Method for simultaneously detecting mussel mucoprotein and recombinant III-type collagen

Abstract

The invention relates to the technical field of detection, in particular to a method for simultaneously detecting mussel mucoprotein and recombinant III type collagen, which adopts reverse high performance liquid chromatography for separation and detection, and specifically comprises the following steps: preparing a derivative standard solution of mussel mucoprotein and recombinant III type collagen; the method comprises the following steps: mixing a mussel mucoprotein standard substance, a recombinant III type collagen standard substance and EDAC, adding a buffer solution to prepare a mixed stock solution with the pH value of 5.8-6.5, and performing ultrasonic treatment for 5-10 minutes to prepare a derivative standard substance solution; putting the derivative standard substance solution into ultrapure water, uniformly shaking, and standing to obtain a stock solution; detecting the absorption wavelength of the stock solution by using a diode array detector; diluting the stock solution into control solutions with different concentrations, detecting by using a high performance liquid chromatograph to obtain liquid chromatograms of the control solutions, and detecting and calculating other products by using the chromatograms. The test method is high in sensitivity and low in detection cost.

Description

Technical Field

[0001] The present invention relates to the field of detection technologies, and particularly to a method for simultaneously detecting mussel adhesive protein and recombinant type III collagen. Background Art

[0002] Mussel adhesive protein is a natural protein extracted from the byssal gland of mussels. It can promote wound healing, and has excellent adhesiveness and good biocompatibility. Recombinant type III collagen can be produced by genetic engineering technology. It can promote cell adhesion, proliferation and differentiation, and has biological activity. Mussel adhesive protein and recombinant type III collagen are complementary in functional roles, and this complementarity enables mussel adhesive protein and recombinant type III collagen to exert better effects when used in combination.

[0003] In biomedicine and tissue engineering, mussel adhesive protein and recombinant type III collagen can be used to construct more stable and biocompatible scaffolds. Among them, the strong adhesion performance provided by mussel adhesive protein helps the integration of the scaffold with host tissues, while recombinant type III collagen can provide a structure similar to the natural extracellular matrix to promote cell migration and proliferation. In the field of drug delivery, mussel adhesive protein can be used as a carrier to improve the stability and targeting of drugs, while recombinant type III collagen can be used as a sustained-release material to control the drug release rate. In the development of biomaterials, the combination of mussel adhesive protein and recombinant type III collagen can create new materials with excellent mechanical properties and biocompatibility for medical devices, implants and adhesives.

[0004] In the combined application of mussel adhesive protein and recombinant type III collagen, in order to ensure that the final product can achieve the expected effects and performance, the content ratio of the two is crucial. Existing experimental techniques that can be used to separate and quantify mussel adhesive protein and recombinant type III collagen include high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and capillary electrophoresis (CE). However, for low-abundance proteins, HPLC cannot provide sufficient sensitivity for accurate quantification; the sensitivity and specificity of ELISA highly depend on the antibodies used, and high-quality antibodies are difficult to obtain and are costly; while the detection sensitivity of CE is limited by the detector used. Therefore, there is an urgent need for a detection method for mussel adhesive protein and recombinant type III collagen with high sensitivity and low detection cost. Summary of the Invention

[0005] In order to solve the problems in the above background art, the present invention provides a method for simultaneously detecting mussel adhesive protein and recombinant type III collagen. This method can be used for separating and quantifying mussel adhesive protein and recombinant type III collagen simultaneously, and has high detection sensitivity and accuracy, and low required cost.

[0006] The solution adopted by the present invention to solve its technical problems is as follows: A method for simultaneously detecting mussel adhesive protein and recombinant type III collagen, which is separated and detected by reverse high performance liquid chromatography, specifically includes the following steps:

[0007] Step 1, prepare derivative standard solutions of mussel adhesive protein and recombinant type III collagen;

[0008] Obtain a mussel adhesive protein standard, a recombinant type III collagen standard, and EDAC. Among them, the ratio of the mass corresponding to the mussel adhesive protein standard, the mass corresponding to the recombinant type III collagen standard, and the mass corresponding to EDAC is 5:5:x, and the value of x ranges from 0.03 to 0.1;

[0009] Mix the mussel adhesive protein standard, the recombinant type III collagen standard, and EDAC to obtain an initial solution A. Obtain a buffer solution for adjusting the PH value, and introduce the buffer solution into the initial solution A to obtain a mixed stock solution. Among them, the PH value of the mixed stock solution is within a preset PH range, and the PH range is: 5.8 to 6.5;

[0010] Perform ultrasonic treatment on the mixed stock solution to derivatize the substances in the mixed stock solution. The ultrasonic time is within a preset range, and the preset range of the ultrasonic time is: 5 to 10 minutes, to obtain a derivative standard solution;

