A method for detecting the integrity of collagen triple helix structure using collagen peptide-coated metal nanoclusters and disulfide nanomaterials
The fluorescence energy transfer detection method of collagen peptide-wrapped metal nanoclusters and disulfide nanomaterials solves the problem of the inability to quantitatively detect the triple helix structure of collagen in the existing technology, and realizes simple and highly sensitive quantitative analysis.
Patent Information
- Application Number
- CN202210878594.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-07-25
- Publication Date
- 2025-09-23
- Estimated Expiration
- 2042-07-25
AI Technical Summary
Existing methods for detecting the triple helix structure of collagen cannot achieve quantitative analysis, and the operation is cumbersome and time-consuming.
Collagen peptides were used to wrap metal nanoclusters and disulfide nanomaterials, and the integrity of the collagen triple helix structure was detected by fluorescence energy transfer. Collagen peptide-wrapped metal nanoclusters were prepared by microwave reaction and mixed with disulfide solution, and quantitative analysis was achieved by combining fluorescence intensity comparison.
The quantitative analysis of the collagen triple helix structure is achieved with simple operation, high sensitivity and short detection time, and is suitable for a wide range of type I collagen detection.
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Figure CN115112904B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biological detection technology, and more specifically, relates to a method for detecting the integrity of collagen triple helix structure by utilizing collagen peptide-wrapped metal nanoclusters and disulfide nanomaterials. Background Art
[0002] Collagen is widely present in connective tissues such as skin, bones, tendons and ligaments. It is responsible for the tensile strength of ligaments and tendons, the elasticity of skin, and the transparency and structural support of the cornea. It also mediates cell adhesion, proliferation, migration and differentiation. Collagen has a unique triple helical structure due to its iconic (Gly-XY)n sequence pattern, which gives collagen functions such as promoting cell regeneration and tissue repair and is widely used in medical beauty, biomaterials, regenerative medicine and other fields. At the same time, the integrity of the collagen triple helical structure is closely related to its molecular behavior and biological properties. The destruction of the triple helical structure will lead to the loss of collagen self-assembly behavior and the weakening of cell adhesion, proliferation, migration and other functions. More importantly, the destruction of the collagen triple helical structure is also closely related to osteoporosis, cirrhosis, thrombosis and cancer. Therefore, it is very important to construct a simple and efficient method to detect the integrity of the collagen triple helical structure.
[0003] Currently, the main methods for detecting collagen triple helix structure include infrared spectroscopy, circular dichroism, and protease digestion. Infrared spectroscopy determines changes in collagen structure by analyzing characteristic absorption peaks of specific functional groups; circular dichroism determines changes in collagen triple helix structure by observing changes in the intensities of positive and negative peaks near 220 nm and 198 nm. However, these two methods can only qualitatively assess the degree of damage to the collagen triple helix structure and cannot quantitatively determine its integrity. Protease digestion utilizes the principle that collagen with an intact triple helix structure is resistant to protease digestion, while collagen with a lost or partially lost triple helix structure is susceptible to protease digestion. Protease digestion analyzes the integrity of the collagen triple helix structure by measuring changes in hydroxyproline content or the number of peptides before and after digestion. While this method allows for quantitative analysis of collagen triple helix integrity, the determination of hydroxyproline content and the number of peptides analyzed by SDS-PAGE are often cumbersome and time-consuming.
[0004] In addition, patent document CN106383232.B discloses a method for detecting the triple helix structure of collagen using disulfide nanomaterials. This technology uses the principle that a polypeptide probe forms a triple helix structure with the polypeptide to be tested to detect the triple helix structure of collagen. However, this technology only detects the formed triple helix structure, not the collagen triple helix structure that already exists in the system. In layman's terms, detecting the formed triple helix structure means that there is no collagen in the test sample, let alone a triple helix structure. The test sample only contains peptides, and the triple helix structure formed by the interaction between the polypeptide probe and the peptide to be tested is detected; detecting the collagen triple helix structure that already exists in the system means that the test sample is collagen with a triple helix structure, and the collagen triple helix structure that already exists in the system. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to construct a new method for detecting the integrity of the triple helix structure of type I collagen. This method can be used for quantitative analysis of the integrity of the collagen triple helix structure and has the advantages of simple operation, high sensitivity, and short detection time.
