Optical detection method of insulin based on graphene oxide / nano-gold composite material

Abstract

The present invention discloses an optical detection method for insulin based on a graphene oxide / nano-gold composite material, belonging to the field of optical sensing systems. The present invention synthesizes the graphene oxide / nano-gold composite material through a one-step method, and successfully uses it for the rapid detection of six types of insulin by utilizing its optical response characteristics to six types of insulin drugs on the market. By mixing and oscillating the graphene oxide / nano-gold composite material with the insulin to be tested for one minute, the displacement-dependent change of the visible light absorbance peak of the insulin to be tested can be exhibited, thereby completing the rapid detection of different concentrations and different types of insulin. The detection method of the present invention has simple steps, a high success rate, is easy to operate, has good detection stability, and is easy to promote and use.

Description

Technical Field

[0001] The present invention belongs to the field of optical sensing systems, and in particular relates to an optical detection method for insulin based on a graphene oxide / nano-gold composite material. Background Art

[0002] Graphene oxide, a functionalized derivative of graphene, is a nearly planar carbon nanosheet material exhibiting a two-dimensional network structure. Graphene oxide's surface contains a variety of oxygen-containing groups, including hydroxyl, epoxy, carbonyl, and carboxyl groups. These functional groups make graphene oxide more hydrophilic than graphene and well dispersed in water. Leveraging these properties, various metal nanoparticles can be combined with the two-dimensional structure of graphene oxide to achieve a combined performance, forming novel nanocomposites. These composite nanomaterials exhibit superior optical, electrical, and chemical properties, and have significant application potential in biosensing and fluorescence detection. Insulin is the only hormone in the body that lowers blood sugar, and hyperglycemia is caused by impaired insulin secretion or its biological effects. Leveraging the exceptional properties of a composite nanomaterial of graphene oxide and gold nanoparticles (graphene oxide / gold nanoparticles), a sensor system based on this composite nanomaterial has been developed for the rapid detection of specific insulin concentrations. This sensor system can complement existing insulin injection devices and has broad application prospects in the management of chronic diseases in patients with diabetes. Summary of the Invention

[0003] The present invention aims to overcome the shortcomings of the existing technology by providing an optical insulin detection method based on a graphene oxide / nano-gold composite material. The sensor system established in this invention enables label-free quantitative detection of different types of commercial insulin drugs and can assist existing insulin injection devices, such as insulin pumps and insulin patches, by providing a remaining drug indication. This method facilitates personalized and refined diabetes treatment, further improving the quality of chronic disease management for patients with diabetes.

[0004] The specific technical solutions adopted in the present invention are as follows:

[0005] The present invention provides an optical detection method for insulin based on a graphene oxide / nano-gold composite material, which is as follows:

[0006] S1: Using different commercial insulins, several groups of insulin standard sample solutions with different concentrations ranging from 5 μmol / L to 20 μmol / L were prepared; the insulin standard sample solutions were then mixed with graphene oxide / nano-gold composite solutions, and visible light absorbance was detected; the graphene oxide / nano-gold composite solution was mixed with water as a control group, and visible light absorbance was also detected;

[0007] S2: Using the average displacement of the absorbance peak of the insulin standard sample solution compared to the absorbance peak of the control group as the ordinate and the concentration of the insulin standard sample solution as the abscissa, a standard concentration dependence curve is drawn, which serves as the calibration curve for subsequent insulin detection;

[0008] S3: The commercial insulin solution to be tested is mixed with the graphene oxide / nanogold composite material solution, and a visible light absorbance test is performed to obtain the displacement average value of the absorbance peak of the solution compared with the absorbance peak of the control group, and the displacement average value is substituted into the corresponding commercial insulin calibration curve obtained in step S2 to obtain the corresponding concentration, thereby realizing optical detection of insulin of the commercial insulin solution to be tested.

[0009] Preferably, the commercial insulin includes detemir insulin, protamine human insulin mixed 30R, recombinant human insulin, aspart insulin, protamine human insulin and neutral insulin.

[0010] Preferably, in step S1, four insulin standard sample solutions of different concentrations are prepared.

[0011] Preferably, in step S1, 0.1 mL of insulin standard sample solutions of different concentrations are mixed with 1.0 mL of graphene oxide / nano-gold composite material solution respectively; 0.1 mL of deionized water is mixed with 1.0 mL of graphene oxide / nano-gold composite material solution.

