Recombinant human type III collagen was obtained by recombinantly introducing the optimized human type III collagen cDNA gene into Pichia pastoris engineered strains, followed by fermentation expression, extraction, and purification. Pigskin gelatin is obtained by extracting pigskin through an alkaline method and then vacuum drying. The solution form of the above-mentioned collagen was obtained by dissolving collagen in a 0.1M acetic acid solution; The above-mentioned sponge-like collagen was obtained by injecting a collagen solution that had been fully dialyzed with pure water into a stainless steel mold and then freeze-drying it.

[0045] Example 1

[0046] This embodiment provides a rapid qualitative and quantitative detection kit for the triple helix structure of collagen, the detection kit comprising: Enzymatic hydrolysis system A for specific collagen non-triple helix structure (a phosphate buffer containing papain, with a papain concentration of 0.5 mg / mL, a total concentration of sodium dihydrogen phosphate and disodium hydrogen phosphate of 0.1 M, and a pH of 6.0). Ultrafiltration centrifuge tubes (molecular weight cutoff of 10 kDa); Enzymatic hydrolysis system B for non-specific enzymatic hydrolysis of small molecule peptides (is a Tris-HCl buffer containing collagenase and trypsin, with a collagenase concentration of 0.5 mg / mL, a trypsin concentration of 0.2 mg / mL, a total concentration of the buffer pairs in the Tris-HCl buffer of 0.1 M, and a pH of 7.4). Colloidal gold test paper; the colloidal gold test paper is provided with a sample pad, a conjugation pad, an NC membrane (nitrocellulose membrane) and an absorbent pad from left to right; the NC membrane is coated with a T line and a C line from left to right; The marker on the binding pad is a gold-labeled antibody prepared by electrostatic coupling of hydroxyproline monoclonal antibody and colloidal gold particles. The method for electrostatic coupling of hydroxyproline monoclonal antibody and colloidal gold particles includes: incubating the hydroxyproline monoclonal antibody and colloidal gold particles together for 60 minutes to obtain a blend; mixing the blend with bovine serum albumin (final concentration of 1%) to block the remaining sites on the surface of colloidal gold particles, and then purifying by high-speed centrifugation to remove unbound antibody and impurities, and collecting the precipitate; resuspending the precipitate in a stabilizing solution to obtain the gold-labeled antibody, wherein the stabilizing solution is a borate buffer containing 1% bovine serum albumin, 0.05% NaN3, and 2% sucrose. The coating on the T-line is a hydroxyproline-bovine serum albumin conjugate with a coupling ratio of 7:1. The preparation method of the hydroxyproline-bovine serum albumin conjugate includes the following steps: hydroxyproline is dissolved in PBS buffer, and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) are added sequentially. The mixture is stirred gently at room temperature for 20 minutes to obtain an activated hydroxyproline solution. The activated hydroxyproline solution is added dropwise to PBS buffer containing bovine serum albumin, and the mixture is stirred slowly at 10°C for 3 hours to obtain a product mixture. The product mixture is dialyzed thoroughly with PBS buffer at 10°C for 3 days to completely remove unreacted small molecules and impurities, thus obtaining the hydroxyproline-bovine serum albumin conjugate. The coating material on line C is goat anti-mouse IgG; The test kit should be stored at a temperature of ≤10℃.

