Application of a composition of nitrogen-doped carbon dots and hemi-xylenol orange in the preparation of bovine insulin fibrosis inhibitor

Abstract

The invention discloses an application of a composition of nitrogen-doped carbon dots and hemi-xylenol orange in preparing a bovine insulin fibrosis inhibitor, belonging to the technical field of medicines. The composition of nitrogen-doped carbon dots and hemi-xylenol orange consists of a nitrogen-doped carbon dot solution and a hemi-xylenol orange solution; the volume ratio of the nitrogen-doped carbon dot solution to the hemi-xylenol orange solution is 9-11:15-17; the concentration of the nitrogen-doped carbon dot solution is 4.5-5.5 mg / mL; the concentration of the hemi-xylenol orange solution is 0.9-1.1 mM; the composition of nitrogen-doped carbon dots and hemi-xylenol orange can improve the inhibitory effect on bovine insulin fibrosis.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to application of a composition of nitrogen-doped carbon dots and hemiclenol orange in the preparation of a bovine insulin fibrosis inhibitor. Background Art

[0002] Insulin is a protein hormone secreted by the pancreatic β-cells in the pancreas. It is an indispensable substance for the body to function. As a drug, insulin has the function of lowering blood sugar and regulating sugar metabolism. When the body secretes insufficient insulin, the utilization of sugar will be impaired, leading to the occurrence of diabetes, while excessive insulin will cause symptoms of hypoglycemia. Therefore, insulin is often used to treat diabetes clinically.

[0003] However, insulin drugs often form amyloid fibers, i.e., insulin fibrillation, during production, transportation, storage, and clinical application. After insulin fibrillation, it will affect the efficacy of insulin drugs. Therefore, inhibiting insulin fibrillation is the key to improving the efficacy of insulin drugs. In addition, since many proteins or peptides can form amyloid fibers under certain conditions, at least more than 20 human diseases such as Alzheimer's disease, Parkinson's disease, familial amyloid neuropathy, and type 2 diabetes are related to amyloid fiber deposition. Insulin, as a protein hormone, is very easy to form amyloid fibers. Therefore, the study of insulin fibrillation inhibitors is of great significance for the screening of anti-fibrotic drugs.

[0004] Currently, the commonly used insulin fibrosis inhibitors mainly include amino acids, proteins, phenolic compounds, inorganic nanoparticles, etc., but they all have the problem of poor inhibitory effect. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides an application of a composition of nitrogen-doped carbon dots and hemi-xylenol orange in the preparation of a bovine insulin fibrosis inhibitor, which can improve the inhibitory effect on bovine insulin fibrosis.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A use of a composition of nitrogen-doped carbon dots and hemi-xylenol orange in the preparation of a bovine insulin fibrosis inhibitor, wherein the composition of nitrogen-doped carbon dots and hemi-xylenol orange consists of a nitrogen-doped carbon dot solution and a hemi-xylenol orange solution;

[0008] The volume ratio of nitrogen-doped carbon dot solution and hemi-xylenol orange solution is 9-11:15-17;

[0009] The concentration of the nitrogen-doped carbon dot solution is 4.5-5.5 mg / mL;

[0010] The concentration of the hemi-xylenol orange solution is 0.9-1.1 mM;

[0011] The preparation method of nitrogen-doped carbon dots in the nitrogen-doped carbon dot solution is as follows: dissolving citric acid and urea in deionized water, transferring the solution into a reaction device, sealing the reaction device, transferring the reaction device into a heating device, controlling the temperature of the heating device to 170-190° C., cooling the reaction device after 3.5-4.5 hours, filtering the reactants in the reaction device, taking the filtrate, transferring the filtrate into a dialysis bag, adding the dialysis bag into deionized water for dialysis, and then drying the liquid in the dialysis bag to obtain nitrogen-doped carbon dots;

[0012] The mass ratio of citric acid to urea is 4.1-4.3:3.5-3.7;

