Visual modified nanogold for high insulin concentration and preparation method thereof

Abstract

The invention relates to the technical field of nano-materials, and discloses visual modified nano-gold for high insulin concentration and a preparation method of the visual modified nano-gold. Ligand molecules which have high affinity to insulin and have amino groups are screened out through a molecular docking technology, nanogold is prepared through a sodium citrate reduction method, and a specific dose of ligand molecules are modified to the surface of nanogold through Au-S bonds. The modified nanogold is mainly applied to visualization of high insulin concentration. The insulin detection concentration range of the prepared modified nanogold can be increased to 1793 times of that of unmodified nanogold, and the modified nanogold can be used for monitoring the insulin release process.

Description

Technical Field

[0001] The invention relates to the technical field of nanomaterials, and in particular to modified nanogold for visualization of high insulin concentration and a preparation method thereof. Background Art

[0002] Patients with type 1 and most advanced type 2 diabetes need to inject exogenous insulin to control blood sugar in their daily blood sugar management. However, the injection method can cause pain, tissue damage and other problems, leading to poor patient compliance. In addition, incorrect dosage judgment may also lead to ineffective blood sugar control and even severe hypoglycemia. Especially for elderly patients, this non-automated operation undoubtedly increases their burden.

[0003] Therefore, a variety of glucose-sensitive microneedle patches have been developed to simulate natural insulin secretion to achieve long-term blood sugar control. However, the following problems still exist during the use of glucose-sensitive microneedle patches:

[0004] (1) Unclear starting point: Microneedle patches need to penetrate the skin to work, but in actual use, it is often difficult to judge whether they have penetrated effectively or whether they have started working.

[0005] (2) Unpredictable endpoints: Since drug release depends on the patient's specific physiological conditions (such as blood glucose levels, local pH or chemical concentration changes), this process is highly individualized and affected by multiple factors such as dietary intake, overall health status, physical activity, sleep, and stress.

[0006] To solve the above problems, it is recommended to introduce a color change mechanism based on the sugar-sensitive microneedle patch to remind patients of the treatment progress. Considering the convenience, low cost and biocompatibility of the microneedle patch, gold nanoparticles with high extinction coefficients that do not require other reagents or energy have become a good choice. The surface of gold nanoparticles can be easily modified by Au-S bonds or charge effects, and there are literature reports on the use of color changes caused by nanogold aggregation to detect small molecules, metal ions, enzymes, proteins, etc.

[0007] Gold nanoparticles have significant local surface plasmon resonance properties and can show color changes visible to the naked eye. In the colloidal state, nanogold solutions usually appear red. When the distance between particles decreases, the color of the solution changes from red to blue. However, the current insulin concentration range that causes nanogold to change color is very narrow, only 0.35-1.4μg / mL, which limits its practical application in monitoring insulin release.

[0008] Therefore, further modification and improvement of gold nanoparticles are needed to expand their color response range in the high insulin concentration range, thereby achieving more effective insulin release monitoring and patient treatment guidance. Summary of the invention

[0009] The purpose of the present invention is to provide a modified nano-gold for visualization of high insulin concentration and a preparation method thereof, which is mainly used to solve the problem that the current nano-gold only produces color response in the low insulin concentration range.

[0010] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0011] A method for preparing modified nanogold for visualization of high insulin concentration comprises the following steps:

[0012] S1: Autodock Qvina-W was used to perform molecular docking simulations on ligand molecules with thiol groups and insulin, and the affinity was calculated;

[0013] S2: Screen out molecules with amino groups from the top affinity ligands;

[0014] S3: Clean the glass bottle and stirring bar with ultrapure water, dissolve 12-48 mg of sodium citrate dihydrate in ultrapure water to prepare a 1-4 mM sodium citrate solution;

[0015] S4: Take 6-10 mL of the above sodium citrate solution and add it into a glass bottle, then add ultrapure water, stir and heat to 100-105°C;

[0016] S5: Dissolve 10-20 mg of chloroauric acid tetrahydrate in ultrapure water to prepare a chloroauric acid solution;

[0017] S6: Take 100-300 μL of chloroauric acid solution and quickly add it to the sodium citrate solution. After keeping heating, naturally cool it to room temperature to obtain a nano-gold dispersion, and store it in a refrigerator;

[0018] S7: Take 10-30 mg of the ligand molecule and dissolve it in NaOH solution;

[0019] S8: Take the nanogold dispersion, add 0.5-1mL of ligand molecule solution respectively, and mix well;

[0020] S9: After modification for 15-45 minutes, the supernatant was removed by centrifugation and washed 2-3 times with NaOH solution to obtain a modified nano-gold dispersion.