[0012] Preferably, in the visible light absorbance detection process, the spectrum detection parameters are set as follows: integration time is 100ms, averaging times is 10 times, scanning range is 180nm to 800nm, and smoothness is 5.

[0013] Preferably, in step S1, the mixed solution is shaken for 1 minute before the visible light absorbance detection is performed.

[0014] Preferably, the preparation method of the graphene oxide / nano-gold composite material solution is as follows:

[0015] 1.225 mL of a 1% by mass chloroauric acid solution and 3 mL of a graphene oxide dispersion were mixed, 150 mL of deionized water was added, and the mixture was allowed to stand in the dark for 30 minutes, then stirred continuously and heated to 80° C.; 1.0-2.5 mL of a 1% by mass sodium citrate solution was then added to the mixture at one time, and stirring and heating at 80° C. were continued for 1 hour; heating was then stopped, stirring was continued for 15 minutes, and the obtained solution was allowed to stand in the dark to room temperature to obtain a graphene oxide / nano-gold composite material solution.

[0016] Furthermore, the graphene oxide / nano-gold composite material solution is placed in a 4° C. refrigerator away from light for future use.

[0017] Furthermore, the amount of sodium citrate solution added is 1.8 mL.

[0018] Furthermore, the concentration of the graphene oxide dispersion is 1 mg / mL.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The rapid insulin optical sensing system of the present invention enables high-precision detection of trace insulin concentrations. Compared with existing methods, it has advantages such as high sensitivity, good visualization, simple operation, simple structure and easy preparation, and can be used in clinical diabetes health management related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the absorption spectrum characterization diagram of the graphene oxide / nano-gold composite material obtained in Example 1;

[0022] Figure 2 Figure 1 shows the graphene oxide / nano-gold composite material sensing and detecting insulin, as well as the absorption spectrum characterization diagram after reacting with insulin;

[0023] Figure 3 This is a diagram showing the effectiveness of the graphene oxide / nano-gold composite nanomaterial standard insulin solution sensing system obtained in Example 1;

[0024] Figure 4 The graphs of the graphene oxide / nano-gold composite nanomaterial obtained in Example 1, respectively, for different concentrations of commercial insulin, show the relationship between the peak shift of absorbance and the concentration of the substance. For example, for insulin detemir injection, y = 0.88x - 2.67, R 2 =0.9966; b. Protamine human insulin mixed injection 30R, y = 1.11x-2.23, R 2 =0.9912; c. Recombinant human insulin injection, y = 0.24x + 0.93, R 2 =0.9956; d. Insulin aspart injection, y = 0.08x + 1.2, R 2 =0.9775; e. Protamine human insulin injection, y = x-1.48, R 2 =0.9974; f. Neutral insulin injection, y = 0.12x + 0.66, R 2 =0.9879;

[0025] Figure 5 Schematic diagram of commercial insulin concentration detection with known species and unknown concentration. DETAILED DESCRIPTION

[0026] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention may be combined accordingly, provided that there is no conflict between them.

[0027] Example 1

[0028] This embodiment realizes rapid optical detection of insulin based on graphene oxide / nano-gold composite material, and the method includes the following steps:

[0029] 1. One-step synthesis of graphene oxide / nano-gold composite material solution

[0030] All glassware to be used needs to be soaked in deionized water for 10 minutes and then washed to prevent the introduction of impurities that affect the synthesis. Then mix 1.225mL of a 1% mass fraction of chloroauric acid solution and 3mL of a 1mg / mL graphene oxide dispersion (graphene oxide sheet thickness 1-5 layers), add 150mL of deionized water, and start stirring and heating after standing for 30 minutes in a dark environment. After heating to 80°C, 1.8mL of a 1% mass fraction of sodium citrate solution is rapidly added to the mixed solution, and the temperature is maintained at 80°C and continued to stir for one hour. Stop heating afterwards, and stop stirring after stirring for another 15 minutes. After the solution is placed in the dark to room temperature, a graphene oxide / nano-gold composite material solution is obtained, which is placed in 4°C dark conditions for standby use.

[0031] The ultraviolet-visible absorption spectrum of the graphene oxide / nano-gold composite material solution finally obtained was scanned as follows:

[0032] 1 mL of the obtained graphene oxide / nano-gold composite material solution was added to the quartz cuvette of the spectrometer. The spectrometer used was the USB2000+ product of Ocean Optics. The specific test parameters were set as integration time 100 ms, average number 10 times, smoothness 5, and scanning wavelength range 180 nm to 800 nm to obtain the UV-visible absorption spectrum of graphene oxide. Figure 1 As shown, a characteristic peak appears at 251 nm and 521 nm, corresponding to the characteristic peaks of graphene oxide and nano-gold, respectively, indicating that graphene oxide and nano-gold colloid do exist in the solution, proving that the graphene oxide / nano-gold composite nanomaterial for insulin detection of the present invention is successfully synthesized.