[0013] The mass volume ratio of citric acid and deionized water is 4.1-4.3 g:38-42 mL;

[0014] The filter membrane used in the filtration is a microporous filter membrane with a filtration accuracy of 0.22 μm;

[0015] The molecular weight cut-off of the dialysis bag is 900-1100;

[0016] The temperature during the drying is 70-80°C.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The composition of nitrogen-doped carbon dots and hemi-xylenol orange of the present invention, when used in the preparation of a bovine insulin fibrosis inhibitor, can inhibit the initial stage of bovine insulin protein fibrosis with good inhibitory effect, and the inhibitory effect is better than that of using nitrogen-doped carbon dots or hemi-xylenol orange alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the fluorescence test result in Experimental Example 1;

[0020] in, Figure 1 a is the fluorescence test result of the control in Experimental Example 1, Figure 1 b is the fluorescence test result of the sample in Experimental Example 1;

[0021] Figure 2 The fluorescence test results in Experimental Example 2;

[0022] Figure 3 The fluorescence test results in Experimental Example 3;

[0023] in, Figure 3 a is the fluorescence test result of the control in Experimental Example 3, Figure 3 b is the fluorescence test result of the sample in Experimental Example 3;

[0024] Figure 4 is the transmission electron microscope image in Experimental Example 4;

[0025] in, Figure 4 a is a transmission electron microscope image of the mixture in Example 2 after being in a water bath for 360 min; Figure 4 b is a transmission electron microscope image of the mixture of the mixed sample to which 26 μL of the mixed solution of nitrogen-doped carbon dots and hemi-xylenol orange was added in Experimental Example 3 after heating for 360 min;

[0026] Figure 5 is the atomic force microscope image in Experimental Example 4;

[0027] in, Figure 5 a is an atomic force microscope image of the mixture in Example 2 after being in a water bath for 120 min; Figure 5 b is an atomic force microscope image of the mixture in Example 2 after being in a water bath for 240 min; Figure 5 c is an atomic force microscope image of the mixture in Example 2 after being in a water bath for 360 min; Figure 5 d is an atomic force microscope image of the mixture of the mixed sample added with 26 μL of the mixed solution of nitrogen-doped carbon dots and hemi-xylenol orange in Experimental Example 3 after heating for 120 min; Figure 5 e is an atomic force microscope image of the mixture of the mixed sample to which 26 μL of the mixed solution of nitrogen-doped carbon dots and hemi-xylenol orange was added in Experimental Example 3 after heating for 240 min; Figure 5 f is an atomic force microscope image of the mixture of the mixed sample to which 26 μL of the mixed solution of nitrogen-doped carbon dots and hemi-xylenol orange was added in Experimental Example 3 after heating for 360 min;

[0028] Figure 6 is the gel electrophoresis result in Experimental Example 5;

[0029] in, Figure 6 a is the SDS-PAGE gel electrophoresis result of the mixture after 0 min, 30 min, 60 min, 90 min, 120 min, and 240 min of water bath in Example 2, the 1st lane is a marker, and the 2nd to 7th lanes correspond to the mixture after 0 min, 30 min, 60 min, 90 min, 120 min, and 240 min of water bath; Figure 6b is the SDS-PAGE gel electrophoresis result of the mixture of the mixed sample of Experimental Example 3 to which 26 μL of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange was added after heating for 0 min, 30 min, 60 min, 90 min, 120 min, and 240 min, wherein the first lane is a marker, and lanes 2-7 correspond to the mixture after heating for 0 min, 30 min, 60 min, 90 min, 120 min, and 240 min; Figure 6 c is the PAGE gel electrophoresis result of the mixture after 0 min, 30 min, 60 min, 90 min, 120 min, and 240 min of water bath in Example 2, and lanes 1-6 correspond to the mixture after 0 min, 30 min, 60 min, 90 min, 120 min, and 240 min of water bath; Figure 6 d is the PAGE gel electrophoresis result of the mixture of the mixed sample of Experimental Example 3 to which 26 μL of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange was added after heating for 0 min, 30 min, 60 min, 90 min, 120 min, and 240 min, and lanes 1-6 correspond to the mixture after heating for 0 min, 30 min, 60 min, 90 min, 120 min, and 240 min;