[0021] As a preferred technical solution, the ligand molecules used in step S1 include small molecules and macromolecules.

[0022] As a preferred technical solution, the ligand molecules with high affinity and amino groups screened in step S2 include but are not limited to one or more of 2-amino-4-(trifluoromethyl)benzenethiol, 2-amino-3-methylbenzenethiol, and 2-(tert-butoxycarbonylamino)ethanethiol.

[0023] As a preferred technical solution, the volume of ultrapure water in step S3 is 30-50 mL.

[0024] As a preferred technical solution, the volume of ultrapure water in step S4 is 5-15 mL.

[0025] As a preferred technical solution, the volume of ultrapure water in step S5 is 1-2 mL.

[0026] As a preferred technical solution, the volume of the NaOH solution in step S7 is 2-3 mL and the concentration is 1 M.

[0027] As a preferred technical solution, the volume of the nanogold dispersion in step S8 is 1-3 mL.

[0028] As a preferred technical solution, the speed of centrifugation to remove the supernatant in step S9 is 4000-8000 rpm and the time is 15-30 min.

[0029] The present invention provides a modified nano-gold for visualization of high insulin concentration. The modified nano-gold is mixed with insulin solutions of different concentrations to produce a color response visible to the naked eye, and the responding insulin concentration range can reach 9.87-2510 μg / ml.

[0030] Compared with similar technologies widely used in the current market, this invention has unprecedented technical advantages in multiple dimensions. These advantages not only profoundly affect the technical landscape of high insulin concentration detection and visualization, but also bring revolutionary changes to diabetes management, drug release monitoring and other fields, which are specifically reflected in the following four aspects:

[0031] (1) The present invention innovatively introduces molecular docking technology, a high-precision computational simulation method, which plays a key role in the screening process of modified nanogold ligand molecules. By accurately simulating the interactions between molecules, we can quickly and efficiently identify those nanogold modified ligands that have a strong affinity for insulin and carry amino groups. This process not only greatly simplifies the cumbersome steps of traditional experimental screening, saving a lot of time, manpower and material costs, but also avoids errors and uncertainties that may occur during the experiment, ensuring the accuracy and reliability of the screening results. The introduction of molecular docking technology is undoubtedly a highlight of the present invention in terms of technological innovation, which marks an important step forward in the direction of intelligentization and precision of nanogold modification technology.

[0032] (2) The preparation method of the present invention is not only simple and efficient, but also the selected nanogold material has excellent biocompatibility. This excellent biocompatibility means that in biomedical applications, nanogold can minimize damage to human cells and improve the safety of treatment. At the same time, the local surface plasmon resonance characteristics unique to nanogold enable it to produce obvious color changes through agglomeration mechanisms under specific conditions. This color change is intuitive and easy to observe, making it possible to achieve visual detection of insulin concentration. In addition, the stability and repeatability of the method have also been fully verified, ensuring that the modified nanogold products produced have consistent high quality.

[0033] (3) The modified gold nanoparticles prepared by the present invention exhibit amazing sensitivity in responding to insulin concentrations. The response concentration range covers a wide range from as low as 9.87 μg / ml to as high as 2510 μg / ml, which is 1792 times higher than that of unmodified gold nanoparticles. This means that the modified gold nanoparticles can respond to a wider range of insulin concentrations in color. This high sensitivity not only provides doctors with a more accurate basis for diagnosis, but also brings great convenience to patients in self-monitoring of blood sugar levels.

[0034] (4) The modified nanogold of the present invention does not need to rely on any exogenous reagent or external energy input when responding to changes in insulin concentration. This feature greatly simplifies the detection process and reduces the difficulty and cost of operation. At the same time, its small size and high extinction coefficient enable it to perform well in various application scenarios, especially in the real-time insulin release process monitoring of new drug delivery systems such as sugar-sensitive microneedle patches, which shows great application potential. By monitoring the release of insulin in real time, doctors can adjust the treatment plan more accurately, improve the treatment effect, and bring better treatment experience and quality of life to patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 This is a graph showing the color change of unmodified gold nanoparticles in response to insulin concentration.

[0037] Figure 2 This is the UV absorption spectrum of unmodified gold nanoparticles in response to insulin concentration.

[0038] Figure 3 This is the ratio of the UV absorption A680 / A519 of unmodified gold nanoparticles in response to insulin concentration.

[0039] Figure 4 The diagram is of the first ten thiol-containing ligand molecules with strong affinity to insulin found by using the molecular docking technology in the present invention.