[0011] Introduce the derivative standard solution into pre-obtained ultrapure water, and shake the ultrapure water after introducing the derivative standard solution to obtain a solution to be left standing. Leave the solution to be left standing to obtain a stock solution, and the preset time for leaving the solution to be left standing is 3 to 8h;

[0012] Step 2, prepare a diode array detector and a high performance liquid chromatograph, detect the absorption wavelength of the stock solution through the diode array detector, and set the detection wavelength of the high performance liquid chromatograph according to the absorption wavelength of the stock solution;

[0013] Step 3, dilute the stock solution into control solution A, control solution B, control solution C, control solution D, control solution E, and control solution F. The concentrations of the substances contained in control solution A, control solution B, control solution C, control solution D, control solution E, and control solution F are different. Use the high performance liquid chromatograph after setting the detection wavelength to inject and gradient elute the above control solutions to obtain the liquid chromatograms of the control solutions;

[0014] Step 4, use the mass concentration of the mussel adhesive protein standard and the recombinant type III collagen standard as the abscissa, and the peak areas in the liquid chromatograms of the mussel adhesive protein standard and the recombinant type III collagen standard as the ordinate to obtain the mass concentration-peak area regression equations of the mussel adhesive protein and the recombinant type III collagen;

[0015] Step Five: Obtain the mussel adhesive protein to be tested, the recombinant type III collagen to be tested, and EDAC. Among them, the ratio of the mass corresponding to the mussel adhesive protein to be tested, the mass corresponding to the recombinant type III collagen to be tested, and the mass corresponding to EDAC is 5:5:0.1;

[0016] Mix the mussel adhesive protein to be tested, the recombinant type III collagen to be tested, and EDAC to obtain the initial solution B. Obtain a buffer solution for adjusting the pH value, and introduce the buffer solution into the initial solution B to obtain a mixed test solution. Among them, the pH value of the mixed test solution is within a preset pH range, and the pH range is: 5.8 to 6.5;

[0017] Perform ultrasonic treatment on the mixed test solution to derivatize the substances in the mixed test solution. The ultrasonic time is within a preset range, and the preset range of the ultrasonic time is: 5 to 10 minutes, to obtain a derivative test solution;

[0018] Introduce the derivative test solution into pre-obtained ultrapure water, and perform a shaking operation on the ultrapure water after introducing the derivative test solution to obtain a solution to be left standing. Let the solution to be left standing stand to obtain a test sample, and the preset standing time for the solution to be left standing is 3 to 8 h;

[0019] Step Six: Using the mass concentration - peak area regression equation of the mussel adhesive protein and recombinant type III collagen as a standard, use a high-performance liquid chromatograph with a set detection wavelength to inject the test sample and perform gradient elution, and calculate the mass concentrations of the mussel adhesive protein and recombinant type III collagen in the mixed test sample according to the peak areas of the liquid chromatogram of the test sample;

[0020] The chromatographic column of the high-performance liquid chromatograph is an Xtimate SEC-300 chromatographic column. The inner diameter of the Xtimate SEC-300 chromatographic column is 7.8 mm, and the length is 300 mm. The column temperature of the Xtimate SEC-300 chromatographic column is 30 °C. The detection wavelength of the high-performance liquid chromatograph is 220 nm. The aqueous solvent of the high-performance liquid chromatograph is trifluoroacetic acid.

[0021] Further, the mobile phase A of the high-performance liquid chromatograph is an aqueous solution of 0.1% concentration trifluoroacetic acid, and the mobile phase B is an acetonitrile solution of 0.1% concentration trifluoroacetic acid.

[0022] Further, the linear ranges of the mass concentration - peak area regression equations of the mussel adhesive protein and recombinant type III collagen are both: the concentration is 2 μg / ml - 500 μg / ml.

[0023] Further, the buffer solution is an acetic acid - sodium acetate buffer solution.

[0024] Further, when injecting the sample to be measured, the injection volume is 5 - 20 μl.

[0025] Further, photocatalytic SiO₂ is added to the initial solution A in Step 1, 2 and the mass ratio of the added photocatalytic SiO₂ 2 to EDAC is 5:1; photocatalytic SiO₂ 2 is added to the initial solution B in Step 5, 2 and the mass ratio of the added photocatalytic SiO₂

[0026] to EDAC is 5:1. 2 Further, the photocatalytic SiO₂ 2 comprises SiO₂ nanoparticles and a TiO₂ coating layer on the outer surface of the SiO₂ nanoparticles. 2 2

[0027] The photocatalytic SiO₂ 2 generates reactive oxygen species under blue light irradiation, which can promote the reaction between EDAC and proteins, accelerate the cross-linking reaction between EDAC and proteins, and improve the efficiency of the derivatization reaction; meanwhile, the by-products generated during the reaction process and the residual EDAC are degraded by ultraviolet light source irradiation subsequently, shortening the EDAC degradation time and preventing protein denaturation.