[0030] Figure 7 The circular dichroism test results in Experimental Example 6;

[0031] in, Figure 7 a is the circular dichroism test result of the mixture obtained after water bathing for 0 min, 60 min, 90 min, 120 min, 180 min, 240 min, and 300 min in Example 2; Figure 7 b is the circular dichroism test results of the mixture obtained by heating the mixed sample of 26 μL of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange in Experimental Example 3 for 0 min, 60 min, 90 min, 120 min, 180 min, 240 min, 300 min, and 360 min.

[0032] Figure 8 is the fluorescence curve in Experimental Example 6;

[0033] in, Figure 8 a is the fluorescence curve of bovine insulin protein fibrosis in Example 2; Figure 8 b is the fluorescence curve obtained after adding 26 μL of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange at 0 min; Figure 8 c is the fluorescence curve obtained after adding 26 μL of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange 30 min later; Figure 8 d is the fluorescence curve obtained after adding 26 μL of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange after 60 min. DETAILED DESCRIPTION

[0034] In order to have a clearer understanding of the technical features, purposes and effects of the present invention, the specific implementation methods of the present invention are now described.

[0035] Example 1 Preparation of nitrogen-doped carbon dots and nitrogen-doped carbon dot solution

[0036] 4.20 g of citric acid and 3.60 g of urea were dissolved in 40.0 mL of deionized water, and then transferred to a 100 mL polytetrafluoroethylene stainless steel reactor. After sealing, the reactor was placed in an oven, the oven was turned off, and the temperature of the oven was controlled to 180°C. After 4 hours, the reactor was naturally cooled to room temperature, filtered using a microporous filter membrane with a filtration accuracy of 0.22 μm, and the filtrate was taken. The filtrate was transferred to a dialysis bag with a molecular weight cutoff of 1000, and dialyzed with deionized water while stirring. Then, the liquid in the dialysis bag was placed in an oven, the oven was turned off, the temperature of the oven was controlled to 70°C, and nitrogen-doped carbon dots were obtained after drying.

[0037] The nitrogen-doped carbon dots were added into deionized water to prepare a nitrogen-doped carbon dot solution with a concentration of 5 mg / mL.

[0038] Example 2 Preparation of fibrotic bovine insulin

[0039] 0.028 g of bovine insulin powder was placed in a beaker, and 1 mL of NaCl / HCl solution was added to the beaker. The bovine insulin powder was stirred until it was completely dissolved. The solution was then filtered using a filter with a filtration accuracy of 0.45 μm. The filtrate was taken, and the temperature of the constant temperature water bath was controlled to 65°C. The filtrate was added to the constant temperature water bath for a 360-min water bath. During the water bath, samples were taken every 30 minutes for subsequent tests.

[0040] The NaCl / HCl solution used in this example was prepared by dissolving NaCl in deionized water to prepare a 25 mM NaCl solution, and then adding an HCl aqueous solution to adjust the pH to 1.6;

[0041] The mass fraction of the HCl aqueous solution is 37%.

[0042] Test Example 1 Test on the inhibitory effect of hemi-xylenol orange (SO)

[0043] The purpose of this test example is to explore the inhibitory effect of the existing bovine insulin fibrosis inhibitor hemi-xylenol orange SO on bovine insulin fibrosis, and to compare it with the inhibitory effect of the nitrogen-doped carbon dots prepared in Example 1 on bovine insulin fibrosis.

[0044] The pH of the PBS buffer used in this test example was 7.4;

[0045] The NaCl / HCl solution used in this test example was prepared by dissolving NaCl in deionized water to prepare a 25 mM NaCl solution, and then adding an HCl aqueous solution to adjust the pH to 1.6;

[0046] The mass fraction of the HCl aqueous solution is 37%.