[0047] This embodiment also provides a rapid qualitative and quantitative detection method for the triple helix structure of collagen. The detection method uses the detection kit of this embodiment and includes the following steps: S1: 0.5 mL of bovine Achilles tendon type I collagen solution sample (solvent is 0.1 mol / L acetic acid aqueous solution, concentration 2.0 mg / mL, known triple helix structure content is 86.5%, this example is to verify the accuracy of the method of the present invention) and 0.5 mL of specific enzymatic hydrolysis system solution A for non-triple helix structure collagen were vortexed and mixed, and then placed in a 25℃ water bath shaker for 15 minutes to complete the first step of enzymatic hydrolysis, and obtained enzymatic hydrolysate containing small molecule polypeptides; S2: Using the above-mentioned ultrafiltration centrifuge tube, the enzymatic hydrolysate containing small molecule polypeptides is subjected to ultrafiltration centrifugation at 4°C and 5000g centrifugation force for 10 minutes to obtain the filtrate; S3: The filtrate is mixed with an equal volume of non-specific enzymatic hydrolysis system B for small molecule peptides by vortexing, and then placed in a 37°C constant temperature shaker for 15 minutes to complete the second enzymatic hydrolysis and obtain an enzymatic hydrolysate containing free hydroxyproline. S4: Using a pipette, draw 150 μL of the enzyme digest containing free hydroxyproline and add it vertically to the sample pad of the colloidal gold test paper. Then, allow it to stand at room temperature (25°C) for 15 minutes. The T line will show a light color, and the C line will show normal color. This is visually identified as a weak positive result. Using a colloidal gold reader, read the T / C ratio (the ratio of the signal intensity of the detection line (T line) to the control line (C line). Substitute this value into the linear standard curve of the triple helix structure content versus the T / C ratio to obtain a relative triple helix structure content of 87.2% in the 0.5 mL bovine Achilles tendon type I collagen solution sample obtained by the method in Example 1.

[0048] Example 2

[0049] This embodiment provides a rapid qualitative and quantitative detection kit for the triple helix structure of collagen, the detection kit comprising: Enzymatic hydrolysis system A for specific collagen non-triple helix structures (is an acetate-sodium acetate buffer containing pepsin, the concentration of pepsin in hydrolysis system A is 1 mg / mL, the sum of the concentrations of acetic acid and sodium acetate in the acetate-sodium acetate buffer is 0.1 M, and the pH of hydrolysis system A is 2.5). Ultrafiltration centrifuge tubes (molecular weight cutoff of 30kDa); Enzymatic hydrolysis system B for nonspecific enzymatic hydrolysis of small molecule peptides (MES buffer (2-morpholinoethanesulfonic acid) containing matrix metalloproteinase MMP-1 and papain, MMP-1 concentration in hydrolysis system B is 1.0 mg / mL, papain concentration in hydrolysis system B is 2 mg / mL, MES buffer concentration is 0.05 M, pH of hydrolysis system B is 6.5). Colloidal gold test paper; the colloidal gold test paper is provided with a sample pad, a conjugation pad, an NC membrane (nitrocellulose membrane) and an absorbent pad from left to right; the NC membrane is coated with a T line and a C line from left to right; The markings on the conjoint pad are the same as those in Example 1; The coating on the T-line is a hydroxyproline-ovalbumin conjugate with a coupling ratio of 8:1. The preparation method of the hydroxyproline-ovalbumin conjugate includes the following steps: hydroxyproline is dissolved in PBS buffer, and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) are added sequentially. The mixture is stirred gently at room temperature for 30 minutes to obtain an activated hydroxyproline solution. The activated hydroxyproline solution is added dropwise to PBS buffer containing ovalbumin, and the mixture is stirred slowly at 10°C for 4 hours to obtain a product mixture. The product mixture is dialyzed thoroughly with PBS buffer at 10°C for 3 days to completely remove unreacted small molecules and impurities, thus obtaining the hydroxyproline-ovalbumin conjugate. The coating material on line C is rabbit anti-mouse IgG; The test kit should be stored at a temperature of ≤10℃.

[0050] This embodiment also provides a rapid qualitative and quantitative detection method for the triple helix structure of collagen. The detection method uses the detection kit of this embodiment and includes the following steps: S1: Mix 2 mg of fish scale type I collagen sponge sample with 1 mL of specific enzymatic hydrolysis system A for collagen non-triple helix structure by manual vigorous shaking, and then place it in a 15℃ environment for 20 minutes to complete the first step of enzymatic hydrolysis, and obtain enzymatic hydrolysate containing small molecule polypeptides. S2: Using the above-mentioned ultrafiltration centrifuge tube, the enzymatic hydrolysate containing small molecule polypeptides is subjected to ultrafiltration centrifugation at 25°C and 3000g centrifugation force for 10 minutes to obtain the filtrate; S3: The filtrate is mixed with an equal volume of non-specific enzymatic hydrolysis system B for small molecule peptides by vortexing, and then placed in a 25°C water bath for 15 minutes to complete the second enzymatic hydrolysis, yielding an enzymatic hydrolysate containing free hydroxyproline. S4: Using a pipette, 100 μL of enzyme hydrolysate containing free hydroxyproline is vertically added to the sample pad of the colloidal gold test paper. The sample is then allowed to stand at 20°C for 10 minutes. The C line develops normally, and the T line develops a color similar to the C line, showing obvious negative reaction characteristics. By visual qualitative judgment, the sponge sample in this embodiment has a highly intact triple helix structure.