[0028] In summary, the beneficial effects of the present invention are as follows:

[0029] EDAC derivatization can change the charge and hydrophobicity of proteins, facilitating the separation of mussel adhesive protein and recombinant type III collagen;

[0030] EDAC derivatization can increase the ultraviolet absorption of groups, contributing to improving the detection selectivity of the diode array detector for mussel adhesive protein and recombinant type III collagen;

[0031] The mussel adhesive protein and recombinant type III collagen after EDAC derivatization have good stability and reproducibility;

[0032] EDAC derivatization can improve the specificity of the detection of mussel adhesive protein and recombinant type III collagen;

[0033] EDAC can activate carboxylic acid groups, enhance their reactivity with amino groups, thereby generating stable derivatives, which helps to maintain the integrity of the derivatives during the detection process of reverse phase high performance liquid chromatography (RP-HPLC), reducing degradation and loss;

[0034] In the detection of recombinant type III collagen, EDAC can be used to activate proline, and then improve its detection specificity and sensitivity in RP-HPLC through derivatization reactions;

[0035] EDAC is a water-soluble carbodiimide derivative, and the excess reagent can be removed by standing in an aqueous solution, which helps to reduce side reactions and improve the purity of the derivatization reaction. Description of the Drawings

[0036] Figure 1 It is the standard curve graph of the mussel adhesive protein standard in Example 1;

[0037] Figure 2 It is the standard curve graph of the recombinant type III collagen standard in Example 1;

[0038] Figure 3 It is the standard curve graph of the mussel adhesive protein standard in Example 2;

[0039] Figure 4 It is the standard curve graph of the recombinant type III collagen standard in Example 2;

[0040] Figure 5 It is the standard curve graph of the mussel adhesive protein standard in Example 3;

[0041] Figure 6 It is the standard curve graph of the recombinant type III collagen standard in Example 3;

[0042] Figure 7 It is the standard curve graph of the mussel adhesive protein standard in Example 4;

[0043] Figure 8 It is the standard curve graph of the recombinant type III collagen standard in Example 4;

[0044] Figure 9 It is the standard curve graph of the mussel adhesive protein standard in Example 5;

[0045] Figure 10 It is the standard curve graph of the recombinant type III collagen standard in Example 5. Detailed Embodiments

[0046] In order to make the content of the present invention easier to be clearly understood, the present invention will be further described below according to specific embodiments.

[0047] The chromatographic columns of the high performance liquid chromatograph used in the following experiments are all Xtimate SEC-300 chromatographic columns. The inner diameter of the Xtimate SEC-300 chromatographic column is 7.8 mm, the length is 300 mm, the column temperature of the Xtimate SEC-300 chromatographic column is 30 °C, the detection wavelength of the high performance liquid chromatograph is 220 nm, the mobile phase A of the high performance liquid chromatograph is 0.1% trifluoroacetic acid aqueous solution, the mobile phase B is 0.1% trifluoroacetic acid acetonitrile mixed solution, photocatalytic SiO 2 including SiO 2 nanoparticles and SiO 2 TiO on the outer surface of the nanoparticles 2 coating layer.

[0048] Stability test of EDAC:

[0049] Prepare the following experimental materials: EDAC powder (purity ≥ 98%), acetic acid-sodium acetate buffer solution A, acetic acid-sodium acetate buffer solution B, acetic acid-sodium acetate buffer solution C, ultrapure water, acetonitrile, among which the pH values of acetic acid-sodium acetate buffer solution A, acetic acid-sodium acetate buffer solution B, and acetic acid-sodium acetate buffer solution C are 5.8, 6.0, and 6.5 respectively.

[0050] Detection instrument: High performance liquid chromatograph (HPLC).

[0051] Experimental steps:

[0052] S1, Dissolve the EDAC powder in ultrapure water to prepare an EDAC solution with a concentration of 10 mg / mL;

[0053] S2, Add the EDAC solution to acetic acid-sodium acetate buffer solution A, acetic acid-sodium acetate buffer solution B, and acetic acid-sodium acetate buffer solution C respectively to prepare test solution A, test solution B, and test solution C;

[0054] S3, Let the above test solutions stand at room temperature, and take samples from each test solution after set time intervals of 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h;

[0055] S4, Use HPLC to quantitatively analyze the sampled EDAC solution to detect the concentration change of EDAC and the generation of degradation products.

[0056] Experimental results: At pH 6.5, the concentration of EDAC gradually decreases after 32 h; at pH 6.0, the concentration of EDAC gradually decreases after 20 h; at pH 5.8, the concentration of EDAC gradually decreases after 2 h.