[0047] The thioflavin T (ThT) used in this test example is a small molecule structure with a single bond that can rotate freely inside the molecule. When amyloid protein becomes fibrillated and the spatial structure changes, the spatial conformation of amyloid protein changes from α-helix to β-fold, and the ThT molecule can bind to the fibrillated protein (Fiber). After binding, the rotation of the ThT molecule is inhibited, but the fluorescence effect is enhanced. Based on the above principle, this test example uses the thioflavin T fluorescence method to mark amyloid protein to determine whether it has become fibrillated.

[0048] Three portions of 0.028 g bovine insulin powder were taken respectively, and completely dissolved with NaCl / HCl solution (the amount of NaCl / HCl solution added was determined by the amount of SO solution added), and then the solutions were filtered using a filter device with a filtration accuracy of 0.45 μm, and the filtrate was taken, and then 0, 16 μL, and 30 μL of 1 mM SO solution were added respectively, so that the total volume of each portion was 1 mL, and three mixed samples were obtained respectively; then the three mixed samples were added to a 65°C water bath and heated, and samples were taken before the start of heating, after 60 min of heating, after 90 min of heating, after 120 min of heating, after 180 min of heating, after 240 min of heating, after 300 min of heating, and after 360 min of heating, 100 μL of sample was taken each time, and then the sample was mixed with 50 μL of 1 mM ThT solution, 850 μL of The PBS buffer was mixed evenly to obtain 1 mL of the test solution, and then the fluorescence test was performed using a fluorescence spectrophotometer model RF-5310PC. At the same time, a control was added and the control was tested for fluorescence. The fluorescence test results are shown in Figure 1 .

[0049] Figure 1 The horizontal axis of a is the wave number of the fluorescence spectrum, and the vertical axis is the luminous intensity of the fluorescence; Figure 1 The horizontal axis in b is the heating time, and the vertical axis is the fluorescence intensity.

[0050] Figure 1In a, the Fiber-SO curve is the fluorescence test result of the test solution obtained by mixing the mixed sample with 30 μL of 1 mM SO solution and heating for 360 min with ThT solution and PBS buffer; the Fiber curve is the fluorescence test result after mixing 100 μL of 1 mM fiber solution, 50 μL of 1 mM ThT solution, and 850 μL PBS buffer; the SO curve is the fluorescence test result after mixing 30 μL of 1 mM SO solution, 50 μL of 1 mM ThT solution, and 920 μL PBS buffer; the Insulin-SO curve is the fluorescence test result after mixing the mixed sample prepared with 30 μL of 1 mM SO solution, 50 μL of 1 mM ThT solution, and 850 μL PBS buffer.

[0051] The preparation method of the fiber solution with a concentration of 1 mM is as follows: 0.028 g of bovine insulin is dissolved in 1 mL of NaCl / HCl solution, and then heated for 6 hours to obtain the fiber solution.

[0052] Depend on Figure 1 a and Figure 1 b It can be seen that when ThT is used as a probe molecule for fluorescence testing, no fluorescence is generated after adding SO, while the control group without SO has strong fluorescence because its protein is converted into fiber. At the same time, it was also tested that pre-fibrosis protein and SO itself do not produce fluorescence. Moreover, when the SO concentration is 30μM, it has a good inhibitory effect.

[0053] Test Example 2 Test on the inhibitory effect of nitrogen-doped carbon dots

[0054] The pH of the PBS buffer used in this test example was 7.4;

[0055] The NaCl / HCl solution used in this test example was prepared by dissolving NaCl in deionized water to prepare a 25 mM NaCl solution, and then adding an HCl aqueous solution to adjust the pH to 1.6;

[0056] The mass fraction of the HCl aqueous solution is 37%.