[0051] Example 3

[0052] This embodiment provides a rapid qualitative and quantitative detection kit for the triple helix structure of collagen, the detection kit comprising: Enzymatic hydrolysis system A for specific enzymatic hydrolysis of collagen with non-triple helix structure (same as in Example 2); Ultrafiltration centrifuge tubes (molecular weight cutoff of 10 kDa); Enzymatic hydrolysis system B for non-specific enzymatic hydrolysis of small molecule peptides (same as in Example 1); The colloidal gold test strip is the same as in Example 1; The test kit should be stored at a temperature of ≤10℃.

[0053] This embodiment also provides a rapid qualitative and quantitative detection method for the triple helix structure of collagen. The detection method uses the detection kit of this embodiment and includes the following steps: S1: Mix 2 mg of human placental type IV collagen with 1 mL of specific enzymatic hydrolysis system A containing non-triple helix collagen by manual vigorous shaking, and then place it in a 25°C environment for 30 minutes to complete the first step of enzymatic hydrolysis, and obtain a hydrolysate containing small molecule polypeptides. S2: Using the above-mentioned ultrafiltration centrifuge tube, the enzymatic hydrolysate containing small molecule polypeptides is subjected to ultrafiltration centrifugation at 4°C and 5000g centrifugation force for 15 minutes to obtain the filtrate; S3: The filtrate is mixed with an equal volume of non-specific enzymatic hydrolysis system B for small molecule peptides by vortexing, and then placed in a 37°C water bath for 20 minutes to complete the second enzymatic hydrolysis, yielding an enzymatic hydrolysate containing free hydroxyproline. S4: Using a pipette, draw 150 μL of enzyme digest containing free hydroxyproline and add it vertically to the sample pad of the colloidal gold test paper. Then, let it stand at 20°C for 10 minutes. The C line develops normally, and the T line develops color similarly to the C line, showing obvious negative reaction characteristics.

[0054] Example 4

[0055] The only difference between this embodiment and Example 3 is that the sample to be tested is recombinant human type III collagen; The C line shows normal color, while the T line shows a significantly lighter color than the C line, which is identified as a weak positive result by visual inspection.

[0056] Example 5

[0057] The only difference between this embodiment and Embodiment 3 is that the sample to be tested is pigskin gelatin; The C line shows normal color, while the T line shows almost no color, indicating a positive result by visual inspection.

[0058] The results of Examples 3-5 demonstrate that the detection kit and detection method of the present invention are effective for collagen samples from different sources and can accurately distinguish between collagen with a natural triple helix structure and collagen analogs without a triple helix structure.

[0059] Examples 6-11

[0060] The only difference between Example 6 and Example 2 is that "2mg fish scale type I collagen sponge sample" is replaced with "2mg bovine Achilles tendon type I collagen sponge sample"; and the temperature of the first enzymatic hydrolysis step is 4℃.

[0061] The only difference between Example 7 and Example 6 is that the temperature of the first step of enzymatic hydrolysis is 10°C.

[0062] The only difference between Example 8 and Example 6 is that the temperature of the first step of enzymatic hydrolysis is 20°C.

[0063] The only difference between Example 9 and Example 6 is that the temperature of the first step of enzymatic hydrolysis is 30°C.

[0064] The only difference between Example 10 and Example 6 is that the temperature of the first enzymatic hydrolysis step is 40°C.

[0065] The only difference between Example 11 and Example 6 is that the temperature of the first enzymatic hydrolysis step is 60°C.