[0006] To achieve the above objectives, the present invention provides a method for detecting the integrity of the collagen triple helix structure using collagen peptide-coated metal nanoclusters and disulfide nanomaterials, the method comprising:
[0007] 1) uniformly mixing the collagen peptide solution, the metal salt solution, and the sodium hydroxide solution, and subjecting the mixture to microwave reaction to obtain the collagen peptide-coated metal nanoclusters;
[0008] 2) mixing the collagen peptide-coated metal nanoclusters obtained in step 1) with a disulfide solution;
[0009] 3) The mixture obtained in step 2) was mixed with equal volumes of collagen standard and collagen sample to be tested, and the maximum fluorescence intensity in the range of 360nm-550nm under 340nm excitation wavelength was measured. The maximum fluorescence intensity of the collagen standard was recorded as F 标 The maximum fluorescence intensity of the collagen sample to be tested is recorded as F A The triple helix structure integrity of the sample was calculated by comparing it with the fluorescence intensity F0 of the blank system under the same test conditions;
[0010] The integrity of the triple helix structure in the sample = (F A -F0) / (F 标 -F0)*100%.
[0011] Here, under the same test conditions, the maximum fluorescence intensity of the blank system in the range of 360nm-550nm at an excitation wavelength of 340nm is used to obtain the fluorescence intensity F0.
[0012] As a preferred embodiment, in step 1), the concentration of the collagen peptide solution is 25-100 mg / mL.
[0013] As a preferred embodiment, in step 1), the concentration of the metal salt solution is 5-15 mmol / L.
[0014] As a preferred embodiment, in step 1), the concentration of the sodium hydroxide solution is 0.8-1.2 mol / L.
[0015] As a preferred embodiment, in step 1), the volume ratio of the collagen peptide solution, the metal salt solution and the sodium hydroxide solution is (10-15):1:1.
[0016] As a preferred embodiment, in step 2), the concentration of the disulfide solution is 30-60 μg / mL.
[0017] As a preferred embodiment, in step 2), the volume ratio of the collagen peptide-wrapped metal nanoclusters to the disulfide solution is (20-50):1.
[0018] As a preferred embodiment, in step 1), the power of the microwave reaction is 200-560 W, and the time of the microwave reaction is 20-60 seconds.
[0019] As a preferred embodiment, in step 1), the collagen peptide is derived from at least one of the skin and bones of mammals and fish, such as mammal skin, mammal bones, fish skin, fish bones, etc., or a mixture of the above substances.
[0020] As a preferred embodiment, in step 1), the metal salt is chloroauric acid, a mixture of chloroauric acid and silver nitrate, or a mixture of chloroauric acid and cupric chloride.
[0021] As a preferred embodiment, in step 2), the disulfide is tungsten disulfide and / or molybdenum disulfide.
[0022] As a preferred embodiment, in step 3), the collagen sample to be tested includes at least one type I collagen extracted from the skin or Achilles tendon tissue of mammals, fish, and amphibians.
[0023] As a preferred embodiment, in step 3), the collagen standard is a standard protein sample with a complete triple helical structure.
[0024] As a preferred embodiment, in step 3), the blank system is the acetic acid solution used to dissolve the collagen sample to be tested.
[0025] As a preferred solution, in step 3), after mixing, the mixture is incubated at room temperature for 5-20 seconds.
[0026] The mechanism of the present invention is as follows: disulfide nanomaterials adsorb collagen peptides to wrap metal nanoclusters, and fluorescence energy transfer occurs, which leads to fluorescence quenching of the metal nanoclusters. After the collagen sample to be tested is introduced, the collagen interacts with the collagen peptides on the surface of the metal nanoclusters, and this interaction force is closely related to the integrity of the collagen triple helix, and causes the metal nanoclusters to break away from the adsorption of the disulfide nanomaterials, and the fluorescence is restored.
[0027] Beneficial effects of the present invention:
[0028] The detection method described in the present invention can quantitatively analyze the integrity of the collagen triple helix structure. Compared with existing technologies, (1) the detection method provided by the present invention has the advantages of simple operation, high sensitivity (it can detect the triple helix structure of slight changes caused by heat treatment for 1 minute), and short detection time (about 5 minutes); (2) it can be used to detect the integrity of the triple helix structure of almost all type I collagen proteins on the market, with a wide detection range.