[0057] Prepare the solution B to be detected again, add photocatalytic SiO to the solution B to be detected 2 , then irradiate and stir the solution B to be detected with a 5000xl ultraviolet light source for 3 h, and then separate the photocatalytic SiO by filtration 2 to obtain the solution B1 to be detected. Let the solution B1 to be detected stand at room temperature, and take samples from the solution B1 at set time intervals of 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h. Use HPLC to quantitatively analyze the sampled EDAC solution to detect the concentration change of EDAC and the generation of degradation products. The experiment shows that when the pH is 6.0, the concentration of EDAC gradually decreases after 2 h.

[0058] Prepare the solution C to be detected again, add photocatalytic SiO to the solution C to be detected 2 , then irradiate and stir the solution C to be detected with a 5000xl ultraviolet light source for 3 h, and then separate the photocatalytic SiO by filtration 2 to obtain the solution C1 to be detected. Let the solution C1 to be detected stand at room temperature, and take samples from the solution C1 at set time intervals of 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h. Use HPLC to quantitatively analyze the sampled EDAC solution to detect the concentration change of EDAC and the generation of degradation products. The experiment shows that when the pH is 6.5, the concentration of EDAC gradually decreases after 4 h.

[0059] Example 1

[0060] Step 1: Prepare a derivative standard solution of mussel adhesive protein and recombinant type III collagen;

[0061] Accurately weigh 5 mg of mussel adhesive protein standard, 5 mg of recombinant type III collagen standard and 0.1 mg of EDAC. Then place the weighed substances in a 10 mL volumetric flask, add 3 ml of ultrapure water and acetic acid-sodium acetate buffer solution, and prepare a mixed stock solution with a pH value of 5.8. Then sonicate for 5 minutes for full derivatization to obtain a derivative standard solution;

[0062] Add ultrapure water to a 10 mL volumetric flask and dilute to the mark, shake well, and then let it stand for 3 h to obtain a stock solution. The concentrations of mussel adhesive protein and recombinant type III collagen in the stock solution are both 500 μg / ml. Among them, the purpose of standing for 3 h is to hydrolyze the remaining EDAC in the derivative standard solution into EDU (N-ethyl-N′-(3-dimethylaminopropyl)urea) to prevent the remaining EDAC in the solution from affecting subsequent detection.

[0063] Step 2: Use a diode array detector to detect the absorption wavelength of the stock solution, and then set the detection wavelength of the high-performance liquid chromatograph according to the absorption wavelength of the stock solution;

[0064] Step 3: Accurately measure the stock solution, dilute it with ultrapure water to prepare a series of concentration control solutions. Measure 10 μl of each of the above control solutions, inject them into the high-performance liquid chromatograph for detection and gradient elution to obtain the liquid chromatogram of the stock solution. Take the mass concentrations of mussel adhesive protein and recombinant type III collagen as the abscissa, and the peak areas of mussel adhesive protein and recombinant type III collagen as the ordinate to obtain the mass concentration-peak area regression equations of mussel adhesive protein and recombinant type III collagen;

[0065] The high-performance liquid chromatography gradient elution process of this example is shown in Table 1:

[0066] Table 1

[0067]

[0068]

[0069] The detection data is shown in Table 2 below:

[0070] Table 2

[0071] Name Peak 1 Area Peak 2 Area Concentration (μg / ml) Control Solution 1 3374.2987 2614.5493 500 Control Solution 2 325.8156 268.4793 50 Control Solution 3 645.9761 521.7093 100 Control Solution 4 1781.4736 1297.3993 250 Control Solution 5 26.0068 24.4108 2.5 Control Solution 6 1008.9786 755.7093 150

[0072] Linear equation 1: y = 6.8049x - 0.0612, correlation coefficient R 2 = 0.9988

[0073] Linear equation 2: y = 5.2122x - 0.59, correlation coefficient R 2 = 0.9998 The above data shows that at pH 5.8, in the detection range of concentration from 2.5 μg / ml to 500 μg / ml, the linear relationship between the peak areas of mussel adhesive protein and recombinant type III collagen and the concentration is good.

[0074] Example 2

[0075] Step 1: Prepare the derivative standard solutions of mussel adhesive protein and recombinant type III collagen;

[0076] Accurately weigh 5 mg of mussel adhesive protein standard, 5 mg of recombinant type III collagen standard and 0.1 mg of EDAC. Then place the weighed substances in a 10 mL volumetric flask, add 3 ml of ultrapure water and acetic acid-sodium acetate buffer solution to prepare a mixed stock solution with a pH of 6.0. Then add 0.5 mg of photocatalytic SiO 2 , irradiate with blue light and stir the mixture containing photocatalytic SiO2 The mixed stock solution was sonicated for 10 min, and the sonication time was 10 minutes to obtain a reaction solution. Subsequently, the reaction solution was irradiated with a 5000x1 ultraviolet light source and stirred for 3 h, and then the photocatalytic SiO 2 was separated out to obtain a derivative standard solution;

[0077] Add ultrapure water to a 10 mL volumetric flask and dilute to the mark, shake well, and then let it stand for 3 h to obtain a stock solution. The concentrations of mussel adhesive protein and recombinant type III collagen in the stock solution are both 500 μg / ml. Among them, the purpose of standing for 3 h is to hydrolyze the remaining EDAC in the derivative standard solution into EDU (N-ethyl-N′-(3-dimethylaminopropyl)urea) to prevent the remaining EDAC in the solution from affecting subsequent detections.