[0066] The results of Examples 6-11 show that: At 4℃, the enzyme activity is insufficient, and the first step of enzymatic hydrolysis is incomplete, resulting in a darker T-line color in the final product. Within the temperature range of 10-30℃, the T line shows stable color development and is comparable to the C line, exhibiting a typical negative reaction; When the temperature rises to 40℃ and 60℃, the high temperature causes the triple helix structure of collagen to denature, lose its specific resistance to proteases, and the first step of enzymatic digestion is overdone, ultimately making the T line color indistinguishable from the positive sample. Examples 6-11 demonstrate that the first-step enzymatic hydrolysis temperature range of 10-30℃ can effectively protect the integrity of the triple helix structure and ensure the specificity of enzymatic hydrolysis, providing experimental basis for limiting the temperature parameters in the detection method of this invention.

[0067] Examples 12-16

[0068] The only difference between Example 12 and Example 1 is that the concentration of bovine Achilles tendon type I collagen solution sample is 5.0 mg / mL, which contains 5% (w / v) glycerol as an interfering agent.

[0069] The only difference between Example 13 and Example 1 is that the concentration of bovine Achilles tendon type I collagen solution sample is 5.0 mg / mL, which contains 2% (w / v) sodium hyaluronate as an interfering agent.

[0070] The only difference between Example 14 and Example 1 is that the concentration of bovine Achilles tendon type I collagen solution sample is 5.0 mg / mL, which contains 1% (w / v) carbomer as an interfering agent.

[0071] The only difference between Example 15 and Example 1 is that the concentration of bovine Achilles tendon type I collagen solution sample is 5.0 mg / mL, which contains 1% (w / v) triethanolamine as an interfering agent.

[0072] The only difference between Example 16 and Example 1 is that the concentration of the bovine Achilles tendon type I collagen solution sample is 5.0 mg / mL.

[0073] The results of Examples 12-16 show that: The test samples with added glycerol, sodium hyaluronate and carbomer showed the same test results as the blank control (Example 16), and the T-line color development was consistent with the negative characteristics. The T line of the test sample with added triethanolamine showed a significantly lighter color, indicating a false positive trend; Examples 12-16 demonstrate that, except for strongly alkaline additives, common cosmetic ingredients do not significantly interfere with the detection method of the present invention, verifying the reliability of the detection method of the present invention in actual sample testing.

[0074] Example 17

[0075] A porcine skin type I collagen solution with an intact triple helix structure (concentration 3.0 mg / mL) was used as a standard with 100% triple helix structure content. A sample with the same concentration but completely destroyed by heat treatment at 80°C for 20 minutes was used as a standard with 0% triple helix structure content. The two standards (100% and 0%) were mixed in volume ratio to prepare test samples with triple helix structure contents of 0%, 10%, 20%, 50%, 80%, and 100% (the total collagen concentration of all samples was kept constant at 3.0 mg / mL).

[0076] The detection kit and detection method of Example 1 of this invention were used to detect the test samples with triple helix structure contents of 0%, 10%, 20%, 50%, 80%, and 100%, respectively, and the corresponding T / C ratios were obtained by colloidal gold reader.

[0077] Comparative Example 1

[0078] The triple helix structure contents of the test samples of Example 17 were determined in parallel using circular dichroism spectroscopy, with contents of 0%, 10%, 20%, 50%, 80%, and 100%, respectively.

[0079] The signal value at 220 nm was measured by circular dichroism spectroscopy. The signal value of the test sample with 100% triple helix structure content was 25.50, the signal value of the test sample with 0% triple helix structure content was 0.10, and the signal value of the test sample with intermediate proportions showed a linear change.

[0080] Linear regression analysis was performed on the T / C ratio (x) of Example 17 and the circular dichroism chromatogram signal value (y) of Comparative Example 1, yielding the regression equation: y = 28.09x - 2.50 (R² - 2.50²). 2 =0.997). The regression equation shows a significant positive correlation between the T / C ratio and the circular dichroism chromatographic signal value. As the content of triple helix structure in the sample decreases, the signal value of the circular dichroism chromatogram at 220 nm decreases linearly (from 25.50 to 0.10), while the T / C ratio of the colloidal gold test strip also gradually decreases (from 0.98 to 0.08). This proves that the detection method of the present invention can accurately and semi-quantitatively reflect the integrity of the triple helix structure of collagen, and is highly consistent with the results of the circular dichroism chromatographic method.