[0029] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 The fluorescence spectrum (A), transmission electron microscopy (B), and X-ray electron spectroscopy (C) of the gold / copper nanoclusters coated with bovine collagen peptide in Example 1 are shown;
[0031] Figure 2 Circular dichroism spectra of bovine Achilles tendon collagen samples under different treatment conditions in Example 2;
[0032] Figure 3 The fluorescence spectra of bovine collagen peptide-coated gold / silver nanoclusters before and after mixing with tungsten disulfide in Example 2;
[0033] Figure 4 The fluorescence spectra of collagen after the complete triple helix structure is introduced and the triple helix structure is destroyed in Example 2;
[0034] Figure 5 These are fluorescence spectra of collagen after the complete triple helix structure is introduced and the triple helix structure is destroyed in Example 3. DETAILED DESCRIPTION
[0035] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. Instead, these embodiments are provided to make the present invention more thorough and complete and to fully convey the scope of the present invention to those skilled in the art.
[0036] In the embodiment of the present invention, the concentration of acetic acid is 0.5 mol / L; collagen sample (mass): acetic acid (volume) = 2 mg: 1 mL.
[0037] Example 1
[0038] 1) Take 5 mL of 75 mg / mL bovine collagen peptide solution, add 0.5 mL of 10 mmol / L chloroauric acid solution, 0.5 mL of 10 mmol / L copper nitrate solution, and 0.5 mL of 1 mol / L sodium hydroxide solution, and react under 400 W microwave for 30 seconds to obtain bovine collagen peptide-coated gold / copper nanoclusters.
[0039] 2) Add 40 μL of molybdenum disulfide solution to 1 mL of the above nanocluster solution.
[0040] 3) The mixture obtained in step 2) was mixed with an equal volume of a collagen standard (grass carp skin collagen with intact triple helix structure) and a collagen sample to be tested (grass carp skin collagen with destroyed triple helix structure), and the maximum fluorescence intensity within the range of 360nm-550nm was measured under an excitation wavelength of 340nm. The maximum fluorescence intensity of the collagen standard was recorded as F 标 The maximum fluorescence intensity of the collagen sample to be tested is recorded as F A The triple helix structure integrity of the sample was calculated by comparing it with the fluorescence intensity F0 of the blank system under the same test conditions;
[0041] F A =328.9, F0=202.0, F 标 =336.7, the integrity of the triple helix structure in the sample = (F A -F0) / (F 标 -F0)*100%=94.2%.
[0042] In this embodiment, the preparation method of grass carp skin collagen with a destroyed triple helix structure is as follows: taking a collagen sample with an intact triple helix structure, dissolving it with acetic acid, and heating it at 35°C for 1 minute to destroy the triple helix structure of the collagen, thereby obtaining a collagen sample to be tested.
[0043] Figure 1 Fluorescence spectrum of bovine collagen peptide-encapsulated gold / copper nanoclusters in Example 1 Figure 1 (A) Transmission electron microscopy Figure 1 (B) X-ray electron spectroscopy Figure 1 (C). Figure 1 In (A), the horizontal axis represents the wavelength, and the vertical axis represents the relative fluorescence intensity. Figure 1 (A) It can be seen that the excitation wavelength of the obtained nanoclusters is 340 nm and the emission wavelength is 410 nm; Figure 1 The scale bar in the lower left corner of (B) is 20 nm; Figure 1 In (C), the horizontal axis represents the electron binding energy, and the vertical axis represents the photoelectron flux intensity. Figure 1 (C) It can be seen that the prepared nanoclusters contain Au and Cu, confirming the successful preparation of the nanoclusters.
[0044] Example 2
[0045] 1) Take 5 mL of 75 mg / mL bovine collagen peptide solution, add 0.5 mL of 10 mmol / L chloroauric acid solution, 0.5 mL of 10 mmol / L silver nitrate solution, and 0.5 mL of 1 mol / L sodium hydroxide solution, and react under 560 W microwave for 20 seconds to obtain bovine collagen peptide-coated gold / silver nanoclusters.
[0046] 2) Add 50 μL of tungsten disulfide solution to 1 mL of the above nanocluster solution.