[0057] Three portions of 0.028 g bovine insulin powder were taken respectively, and completely dissolved with NaCl / HCl solution (the amount of NaCl / HCl solution added was determined by the amount of nitrogen-doped carbon dot solution added), and then the solution was filtered using a filter device with a filtration accuracy of 0.45 μm to take the filtrate, and then 0, 20 μL, and 40 μL of the nitrogen-doped carbon dot solution prepared in Example 1 were added respectively, so that the total volume of each portion was 1 mL, and three mixed samples were obtained respectively; then the three mixed samples were added to a 65° C. water bath and heated, and samples were taken before the start of heating, after heating for 60 min, after heating for 90 min, after heating for 120 min, after heating for 180 min, after heating for 240 min, after heating for 300 min, and after heating for 360 min, 100 μL of sample was taken each time, and then the sample was mixed with 50 μL of 1 mM ThT solution, 850 μL The PBS buffer was mixed evenly to obtain 1 mL of the test solution, and then the fluorescence test was performed using a fluorescence spectrophotometer model RF-5310PC. The fluorescence test results are shown in Figure 2 , Figure 2 The horizontal axis is the heating time, and the vertical axis is the fluorescence intensity.

[0058] Experimental Example 3 Inhibition Effect Test of the Combination of Nitrogen-doped Carbon Dots and Dye SO

[0059] The pH of the PBS buffer used in this test example was 7.4;

[0060] The NaCl / HCl solution used in this test example was prepared by dissolving NaCl in deionized water to prepare a 25 mM NaCl solution, and then adding an HCl aqueous solution to adjust the pH to 1.6;

[0061] The mass fraction of the HCl aqueous solution is 37%.

[0062] Three portions of 0.028 g bovine insulin powder were taken respectively, and completely dissolved with NaCl / HCl solution (the amount of NaCl / HCl solution added was determined by the subsequent amount of addition), and then the solutions were filtered using a filter device with a filtration accuracy of 0.45 μm to obtain the filtrate, and then inhibitors were added respectively, specifically, 10 μL of 16 μM hemi-xylenol orange solution, 10 μL of nitrogen-doped carbon dot solution prepared in Example 1, and 26 μL of a mixture of nitrogen-doped carbon dots and hemi-xylenol orange were added respectively, so that the total volume of each portion was 1 mL, and three mixed samples were obtained respectively; then the three mixed samples were added to a 65° C. water bath and heated, and samples were taken before the start of heating, after heating for 60 min, after heating for 90 min, after heating for 120 min, after heating for 180 min, after heating for 240 min, after heating for 300 min, and after heating for 360 min, 100 μL of sample was taken each time, and then the sample was mixed with 50 μL of 1 mM ThT solution, 850 μL The PBS buffer was mixed evenly to obtain 1 mL of the test solution, and then the fluorescence test was performed using a fluorescence spectrophotometer model RF-5310PC. At the same time, a control was added and the control was tested for fluorescence. The fluorescence test results are shown in Figure 3 .

[0063] Figure 3 The horizontal axis of a is the wave number of the fluorescence spectrum, and the vertical axis is the luminous intensity of the fluorescence; Figure 3 The horizontal axis in b is the heating time, and the vertical axis is the fluorescence intensity.

[0064] Figure 3 In a, the Fiber-CDSO curve is the fluorescence test result of the test solution obtained by mixing the mixed sample with 26 μL of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange after heating for 360 minutes and mixing with ThT solution and PBS buffer; the Fiber curve is the fluorescence test result of mixing 100 μL of 1 mM fiber solution with 50 μL of 1 mM ThT solution and 850 μL PBS buffer; the CDSO curve is the fluorescence test result of mixing 100 μL of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange with 50 μL of 1 mM ThT solution and 920 μL PBS buffer; the Insulin-CDSO curve is the fluorescence test result of mixing the mixed sample of 26 μL of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange with 50 μL of 1 mM ThT solution and 850 μL PBS buffer.