[0081] Comparative Example 2

[0082] This comparative example also provides a method for detecting the triple helix structure of collagen, the method comprising the following steps: S1: Mix 0.5 mL of bovine Achilles tendon type I collagen solution sample (same as in Example 1) with 2 mL of non-specific enzymatic hydrolysis small molecule polypeptide hydrolysis system B (same as in Example 1) by vortexing, and then place it in a 37°C constant temperature incubator for 30 minutes to complete the enzymatic hydrolysis and obtain the comparative enzymatic hydrolysate. S2: Using a pipette, 150 μL of the comparative enzymatic digestion solution was vertically added to the sample pad of the colloidal gold test paper (the same as in Example 1). The mixture was then allowed to stand at room temperature (25°C) for 15 minutes. The T line showed almost no color, while the C line showed normal color, similar to the color development result of pigskin gelatin. The result was identified as positive by visual inspection.

[0083] The comparison of the results of Example 1 and Comparative Example 2 shows that, without the first step of enzymatic hydrolysis and ultrafiltration separation, collagen with an intact triple helix structure is completely degraded by the enzymatic hydrolysis system solution B, making it impossible to distinguish from collagen analogs (such as gelatin) that have lost their triple helix structure, resulting in false positive results. This proves the necessity of the first step of enzymatic hydrolysis and ultrafiltration in this invention for protecting active collagen and achieving specific detection.

[0084] Comparative Example 3

[0085] Weigh 2 mg of the same sample as in Example 2, but do not use the detection method of the present invention. Instead, use the traditional hydrochloric acid hydrolysis-hydroxyproline chemical colorimetric method for determination.

[0086] The specific procedure is as follows: Place the sample to be tested in an ampoule containing 6M HCl and hydrolyze it in an oven at 110℃ for 16 hours. After neutralizing and filtering the hydrolysate, treat it using chloramine-T oxidation and the colorimetric reaction of p-dimethylaminobenzaldehyde. Finally, measure the absorbance at 558nm using a UV-Vis spectrophotometer and analyze it using a standard curve. Figure 6 Calculate the total hydroxyproline content of the sample to be tested, and then estimate the total collagen content.

[0087] The entire process took more than 18 hours and could not distinguish between active collagen with a triple helix structure and denatured collagen such as gelatin. This comparative example 3 demonstrates that although traditional chemical colorimetric methods can be used to determine total collagen content, they have inherent defects such as extremely cumbersome procedures, extremely long processing times, and complete inability to reflect the integrity of the collagen triple helix structure. This highlights the significant advancement of this invention in rapidly and specifically assessing the structural integrity of collagen.

[0088] Comparative Example 4

[0089] Take 0.5 mL of the same bovine Achilles tendon type I collagen solution sample as in Example 1, dissolve it in 5 mL of 0.5 M acetic acid aqueous solution, and adjust the pH to 3.7 with 0.5 M NaOH aqueous solution to obtain a mixed system. Add 1 mL of trypsin solution (the mass ratio of trypsin to the total protein in the bovine Achilles tendon type I collagen solution sample is 1:40) to the mixed system, mix well, and obtain the enzymatic hydrolysis system solution.

[0090] Take 2 mL of the enzymatic hydrolysis system solution, add 5 mL of 8.4 M hydrochloric acid aqueous solution, and hydrolyze at 110 °C for 24 hours to obtain the hydrolysate. The hydroxyproline content in the hydrolysate is determined by an amino acid analyzer to obtain the total hydroxyproline content before enzymatic hydrolysis.