[0040] Figure 5 The electron microscope image and energy spectrum image of the 2-amino-4-(trifluoromethyl)benzenethiol modified nano-gold of the present invention are shown.

[0041] Figure 6 This is a color change diagram of the 2-amino-4-(trifluoromethyl)benzenethiol-modified nanogold in response to insulin concentration of the present invention.

[0042] Figure 7 It is the ultraviolet absorption spectrum of 2-amino-4-(trifluoromethyl)benzenethiol modified nano-gold in response to insulin concentration of the present invention.

[0043] Figure 8 It is a ratio diagram of ultraviolet absorption A680 / A519 of 2-amino-4-(trifluoromethyl)benzenethiol-modified nano-gold in response to insulin concentration of the present invention. DETAILED DESCRIPTION

[0044] The present invention is described in more detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited thereto. For those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention. The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in the field.

[0045] The embodiments of the present invention are as follows:

[0046] Embodiment 1:

[0047] A method for preparing modified nanogold for visualization of high insulin concentration comprises the following steps:

[0048] (1) Molecular docking simulation: All simulation results were obtained by running Autodock Qvina-W. Each simulation process involved insulin and ligand molecules. The structural information of insulin was obtained from RCSB PDB (ID: 2ZP6, www.rcsb.org), and water molecules and ligands were removed. The ligand molecules were selected as molecules with thiol groups, and all related structural information was obtained from PubChem (pubchem.ncbi.nlm.nih.gov). In each docking process, a 30.28 nm 3The (2.9nm×2.9nm×3.6nm) box is used as the simulation area, which can completely include the target insulin molecule. The calculated affinities are collected for analysis. Figure 4 A diagram of the top 10 thiol-containing ligand molecules with strong affinity for insulin is shown, and ligand molecules with strong affinity and positive charge are selected, such as 2-amino-4-(trifluoromethyl)benzenethiol.

[0049] (2) Preparation of gold nanoparticles: Clean the glass bottle and stirring bar with ultrapure water. Take 23.52 mg of sodium citrate dihydrate and dissolve it in 40 mL of ultrapure water to prepare a 2 mM sodium citrate solution. Take 8 mL of 2 mM sodium citrate solution and add it to a glass bottle, then add 10 mL of ultrapure water, stir and heat to 102 °C. Take 14.3 mg of chloroauric acid tetrahydrate and dissolve it in 1.5 mL of ultrapure water to prepare a 25 mM chloroauric acid solution. Take 200 μL of 25 mM chloroauric acid solution and quickly add it to the boiling sodium citrate solution. The color of the solution changes from light black to purple-black, then to purple-red, and finally to wine red. Keep heating for 15 minutes, cool naturally to room temperature, and store in a refrigerator.

[0050] (3) Response of gold nanoparticles to insulin concentration: 30 mg of insulin was first dissolved in 200 μL pH1 HCl, then 5 mL 0.01 M PBS was added, and then 200 μL pH13 NaOH was used to neutralize the solution, and then the solution was diluted gradually. 150 μL of insulin solution was taken and mixed with 50 μL of gold nanoparticles, and then UV light was measured. Figure 1 This is a graph showing the color change of unmodified gold nanoparticles in response to insulin concentration. Figure 2 is the UV absorption spectrum of unmodified gold nanoparticles in response to insulin concentration. Figure 3 It is the ratio of the ultraviolet absorption A680 / A519 of unmodified nanogold in response to insulin concentration. It can be seen that the concentration response range of unmodified nanogold to insulin is 0.35-1.41μg / mL.

[0051] (4) Preparation of 2-amino-4-(trifluoromethyl)benzenethiol-modified nanogold: 20 mg of 2-amino-4-(trifluoromethyl)benzenethiol was dissolved in 2.8 mL of 1M NaOH. 2 mL of nanogold dispersion was added with 0.5 mL of 2-amino-4-(trifluoromethyl)benzenethiol solution and mixed evenly. After modification for 30 min, the supernatant was removed by centrifugation at 4000 rpm for 15 min, and the mixture was washed twice with 1M NaOH solution to obtain 2-amino-4-(trifluoromethyl)benzenethiol-modified nanogold dispersion. Figure 5This is the electron microscope image and energy spectrum of 2-amino-4-(trifluoromethyl)benzenethiol modified gold nanoparticles. From the energy spectrum, we can see that the N, S, and F elements contained in 2-amino-4-(trifluoromethyl)benzenethiol are enriched on the surface of gold nanoparticles, indicating that 2-amino-4-(trifluoromethyl)benzenethiol is successfully modified on the surface of gold nanoparticles.