[0065] The preparation method of the mixed solution of nitrogen-doped carbon dots and hemi-xylenol orange is as follows: 10 μL of a nitrogen-doped carbon dot solution with a concentration of 5 mg / mL and 16 μL of a hemi-xylenol orange solution with a concentration of 1 mM are mixed evenly.

[0066] The preparation method of the fiber solution with a concentration of 1 mM is as follows: 2.8 mg of bovine insulin is dissolved in 1 mL of NaCl / HCl solution, and then heated for 6 hours to obtain the fiber solution.

[0067] From the above results, it can be seen that the mixed solution of nitrogen-doped carbon dots and hemi-xylenol orange has a better inhibition effect.

[0068] Test Example 4 Morphology Characterization

[0069] 1. The mixture obtained after 360 min of water bath in Example 2 and the mixture obtained after heating for 360 min by adding 26 μL of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange in Experimental Example 3 were analyzed by transmission electron microscopy. The obtained transmission electron microscopy images are shown in FIG. Figure 4 .

[0070] The method of transmission electron microscopy analysis is as follows: after adding the sample to deionized water and diluting it 1000 times, 3 μL is aspirated and dropped on a copper grid to be dried, and then stained with 3 μL of 1 mg / mL phosphotungstic acid solution, and then a transmission electron microscope is used to collect images.

[0071] Figure 4 In a, we can clearly see the fibrous protein, which is several tens of micrometers long and about 2 nm in diameter; Figure 4 In b, the production of fiber-free protein can be clearly seen, indicating that the mixed solution of nitrogen-doped carbon dots and hemi-xylenol orange has a better inhibitory effect.

[0072] 2. Atomic force microscopy analysis was performed on the mixture obtained after 120 min, 240 min, and 360 min of water bath in Example 2 and the mixture obtained after 120 min, 240 min, and 360 min of heating by adding 26 μL of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange in Experimental Example 3. The obtained atomic force microscopy images are shown in FIG. Figure 5 .

[0073] The method of atomic force microscopy analysis is: after adding the sample to deionized water and diluting it 1000 times, 3 μL is aspirated and dropped on a silicon wafer and allowed to dry, and then an atomic force fiberscope is used to collect images.

[0074] Figure 5 This further illustrates that the mixed solution of nitrogen-doped carbon dots and hemi-xylenol orange has a better inhibitory effect.

[0075] Experimental Example 5 Gel electrophoresis test

[0076] Gel electrophoresis analysis was performed on the mixture obtained after water bathing in Example 2 and the mixture obtained after heating the mixed sample in Experimental Example 3 to which 26 μL of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange was added. The gel electrophoresis results are shown in FIG. Figure 6 Among them, SDS-PAGE is polyacrylamide gel electrophoresis with the addition of protein denaturant SDS, and PAGE is polyacrylamide gel electrophoresis.

[0077] Depend on Figure 6 It can be seen that as the heating time increases, Figure 6 The protein in a has a tail. Figure 6 The state of the protein in b remains in its initial state.

[0078] Test Example 6 Circular dichroism test

[0079] The mixture obtained after 0min, 60min, 90min, 120min, 180min, 240min, and 300min of water bath in Example 2 and the mixed sample of Experimental Example 3 to which 26μL of the mixed solution of nitrogen-doped carbon dots and hemi-xylenol orange were added were heated for 0min, 60min, 90min, 120min, 180min, 240min, 300min, and 360min to obtain the mixture were subjected to circular dichroism test. The circular dichroism test results are shown in FIG. Figure 7 , Figure 7 a and Figure 7 The horizontal axis in b is the wavelength of the plane polarized light, and the vertical axis is the ellipticity.

[0080] Circular dichroism is used to measure the optical rotation of a substance to light of different wavelengths. It has the advantages of high resolution, high sensitivity and non-destructiveness, and can reflect important data on the secondary structure of proteins.