[0047] 3) The mixture obtained in step 2) was mixed with equal volumes of a collagen standard (bovine Achilles tendon collagen with intact triple helical structure) and a test collagen sample (bovine Achilles tendon collagen with disrupted triple helical structure), and the maximum fluorescence intensity within the range of 360 nm to 550 nm was measured under an excitation wavelength of 340 nm. The maximum fluorescence intensity of the collagen standard was recorded as F 标 The maximum fluorescence intensity of the collagen sample to be tested is recorded as F A The triple helix structure integrity of the sample was calculated by comparing it with the fluorescence intensity F0 of the blank system under the same test conditions;
[0048] F0=225.5,F 标 =342.4, F of samples prepared under different heat treatment conditions A =338.8 (40℃1 min), 323.6 (40℃10 min), 315.7 (40℃15 min), 298.2 (40℃20 min), 233.2 (70℃20 min), the integrity of the triple helix structure in different samples = (F A -F0) / (F 标 -F0)*100%=96.9% (40℃1 minute), 83.9% (40℃10 minutes), 77.2% (40℃15 minutes), 62.2% (40℃20 minutes), 6.6% (70℃20 minutes).
[0049] In this embodiment, the preparation method of bovine Achilles tendon collagen with a destroyed triple helix structure is as follows: take a collagen sample with an intact triple helix structure, dissolve it with acetic acid, and heat it at 40°C or 70°C for 1-20 minutes to destroy the triple helix structure of the collagen, thereby obtaining a collagen sample to be tested.
[0050] Figure 2Circular dichroism spectra of bovine Achilles tendon collagen samples under different treatment conditions in Example 2. Figure 2 The horizontal axis represents the wavelength, and the vertical axis represents the peak intensity. Figure 2 It can be seen that the complete triple helical structure collagen has a positive peak and a negative peak near 220nm and 197nm respectively. With the extension of heat treatment time or the increase of temperature, the positive and negative peaks gradually decrease, indicating that the triple helical structure of the collagen sample has been destroyed, and the heat treatment conditions will affect the integrity of the triple helical structure.
[0051] Figure 3 This is the fluorescence spectrum of the bovine collagen peptide-encapsulated gold / silver nanoclusters before and after mixing with tungsten disulfide in Example 2. Figure 2 The horizontal axis represents the wavelength, and the vertical axis represents the fluorescence intensity. Figure 3 It can be seen that the introduction of tungsten disulfide nanomaterials significantly reduced the fluorescence intensity of bovine collagen peptide-wrapped gold / silver nanoclusters, confirming that tungsten disulfide nanomaterials have a strong fluorescence quenching effect.
[0052] Figure 4 These are fluorescence spectra of collagen after the complete triple helix structure is introduced and the triple helix structure is destroyed in Example 2. Figure 4 The horizontal axis represents the wavelength, and the vertical axis represents the fluorescence intensity. Figure 4 The introduction of collagen restored the fluorescence signal of the bovine collagen peptide-coated gold / silver nanoclusters. Compared to the heat-treated collagen sample, the addition of collagen with an intact triple helical structure resulted in a stronger fluorescence signal. Furthermore, the fluorescence signals elicited by samples under different heat treatment conditions varied, indicating that fluorescence intensity is correlated with the integrity of the collagen triple helical structure.
[0053] Example 3
[0054] 1) Take 5 mL of 75 mg / mL bovine collagen peptide solution, add 0.5 mL of 10 mmol / L chloroauric acid solution, 0.5 mL of 10 mmol / L silver nitrate solution, and 0.5 mL of 1 mol / L sodium hydroxide solution, and react under 560 W microwave for 20 seconds to obtain bovine collagen peptide-coated gold / silver nanoclusters.
[0055] 2) Add 50 μL of tungsten disulfide solution to 1 mL of the above nanocluster solution.