[0078] Step 2: Use a diode array detector to detect the absorption wavelength of the stock solution, and then set the detection wavelength of the high-performance liquid chromatograph according to the absorption wavelength of the stock solution;

[0079] Step 3: Accurately measure the stock solution, dilute it with ultrapure water to prepare a series of concentration control solutions. Measure 10 μl of each of the above control solutions, inject them into the high-performance liquid chromatograph for detection and gradient elution to obtain the liquid chromatogram of the stock solution. Respectively, with the mass concentration of mussel adhesive protein and recombinant type III collagen as the abscissa and the peak area of mussel adhesive protein and recombinant type III collagen as the ordinate, obtain the mass concentration-peak area regression equation of mussel adhesive protein and recombinant type III collagen;

[0080] The high-performance liquid chromatography gradient elution process of this example is shown in Table 3:

[0081] Table 3

[0082]

[0083]

[0084] The detection data are shown in Table 4 below:

[0085] Table 4

[0086] Name Peak 1 Area Peak 2 Area Concentration (μg / ml) Control Solution 1 1184.6187 1965.1681 500 Control Solution 2 156.4587 146.7581 50 Control Solution 3 263.6987 453.2481 100 Control Solution 4 558.4187 1082.7181 250 Control Solution 5 8.3807 61.0926 2.5 Control Solution 6 273.6987 453.2481 150

[0087] Linear equation 1: y = 2.3222x + 0.1928, correlation coefficient R 2 = 0.9896

[0088] Linear equation 2: y = 3.9535x + 0.2019, correlation coefficient R 2 = 0.9856

[0089] The above data indicate that when the pH value is 6.0, within the detection range of the concentrations of mussel adhesive protein and recombinant type III collagen from 2.5 μg / ml to 500 μg / ml, the linear relationship between the peak area and the concentration is good.

[0090] Example 3

[0091] Step 1: Prepare a derivative standard solution of mussel adhesive protein and recombinant type III collagen;

[0092] Accurately weigh 5 mg of mussel adhesive protein standard, 5 mg of recombinant type III collagen standard, and 0.1 mg of EDAC. Then place the weighed substances in a 10 mL volumetric flask, add 3 ml of ultrapure water and acetic acid-sodium acetate buffer solution to prepare a mixed stock solution with a pH value of 6.5. Then add 0.5 mg of photocatalytic SiO 2 , irradiate with blue light and stir the mixed stock solution containing photocatalytic SiO 2 for 10 min, then perform ultrasonic treatment for 10 minutes to obtain a reaction solution. Then irradiate and stir the reaction solution with a 5000x l ultraviolet light source for 3 h, and then separate the photocatalytic SiO 2 by filtration to obtain the derivative standard solution;

[0093] Add ultrapure water to a 10 mL volumetric flask and dilute to the mark, shake well, and then let it stand for 5 h to obtain a stock solution. The concentrations of mussel adhesive protein and recombinant type III collagen in the stock solution are both 500 μg / ml. Among them, the purpose of standing for 5 h is to hydrolyze the remaining EDAC in the derivative standard solution into EDU (N-ethyl-N′-(3-dimethylaminopropyl)urea) to prevent the remaining EDAC in the solution from affecting subsequent detections.

[0094] Step 2: Use a diode array detector to detect the absorption wavelength of the stock solution, and then set the detection wavelength of the high performance liquid chromatograph according to the absorption wavelength of the stock solution;

[0095] Step 3: Accurately measure the stock solution, dilute it with ultrapure water to prepare a series of concentration control solutions. Measure 10 μl of each of the above control solutions, inject them into the high performance liquid chromatograph for detection and gradient elution to obtain the liquid chromatogram of the stock solution. Respectively, take the mass concentration of mussel adhesive protein and recombinant type III collagen as the abscissa, and the peak area of mussel adhesive protein and recombinant type III collagen as the ordinate to obtain the mass concentration-peak area regression equation of mussel adhesive protein and recombinant type III collagen;

[0096] The high performance liquid chromatography gradient elution process of this example is shown in Table 5:

[0097] Table 5

[0098]

[0099] The detection data are shown in Table 6 below:

[0100] Table 6

[0101] Name Peak 1 Area Peak 2 Area Concentration (μg / ml) Control Solution 1 2766.1781 4162.7168 500 Control Solution 2 350.9131 486.6118 50 Control Solution 3 521.4981 745.9568 100 Control Solution 4 1653.2531 2363.9918 250 Control Solution 5 19.8573 112.434 2.5 Control Solution 6 641.4981 916.9568 150

[0102] Linear equation 1: y = 5.6365x + 3.4553, correlation coefficient R 2 = 0.9791

[0103] Linear equation 2: y = 8.3953x - 7.8977, correlation coefficient R 2 = 0.9820

[0104] The above data show that when the pH value is 6.5, within the detection range of the concentrations of mussel adhesive protein and recombinant type III collagen from 2.5 μg / ml to 500 μg / ml, the linear relationship between the peak area and the concentration is good.

[0105] It can be seen from Example 1, Example 2, and Example 3 that within the detection range of the concentrations of mussel adhesive protein and recombinant type III collagen from 2.5 μg / ml to 500 μg / ml, when the derivatized pH value is 5.8, the linear relationship between the peak area and the concentration is the best. Therefore, the derivatized pH value set for the following experiments is 5.8.

[0106] Example 4

[0107] Step 1, prepare a derivative standard solution of mussel adhesive protein and recombinant type III collagen;

[0108] Accurately weigh 5 mg of mussel adhesive protein standard, 5 mg of recombinant type III collagen standard, and 5 mg of EDAC. Subsequently, place the weighed substances in a 10 mL volumetric flask, add 3 ml of ultrapure water and acetic acid - sodium acetate buffer solution, prepare a mixed stock solution with a pH value of 5.8, and then ultrasonicate for 10 minutes for full derivatization to obtain a derivative standard solution;

[0109] Add ultrapure water to the 10 mL volumetric flask and dilute to the mark, shake well, and then let it stand for 4 h to obtain a stock solution. The concentrations of mussel adhesive protein and recombinant type III collagen in the stock solution are both 500 μg / ml. Among them, the purpose of standing for 4 h is to hydrolyze the remaining EDAC in the derivative standard solution into EDU (N - ethyl - N′-(3 - dimethylaminopropyl)urea) to prevent the remaining EDAC in the solution from affecting subsequent detections.

[0110] Step 2: Use a diode array detector to detect the absorption wavelength of the stock solution, and then set the detection wavelength of the high-performance liquid chromatograph according to the absorption wavelength of the stock solution;

[0111] Step 3: Accurately measure the stock solution, dilute it with ultrapure water to prepare a series of concentration control solutions. Measure 10 μl of each of the above control solutions, inject them into the high-performance liquid chromatograph for detection and gradient elution to obtain the liquid chromatogram of the stock solution. Using the mass concentration of mussel adhesive protein and recombinant type III collagen as the abscissa and the peak area of mussel adhesive protein and recombinant type III collagen as the ordinate, obtain the mass concentration-peak area regression equation of mussel adhesive protein and recombinant type III collagen;

[0112] The high-performance liquid chromatography gradient elution process of this example is shown in Table 7:

[0113] Table 7

[0114]

[0115] The detection data is shown in Table 8 below:

[0116] Table 8

[0117] Name Peak 1 Area Peak 2 Area Concentration (μg / ml) Control Solution 1 2264.0181 2356.9188 500 Control Solution 2 426.9581 849.9788 50 Control Solution 3 853.2981 1261.6388 100 Control Solution 4 1632.3181 1673.6188 250 Control Solution 5 11.9351 76.3518 2.5 Control Solution 6 253.2981 321.6388 150

[0118] Linear equation 1: y = 4.4831x + 120.57, correlation coefficient R 2 = 0.8549

[0119] Linear equation 2: y = 4.0933x + 372, correlation coefficient R 2 = 0.7476

[0120] The above data shows that when the mass ratio of the mussel adhesive protein standard, recombinant type III collagen standard to EDAC is 1:1:1, the linear relationship between the peak area and concentration of mussel adhesive protein and recombinant type III collagen is poor in the detection range of 2.5 μg / ml to 500 μg / ml.

[0121] Example 5

[0122] Step 1: Prepare a derivative standard solution of mussel adhesive protein and recombinant type III collagen;

[0123] Accurately weigh 5 mg of mussel adhesive protein standard, 5 mg of recombinant type III collagen standard and 0.03 mg of EDAC. Then place the weighed substances in a 10 mL volumetric flask, add 3 ml of ultrapure water and acetic acid-sodium acetate buffer solution to prepare a mixed stock solution with a pH value of 5.8, and then ultrasonicate for 5 minutes for full derivatization to obtain a derivative standard solution;

[0124] Add ultrapure water to a 10 mL volumetric flask and dilute to the mark. Shake well and then let it stand for 2.5 h to obtain a stock solution. The concentrations of mussel adhesive protein and recombinant type III collagen in the stock solution are both 500 μg / ml. Among them, the purpose of standing for 2.5 h is to hydrolyze the remaining EDAC in the derivative standard solution into EDU (N-ethyl-N′-(3-dimethylaminopropyl)urea) to prevent the remaining EDAC in the solution from affecting subsequent detections.