[0081] The results of circular dichroism tests showed that fibrillation can cause significant changes in the secondary structure of the protein, from α-folding to β-folding, and the peak position changes from 213nm and 225nm to a single peak near 220nm. However, after adding the mixture of nitrogen-doped carbon dots and hemi-xylenol orange, the secondary structure of the protein did not change, proving that the mixture of nitrogen-doped carbon dots and hemi-xylenol orange inhibits the fibrillation of bovine insulin protein at the primary stage. This further proves that the mixture of nitrogen-doped carbon dots and hemi-xylenol orange has an inhibitory effect on amyloid fibrillation.

[0082] In order to further explore the inhibitory effect of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange, a control experiment was set up to explore the effect of addition time on the inhibitory effect through fluorescence monitoring. The obtained fluorescence curve is shown in Figure 2. Figure 8 First, the growth curve of bovine insulin in Example 2 was monitored by fluorescence to obtain Figure 8a, that is, the normal fluorescence curve of bovine insulin protein fibrillation when no carbon dot dye conjugate is added. Then, 26 μL of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange (the preparation method of the mixture is the same as that of Experimental Example 3) was added after 0 min, 30 min, and 60 min, respectively, and then the growth curve of bovine insulin was monitored by fluorescence, and the fluorescence curves obtained were shown in FIG. Figure 8 b. Figure 8 c. Figure 8 d, it was found that only the fluorescence curve of 8b was Figure 8 The fluorescence curve of a is different. Figure 8 In the fluorescence curve of b, the fluorescence intensity hardly changes; Figure 8 c and Figure 8 The fluorescence curve of d Figure 8 a is basically the same, indicating that the inhibitory effect of the mixture of nitrogen-doped carbon dots and hemi-xylenol orange on amyloid protein fibrillation occurs in the initial stage of bovine insulin protein fibrillation.

[0083] Unless otherwise specified, all percentages used in the present invention are by mass.

[0084] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a composition of nitrogen-doped carbon dots and hemi-xylenol orange in the preparation of a bovine insulin fibrosis inhibitor, characterized in that: The composition of nitrogen-doped carbon dots and hemi-xylenol orange consists of a nitrogen-doped carbon dots solution and a hemi-xylenol orange solution; The volume ratio of nitrogen-doped carbon dot solution and hemi-xylenol orange solution is 9-11:15-17; The concentration of the nitrogen-doped carbon dot solution is 4.5-5.5 mg / mL; The concentration of the hemi-xylenol orange solution is 0.9-1.1 mM; The preparation method of nitrogen-doped carbon dots in the nitrogen-doped carbon dot solution is as follows: dissolving citric acid and urea in deionized water, transferring the solution into a reaction device, sealing the reaction device, transferring the reaction device into a heating device, controlling the temperature of the heating device to 170-190° C., cooling the reaction device after 3.5-4.5 hours, filtering the reactants in the reaction device, taking the filtrate, transferring the filtrate into a dialysis bag, adding the dialysis bag into deionized water for dialysis, and then drying the liquid in the dialysis bag to obtain nitrogen-doped carbon dots; The mass ratio of citric acid to urea is 4.1-4.3:3.5-3.7; The mass volume ratio of citric acid and deionized water is 4.1-4.3 g:38-42 mL.

2. Use of the composition of nitrogen-doped carbon dots and hemi-xylenol orange according to claim 1 in the preparation of bovine insulin fibrosis inhibitor, characterized in that: The filter membrane used in the filtration is a microporous filter membrane with a filtration accuracy of 0.22 μm.

3. Use of the composition of nitrogen-doped carbon dots and hemiclenol orange according to claim 1 in the preparation of bovine insulin fibrosis inhibitor, characterized in that: The molecular weight cut-off of the dialysis bag is 900-1100.

4. Use of the composition of nitrogen-doped carbon dots and hemiclenol orange according to claim 1 in the preparation of bovine insulin fibrosis inhibitor, characterized in that: The temperature during the drying is 70-80°C.

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