[0091] The remaining enzymatic hydrolysis solution was placed in a 25℃ constant temperature shaker and enzymatically hydrolyzed for 4 hours. After enzymatic hydrolysis, 2 mL of the hydrolysate was taken, and 1 mL of 30% trichloroacetic acid solution pre-cooled at 4℃ was added. After shaking to mix, the solution was centrifuged at 5000g for 10 minutes at 4℃. The supernatant was discarded, and the precipitate was washed three times with 10% trichloroacetic acid solution pre-cooled at 4℃. 5 mL of 6M hydrochloric acid aqueous solution was added to the precipitate, and the solution was hydrolyzed at 110℃ for 24 hours. The hydroxyproline content in the hydrolysate was determined using an amino acid analyzer, and the triple helix structure content in the comparative sample was calculated.

[0092] Calculation formula: NC = X 2 / X 1 ×100%; In the formula NC —The content of collagen triple helix structure in the comparative sample, expressed in %; X 1—The content of hydroxyproline in the enzymatic hydrolysis system before enzymatic hydrolysis. X 2—The content of hydroxyproline in the enzymatic hydrolysis system after enzymatic hydrolysis.

[0093] The comparative method ultimately determined the triple helix structure content in the sample to be 85.5%, which is similar to the result of Example 1 of this invention. However, the entire detection process of this comparative method takes more than 50 hours and involves multiple acid-base adjustments, prolonged high-temperature hydrolysis, and analysis with large instruments. The operation is cumbersome and the reagent consumption is large, which cannot meet the needs of rapid detection. This comparative example 4 demonstrates that although the existing enzymatic hydrolysis-precipitation method can accurately determine the triple helix structure content, it has obvious drawbacks such as complex procedures and extremely long time consumption. This further highlights the great advantage of the present invention in combining ultrafiltration coupled with enzymatic hydrolysis and colloidal gold rapid detection technology in terms of detection efficiency.

[0094] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A rapid qualitative and quantitative detection kit for the triple helix structure of collagen, characterized in that, The detection kit includes: Enzymatic hydrolysis system A for specific enzymatic hydrolysis of collagen with non-triple helix structure; Ultrafiltration centrifuge tubes; Enzymatic hydrolysis system B for non-specific enzymatic hydrolysis of small molecule peptides; Colloidal gold test paper; the colloidal gold test paper is provided with a sample pad, a conjugate pad, an NC membrane and an absorbent pad from left to right; the NC membrane is coated with a T line and a C line from left to right; The marker on the binding pad is a gold-labeled antibody prepared by electrostatic coupling of hydroxyproline monoclonal antibody and colloidal gold particles; The coating on the T-line is a hydroxyproline-carrier protein conjugate.

2. The rapid qualitative and quantitative detection kit for the triple helix structure of collagen according to claim 1, wherein, The specific enzymatic hydrolysis system A for non-triple helix collagen is obtained by mixing an enzyme that specifically hydrolyzes non-triple helix collagen with a first buffer solution. The enzyme that specifically hydrolyzes non-triple-helix collagen is at least one of pepsin, trypsin, figase, and papain. The first buffer solution is at least one of glycine-hydrochloric acid, citrate-sodium citrate, acetic acid-sodium acetate, Tris-HCl, sodium dihydrogen phosphate-disodium hydrogen phosphate, 2-morpholine ethanesulfonic acid, 3-morpholine propanesulfonic acid, piperazine-N,N'-bis(2-ethanesulfonic acid), N-(2-acetamido)-2-aminoethanesulfonic acid, and triethanolamine-hydrochloric acid; The pH of the specific enzymatic hydrolysis system A for collagen with a non-triple helix structure is 1-9.

3. The rapid qualitative and quantitative detection kit for the triple helix structure of collagen according to claim 1, wherein, The enzymatic hydrolysis system B for non-specific enzymatic hydrolysis of small molecule peptides is obtained by mixing the enzyme for non-specific enzymatic hydrolysis of small molecule peptides and the second buffer solution. The enzyme used for non-specific enzymatic hydrolysis of small molecule polypeptides is at least one of collagenase, pepsin, trypsin, figase, papain, proline endopeptidase, and matrix metalloproteinase. The second buffer solution is at least one of glycine-hydrochloric acid, citrate-sodium citrate, acetic acid-sodium acetate, Tris-HCl, sodium dihydrogen phosphate-disodium hydrogen phosphate, 2-morpholine ethanesulfonic acid, 3-morpholine propanesulfonic acid, piperazine-N,N'-bis(2-ethanesulfonic acid), N-(2-acetamido)-2-aminoethanesulfonic acid, and triethanolamine-hydrochloric acid; The pH of the enzymatic hydrolysis system B for non-specific enzymatic hydrolysis of small molecule peptides is 1-9.