[0125] Step 2: Use a diode array detector to detect the absorption wavelength of the stock solution, and then set the detection wavelength of the high-performance liquid chromatograph according to the absorption wavelength of the stock solution;

[0126] Step 3: Accurately measure the stock solution and dilute it with ultrapure water to prepare a series of control solutions with different concentrations. Measure 10 μl of each of the above control solutions, inject them into the high-performance liquid chromatograph for detection and gradient elution to obtain the liquid chromatogram of the stock solution. Use the mass concentration of mussel adhesive protein and recombinant type III collagen as the abscissa and the peak area of mussel adhesive protein and recombinant type III collagen as the ordinate to obtain the mass concentration-peak area regression equation of mussel adhesive protein and recombinant type III collagen;

[0127] The high-performance liquid chromatography gradient elution process of this example is shown in Table 9:

[0128] Table 9

[0129]

[0130] The detection data are shown in Table 10 below:

[0131] Table 10

[0132]

[0133]

[0134] Linear equation 1: y = 2.5676x - 3.3239, correlation coefficient R 2 = 0.9864

[0135] Linear equation 2: y = 3.6119x - 7.6862, correlation coefficient R 2 = 0.9852

[0136] The above data show that when the mass ratio of mussel adhesive protein standard, recombinant type III collagen standard to EDAC is 5:5:0.03, within the detection range of the concentration of mussel adhesive protein and recombinant type III collagen from 2.5 μg / ml to 500 μg / ml, the linear relationship between the peak area and the concentration is good.

[0137] Example 6

[0138] S1, Prepare the test samples of derivatives of mussel adhesive protein and recombinant type III collagen;

[0139] S2, Accurately weigh 8 mg of medical recombinant mussel adhesive protein repair dressing and 0.1 mg of EDAC. Then place the weighed substances in a 10 mL volumetric flask, add 3 mL of acetic acid-sodium acetate buffer solution to prepare a mixed test sample with a pH value of 5.8; sonicate for 5 minutes for full derivatization to prepare a derivative test sample; add ultrapure water to the derivative test sample and dilute to the scale, shake well, and let stand for 2.5 h to obtain the test sample.

[0140] S3, Using the mass concentration-peak area regression equation of mussel adhesive protein and recombinant type III collagen in Experiment 1 as the standard, use a high-performance liquid chromatograph to detect, inject samples, and perform gradient elution on the test samples, and calculate the mass concentrations of mussel adhesive protein and recombinant type III collagen in the mixed test samples according to the peak areas of the test samples;

[0141] The detection data are shown in Table 11 below:

[0142] Table 11

[0143]

[0144] According to the linear equation 1: y = 6.8049x - 0.0612, the concentration of mussel adhesive protein in the test sample is calculated to be 4.2 μg / ml; then there are 42 μg of mussel adhesive protein in 8 mg of freeze-dried dressing

[0145] According to the linear equation 2: y = 5.2122x - 0.59, the concentration of recombinant type III collagen in the test sample is calculated to be 37.5 μg / ml; then there are 375 μg of recombinant type III collagen in 8 mg of freeze-dried dressing.

[0146] The above-described embodiments are only the preferred embodiments of the present invention and cannot be used to limit the protection scope of the present invention. Any non-substantive changes and modifications made by those skilled in the art based on the present invention fall within the protection scope of the present invention.