[0052] (5) Response of 2-amino-4-(trifluoromethyl)benzenethiol-modified gold nanoparticles to insulin concentration: 30 mg of insulin was first dissolved in 200 μL pH1 HCl, then 5 mL 0.01 M PBS was added, and then 200 μL pH13 NaOH was used for neutralization, and then the solution was diluted step by step. 150 μL of insulin solution was taken and mixed with 50 μL of 2-amino-4-(trifluoromethyl)benzenethiol-modified gold nanoparticles, and then UV light was measured. Figure 6 This is a graph showing the color change of 2-amino-4-(trifluoromethyl)benzenethiol-modified gold nanoparticles in response to insulin concentration. Figure 7 This is the UV absorption spectrum of 2-amino-4-(trifluoromethyl)benzenethiol-modified gold nanoparticles in response to insulin concentration. Figure 8 It is the ratio of ultraviolet absorption A680 / A519 of 2-amino-4-(trifluoromethyl)benzenethiol-modified gold nanoparticles in response to insulin concentration. It can be seen that the concentration response range of 2-amino-4-(trifluoromethyl)benzenethiol-modified gold nanoparticles to insulin is 9.87-2510 μg / mL, which is 1793 times higher than that of unmodified gold nanoparticles.

[0053] Embodiment 2:

[0054] A method for preparing modified nanogold for visualization of high insulin concentration comprises the following steps:

[0055] (1) Molecular docking simulation: All simulation results were obtained by running Autodock Qvina-W. Each simulation process involved insulin and ligand molecules. The structural information of insulin was obtained from RCSB PDB (ID: 2ZP6, www.rcsb.org), and water molecules and ligands were removed. The ligand molecules were selected as molecules with thiol groups, and all related structural information was obtained from PubChem (pubchem.ncbi.nlm.nih.gov). In each docking process, a 30.28 nm 3 The (2.9 nm × 2.9 nm × 3.6 nm) box is used as the simulation area, which can completely include the target insulin molecule. The calculated affinities are collected for analysis, and ligand molecules with strong affinity and positive charge are selected, such as 2-amino-3-methylbenzenethiol.

[0056] (2) Preparation of gold nanoparticles: Clean the glass bottle and stirring bar with ultrapure water. Take 12 mg of sodium citrate dihydrate and dissolve it in 40 mL of ultrapure water to prepare a 1 mM sodium citrate solution. Take 6 mL of 1 mM sodium citrate solution and add it to a glass bottle, then add 10 mL of ultrapure water, stir and heat to 100°C. Take 10 mg of chloroauric acid tetrahydrate and dissolve it in 1.5 mL of ultrapure water to prepare a 25 mM chloroauric acid solution. Take 100 μL of chloroauric acid solution and quickly add it to the boiling sodium citrate solution. The color of the solution changes from light black to purple-black, then to purple-red, and finally to wine red. Keep heating for 15 minutes, cool naturally to room temperature, and store in the refrigerator.

[0057] (3) Preparation of 2-amino-3-methylbenzenethiol-modified nanogold: 10 mg of 2-amino-3-methylbenzenethiol was dissolved in 2.8 mL of 1M NaOH. 2 mL of nanogold dispersion was added with 0.6 mL of 2-amino-3-methylbenzenethiol solution and mixed evenly. After modification for 15 min, the supernatant was removed by centrifugation at 4000 rpm for 15 min, and the mixture was washed twice with 1M NaOH solution to obtain 2-amino-3-methylbenzenethiol-modified nanogold dispersion.

[0058] Embodiment 3:

[0059] A method for preparing modified nanogold for visualization of high insulin concentration comprises the following steps:

[0060] (1) Molecular docking simulation: All simulation results were obtained by running Autodock Qvina-W. Each simulation process involved insulin and ligand molecules. The structural information of insulin was obtained from RCSB PDB (ID: 2ZP6, www.rcsb.org), and water molecules and ligands were removed. The ligand molecules were selected as molecules with thiol groups, and all related structural information was obtained from PubChem (pubchem.ncbi.nlm.nih.gov). In each docking process, a 30.28 nm 3 The (2.9 nm × 2.9 nm × 3.6 nm) box was used as the simulation area, which can completely include the target insulin molecule. The calculated affinities were collected for analysis, and ligand molecules with strong affinity and positive charge were selected, such as 2-(tert-butyloxycarbonylamino)ethanethiol.