4. The rapid qualitative and quantitative detection kit for the triple helix structure of collagen according to claim 1, wherein, The ultrafiltration centrifuge tube has a molecular weight cutoff of 3-100 kDa.

5. The rapid qualitative and quantitative detection kit for the triple helix structure of collagen according to claim 1, wherein, The coating material on the C line is at least one of goat anti-mouse IgG, rabbit anti-mouse IgG, and staphylococcal A protein.

6. The rapid qualitative and quantitative detection kit for the triple helix structure of collagen according to claim 1, wherein, The method for electrostatic coupling of the hydroxyproline monoclonal antibody with colloidal gold particles includes: incubating the hydroxyproline monoclonal antibody with colloidal gold particles to obtain a blend; mixing the blend with bovine serum albumin and centrifuging to collect the precipitate; and resuspending the precipitate in a stabilizing solution to obtain the gold-labeled antibody. The colloidal gold particles have a particle size of 15-80 nm.

7. The rapid qualitative and quantitative detection kit for the triple helix structure of collagen according to claim 1, wherein, The coupling ratio of the hydroxyproline-carrier protein conjugate is (5-10):1; The carrier protein is at least one of bovine serum albumin, ovalbumin, and hemocyanin; The preparation method of the hydroxyproline-carrier protein conjugate includes the following steps: mixing and stirring hydroxyproline, PBS buffer, EDC and NHS, and obtaining an activated hydroxyproline solution through a first reaction; adding the activated hydroxyproline solution dropwise to a PBS buffer containing the carrier protein, stirring, and obtaining a product mixture system through a second reaction; and obtaining the hydroxyproline-carrier protein conjugate by dialysis.

8. The rapid qualitative and quantitative detection kit for the triple helix structure of collagen according to claim 1, wherein, The storage temperature of the test kit is ≤10℃; The test kit can detect at least one of the following: collagen solution, collagen gel, collagen sponge, and collagen dressing.

9. A rapid qualitative and quantitative detection method for the triple helix structure of collagen, characterized in that, The detection method uses the detection kit described in any one of claims 1-8 and includes the following steps: S1: Mix the sample to be tested with the specific enzymatic hydrolysis system A containing non-triple helix collagen by shaking, and perform the first enzymatic hydrolysis step to obtain a hydrolysate containing small molecule polypeptides. S2: The enzymatic hydrolysate containing small molecule polypeptides is subjected to ultrafiltration and centrifugation to obtain the filtrate; S3: The filtrate is mixed with the enzymatic hydrolysis system B of the non-specific enzymatic hydrolysis of small molecule peptides by shaking, and the second enzymatic hydrolysis is carried out to obtain the enzymatic hydrolysate containing free hydroxyproline; S4: Add the free hydroxyproline enzymatic hydrolysate to the sample pad of the colloidal gold test paper. After the colloidal gold test paper has developed color, perform qualitative analysis by visual inspection and / or semi-quantitative analysis using a colloidal gold reader.

10. The rapid qualitative and quantitative detection method for the triple helix structure of collagen according to claim 9, wherein, The temperature for the first step of enzymatic hydrolysis is 10-30℃; The temperature for the second step of enzymatic hydrolysis is 10-50℃; The time for the first and second enzymatic hydrolysis steps is independently 10-60 min; The centrifugal force of the ultrafiltration centrifugation is 1000-8000g, the centrifugation temperature is 3-30℃, and the centrifugation time is 8-30min; The colloidal gold test strip uses a competitive inhibition method; the detection environment temperature in step S4 is 4-40℃; the time taken from adding the free hydroxyproline enzyme hydrolysate to the sample pad of the colloidal gold test strip to the completion of color development of the colloidal gold test strip is 5-20 minutes.

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