[0056] 3) The mixture obtained in step 2) was mixed with equal volumes of a collagen standard (pigskin collagen with intact triple helical structure) and a collagen sample to be tested (pigskin collagen with disrupted triple helical structure), and the maximum fluorescence intensity within the range of 360 nm to 550 nm was measured under an excitation wavelength of 340 nm. The maximum fluorescence intensity of the collagen standard was recorded as F 标 The maximum fluorescence intensity of the collagen sample to be tested is recorded as FA The triple helix structure integrity of the sample was calculated by comparing it with the fluorescence intensity F0 of the blank system under the same test conditions;
[0057] F A =261.8, F0=202.3, F 标 =285.2, the integrity of the triple helix structure in the sample = (F A -F0) / (F 标 -F0)*100%=71.8%.
[0058] In this embodiment, the preparation method of pig skin collagen with intact triple helix structure and destroyed triple helix structure is as follows: take a collagen sample with intact triple helix structure, dissolve it with acetic acid, and heat it at 40°C for 15 minutes to destroy the triple helix structure of the collagen, thereby obtaining a collagen sample to be tested.
[0059] Figure 5 These are fluorescence spectra of collagen after the complete triple helix structure is introduced and the triple helix structure is destroyed in Example 3. Figure 5 The horizontal axis represents the wavelength, and the vertical axis represents the fluorescence intensity. Figure 5 It can be seen that the introduction of collagen samples restored the fluorescence signal of bovine collagen peptide-coated gold nanoclusters. Moreover, the collagen samples with intact triple helical structures produced stronger fluorescence signals than the corresponding samples with disrupted triple helices, indicating that the fluorescence intensity is correlated with the integrity of the collagen triple helical structure.
[0060] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not 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 method for detecting the integrity of collagen triple helix structure using collagen peptide-coated metal nanoclusters and disulfide nanomaterials, characterized in that: The method includes: 1) uniformly mixing the collagen peptide solution, the metal salt solution, and the sodium hydroxide solution, and subjecting the mixture to microwave reaction to obtain the collagen peptide-coated metal nanoclusters; In step 1), the concentration of the collagen peptide solution is 25-100 mg / mL; In step 1), the concentration of the metal salt solution is 5-15 mmol / L; In step 1), the concentration of the sodium hydroxide solution is 0.8-1.2 mol / L; In step 1), the volume ratio of the collagen peptide solution, the metal salt solution and the sodium hydroxide solution is (10-15):1:1; In step 1), the power of the microwave reaction is 200-560 W, and the microwave reaction time is 20-60 seconds; In step 1), the collagen peptide is derived from at least one of the skin and bones of mammals and fish; In step 1), the metal salt is chloroauric acid, a mixture of chloroauric acid and silver nitrate, or a mixture of chloroauric acid and cupric chloride; 2) mixing the collagen peptide-coated metal nanoclusters obtained in step 1) with a disulfide solution; In step 2), the concentration of the disulfide solution is 30-60 μg / mL; In step 2), the volume ratio of the collagen peptide-wrapped metal nanoclusters to the disulfide solution is (20-50):1; In step 2), the disulfide is tungsten disulfide and / or molybdenum disulfide; 3) The mixture obtained in step 2) was mixed with equal volumes of collagen standard and collagen sample to be tested, and the maximum fluorescence intensity in the range of 360nm-550nm under 340nm excitation wavelength was measured. The maximum fluorescence intensity of the collagen standard was recorded as F 标 The maximum fluorescence intensity of the collagen sample to be tested is recorded as F A The triple helix structure integrity of the sample was calculated by comparing it with the fluorescence intensity F0 of the blank system under the same test conditions; The integrity of the triple helix structure in the sample = (F A -F0) / (F 标 -F0)*100%.
2. The method for detecting the integrity of the collagen triple helix structure using collagen peptide-coated metal nanoclusters and disulfide nanomaterials according to claim 1, wherein: In step 3), the collagen sample to be tested includes at least one type I collagen extracted from the skin or Achilles tendon tissue of mammals, fish, and amphibians.
3. The method for detecting the integrity of the collagen triple helix structure using collagen peptide-coated metal nanoclusters and disulfide nanomaterials according to claim 1, wherein: In step 3), the collagen standard is a standard protein sample with a complete triple helical structure; In step 3), the blank system is the acetic acid solution used to dissolve the collagen sample to be tested.
4. The method for detecting the integrity of the collagen triple helix structure using collagen peptide-coated metal nanoclusters and disulfide nanomaterials according to claim 1, wherein: In step 3), after mixing, incubate at room temperature for 5-20 seconds.