[0061] (2) Preparation of gold nanoparticles: Clean the glass bottle and stirring bar with ultrapure water. Take 48 mg of sodium citrate dihydrate and dissolve it in 40 mL of ultrapure water to prepare a 4 mM sodium citrate solution. Take 10 mL of 4 mM sodium citrate solution and add it to a glass bottle, then add 10 mL of ultrapure water, stir and heat to 105°C. Take 20 mg of chloroauric acid tetrahydrate and dissolve it in 1.5 mL of ultrapure water to prepare a chloroauric acid solution. Take 300 μL of chloroauric acid solution and quickly add it to the boiling sodium citrate solution. The color of the solution changes from light black to purple-black, then to purple-red, and finally to wine red. Keep heating for 15 minutes, cool naturally to room temperature, and store in the refrigerator.

[0062] (3) Preparation of 2-(tert-butoxycarbonylamino)ethanethiol-modified nanogold: 30 mg of 2-(tert-butoxycarbonylamino)ethanethiol was dissolved in 2.8 mL of 1M NaOH. 2 mL of nanogold dispersion was added with 1 mL of 2-(tert-butoxycarbonylamino)ethanethiol solution and mixed evenly. After modification for 45 min, the supernatant was removed by centrifugation at 4000 rpm for 15 min, and the mixture was washed twice with 1M NaOH solution to obtain 2-(tert-butoxycarbonylamino)ethanethiol-modified nanogold dispersion.

Claims

1. A method for preparing modified gold nanoparticles for visualization of high insulin concentration, characterized in that: The following steps are involved: S1: Autodock Qvina-W was used to perform molecular docking simulations on ligand molecules with thiol groups and insulin, and the affinity was calculated; S2: Screen out molecules with amino groups from the top affinity ligands; S3: Clean the glass bottle and stirring bar with ultrapure water, dissolve 12-48 mg of sodium citrate dihydrate in ultrapure water to prepare a 1-4 mM sodium citrate solution; S4: Take 6-10 mL of the above sodium citrate solution and add it into a glass bottle, then add ultrapure water, stir and heat to 100-105°C; S5: Dissolve 10-20 mg of chloroauric acid tetrahydrate in ultrapure water to prepare a chloroauric acid solution; S6: Take 100-300 μL of chloroauric acid solution and quickly add it to the sodium citrate solution. After keeping heating, naturally cool it to room temperature to obtain a nano-gold dispersion, and store it in a refrigerator; S7: Take 10-30 mg of the ligand molecule and dissolve it in NaOH solution; S8: Take the nanogold dispersion, add 0.5-1mL of ligand molecule solution respectively, and mix well; S9: After modification for 15-45 minutes, the supernatant was removed by centrifugation and washed 2-3 times with NaOH solution to obtain a modified nano-gold dispersion.

2. The method for preparing a modified nano-gold for visualization of high insulin concentration according to claim 1, characterized in that: The ligand molecules used in step S1 include small molecules and macromolecules.

3. The method for preparing a modified gold nanoparticle for visualization of high insulin concentration according to claim 1, characterized in that: The ligand molecules with high affinity and amino groups selected in step S2 include, but are not limited to, one or more of 2-amino-4-(trifluoromethyl)benzenethiol, 2-amino-3-methylbenzenethiol, and 2-(tert-butoxycarbonylamino)ethanethiol.

4. The method for preparing a modified nano-gold for visualization of high insulin concentration according to claim 1, characterized in that: The volume of ultrapure water in step S3 is 30-50 mL.

5. The method for preparing a modified nano-gold for visualization of high insulin concentration according to claim 1, characterized in that: The volume of ultrapure water in step S4 is 5-15 mL.

6. The method for preparing a modified nano-gold for visualization of high insulin concentration according to claim 1, characterized in that: The volume of ultrapure water in step S5 is 1-2 mL.

7. The method for preparing modified nano-gold for visualization of high insulin concentration according to claim 1, characterized in that: The volume of the NaOH solution in step S7 is 2-3 mL, and the concentration is 1 M.

8. The method for preparing modified nano-gold for visualization of high insulin concentration according to claim 1, characterized in that: The volume of the nano-gold dispersion in step S8 is 1-3 mL.

9. The method for preparing modified nano-gold for visualization of high insulin concentration according to claim 1, characterized in that: In the step S9, the speed of centrifugation to remove the supernatant is 4000-8000 rpm, and the time is 15-30 min.

10. A modified nano-gold for visualization of high insulin concentration prepared according to the method of any one of claims 1 to 9, characterized in that: The modified nanogold mixed with insulin solutions of different concentrations produced a color response visible to the naked eye, and the response insulin concentration range could reach 9.87-2510μg / ml.

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