Claims

1. A method for simultaneously detecting mussel mucin and recombinant type III collagen, characterized in that: Reverse phase high performance liquid chromatography is used for separation and detection, which specifically includes the following steps: Step 1, preparing a standard solution of a derivative of mussel mucin and recombinant type III collagen; Obtain a mussel mucin standard, a recombinant type III collagen standard and EDAC, wherein the ratio of the mass corresponding to the mussel mucin standard, the mass corresponding to the recombinant type III collagen standard and the mass corresponding to the EDAC is 5:5:x, and the value of x is 0.03 to 0.1; Mixing the mussel mucin standard, the recombinant type III collagen standard and EDAC to obtain an initial solution A, obtaining a buffer solution for adjusting the pH value, and introducing the buffer solution into the initial solution A to obtain a mixed stock solution, wherein the pH value of the mixed stock solution is within a preset pH range, and the pH range is: 5.8 to 6.5; The mixed stock solution is subjected to ultrasonic treatment to derivatize the substances in the mixed stock solution, and the ultrasonic time is within a preset range, and the preset range of the ultrasonic time is: 5 to 10 minutes, to obtain a derivative standard solution; The derivative standard solution is introduced into the pre-acquired ultrapure water, and the ultrapure water after the derivative standard solution is introduced is shaken to obtain a solution to be left standing, and the solution to be left standing is left standing to obtain a stock solution, and the time for leaving the solution to be left standing is preset to be 3 to 8 hours; Step 2, preparing a diode array detector and a high performance liquid chromatograph, detecting the absorption wavelength of the stock solution by the diode array detector, and setting the detection wavelength of the high performance liquid chromatograph according to the absorption wavelength of the stock solution; Step 3, diluting the stock solution into control solution A, control solution B, control solution C, control solution D, control solution E, and control solution F, wherein the concentrations of the substances contained in the control solution A, control solution B, control solution C, control solution D, control solution E, and control solution F are different, and injecting and gradient eluting the control solutions using a high performance liquid chromatograph with a set detection wavelength to obtain a liquid chromatogram of the control solutions; Step 4, using the mass concentration of the mussel mucin standard and the recombinant type III collagen standard as the abscissa and the peak area in the liquid chromatogram of the mussel mucin standard and the recombinant type III collagen standard as the ordinate, respectively, to obtain the mass concentration-peak area regression equation of mussel mucin and recombinant type III collagen; Step 5, obtaining the mussel mucin to be tested, the recombinant type III collagen to be tested and EDAC, wherein the ratio of the mass corresponding to the mussel mucin to be tested, the mass corresponding to the recombinant type III collagen to be tested and the mass corresponding to EDAC is 5:5:0.1; Mixing the mussel mucin to be tested, the recombinant type III collagen to be tested and EDAC to obtain an initial solution B, obtaining a buffer solution for adjusting the pH value, and introducing the buffer solution into the initial solution B to obtain a mixed solution to be tested, wherein the pH value of the mixed solution to be tested is within a preset pH range, and the pH range is: 5.8 to 6.5; Ultrasonic treatment is performed on the mixed test solution to derivatize the substances in the mixed test solution, and the ultrasonic time is within a preset range, and the preset range of the ultrasonic time is: 5 to 10 minutes, to obtain a derivative test solution; The derivative test solution is introduced into the pre-acquired ultrapure water, and the ultrapure water after the derivative test solution is introduced is shaken to obtain a solution to be left standing, and the solution to be left standing is left standing to obtain a sample to be tested, and the time for leaving the solution to be left standing is preset to be 3 to 8 hours; Step six, using the mass concentration-peak area regression equation of the mussel mucin and recombinant type III collagen as the standard, using a high performance liquid chromatograph with a set detection wavelength to inject and gradient elute the sample to be tested, and calculating the mass concentration of mussel mucin and recombinant type III collagen in the mixed sample to be tested according to the peak area of ​​the liquid chromatogram of the sample to be tested; The chromatographic column of the high performance liquid chromatograph is an Xtimate SEC-300 chromatographic column, the inner diameter of the Xtimate SEC-300 chromatographic column is 7.8 mm, the length is 300 mm, the column temperature of the Xtimate SEC-300 chromatographic column is 30° C., the detection wavelength of the high performance liquid chromatograph is 220 nm, and the aqueous phase solvent of the high performance liquid chromatograph is trifluoroacetic acid.

2. A method for simultaneously detecting mussel mucin and recombinant type III collagen according to claim 1, characterized in that: The mobile phase A of the high performance liquid chromatograph is a 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is a 0.1% trifluoroacetic acid acetonitrile solution.

3. The method for simultaneously detecting mussel mucin and recombinant type III collagen according to claim 1, characterized in that: The linear ranges of the mass concentration-peak area regression equations of the mussel mucin and the recombinant type III collagen are both: concentration of 2 μg / ml-500 μg / ml.

4. The method for simultaneously detecting mussel mucin and recombinant type III collagen according to claim 1, characterized in that: The buffer solution is an acetic acid-sodium acetate buffer solution.

5. The method for simultaneously detecting mussel mucin and recombinant type III collagen according to claim 1, characterized in that: When the sample to be tested is injected, the injection volume is 5-20 μl.

6. The method for simultaneously detecting mussel mucin and recombinant type III collagen according to claim 1, characterized in that: Adding photocatalytic SiO2 to the initial solution A in step 1, wherein the mass ratio of the added photocatalytic SiO2 to EDAC is 5:1; Photocatalytic SiO2 is added to the initial solution B in step 5, and the mass ratio of the added photocatalytic SiO2 to EDAC is 5:

1.

7. A method for simultaneously detecting mussel mucin and recombinant type III collagen according to claim 6, characterized in that: The photocatalytic SiO2 comprises SiO2 nanoparticles and a TiO2 coating layer on the outer surface of the SiO2 nanoparticles.

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