[0033] 2. Verify the effectiveness of the sensing system with standard insulin solution

[0034] Prepare bovine insulin standard sample solutions. Use deionized water as the solvent to prepare four concentration gradients (5 μmol / L, 10 μmol / L, 20 μmol / L, and 30 μmol / L). Add 1 mL of the graphene oxide / nanogold solution and 0.1 mL of the bovine insulin standard sample solutions of various concentrations to a cuvette. After shaking for one minute, the resulting mixtures are then subjected to UV-visible absorption spectroscopy.

[0035] The absorption spectrum of the blank control solution was measured. Deionized water was selected as the target detection object in the blank control experiment. 1 mL of the graphene oxide / nanogold colloid solution was mixed with 0.1 mL of deionized water. After shaking for one minute, the mixture was added to the cuvette of the spectrometer.

[0036] The spectrometer test parameters are the same as in step 1, and the measurement process is as follows: Figure 2 The UV-visible absorbance curve obtained by measurement is plotted in the same coordinate system. By comparison, it can be seen that as the concentration of bovine insulin solution increases, the peak point produces a red shift and the displacement increases, indicating that this sensing system is effective. Figure 3 shown.

[0037] 3. Establish a concentration-dependent curve for commercial insulin detection based on graphene oxide / nano-gold composite nanomaterials, specifically including the following steps:

[0038] 3.1) Preparation of commercial insulin standard sample solutions: For each of the six insulin injections (insulin detemir, protamine human insulin 30R, recombinant human insulin, insulin aspart, protamine human insulin, and neutral insulin), four standard sample solutions with different concentration gradients (5 μmol / L, 10 μmol / L, 15 μmol / L, and 20 μmol / L) were prepared using deionized water as the solvent.

[0039] 3.2) Testing the insulin standard sample solution and establishing a light wavelength-UV absorbance curve, specifically comprising the following sub-steps:

[0040] 3.2.1) Measure the UV absorption spectrum of the blank control solution, and select deionized water as the target detection object in the blank control experiment. In order to eliminate the influence of water on the colloidal system of the graphene oxide / nanogold composite colloidal solution, 1 mL of the graphene oxide / nanogold colloidal solution is mixed with 0.1 mL of deionized water. After one minute of oscillation, the mixture is added to the measurement cavity of the spectrometer. Set the spectrometer measurement parameters: integration time 100 milliseconds, averaging times 10 times, smoothness 5, scanning wavelength 180-800 nm, and use the spectrometer as the instrument platform to perform UV absorption spectrum scanning on the blank control solution. After the measurement is completed, slowly aspirate the above-mentioned measurement solution, then add 3 mL of deionized water, let it stand for 5 minutes, and then slowly aspirate the deionized water. Repeat this step 3 times to clean the residual mixed solution from the previous measurement. Finally, use a pressurized air gun to blow the cuvette dry.

[0041] 3.2.2) Measuring the insulin standard sample solution: Add 1 mL of the graphene oxide / nanogold colloidal solution and 0.1 mL of a 5 μmol / L insulin standard sample solution to the measurement chamber. After shaking for one minute, repeat the above measurement steps on the resulting mixture and perform UV-visible absorption spectroscopy on the sample solution. Repeat the measurement five times, and after each measurement, clean the cuvette according to the cleaning steps in 3.2.1).

[0042] 3.2.3) Repeat the blank measurement and insulin standard sample solution measurement above until all concentrations of insulin standard sample solutions prepared in step 3.1) are measured, and finally obtain UV absorption spectra of the graphene oxide / nano-gold composite nanomaterial for different commercial insulin standard sample solutions at different concentrations;

[0043] 3.2.4) Based on the changes in the spectrogram, the average value of the absorbance peak point displacement is used as the sensing probe of the graphene oxide / nanogold composite sensing platform for rapid insulin detection of the present invention. The standard concentration dependence curve of the specific commercial insulin solution is drawn with the average displacement value as the ordinate and the concentration of the measured insulin standard sample solution as the abscissa. Each concentration of the insulin standard sample solution is sampled and measured 5 times. The absorbance peak point displacement values of each concentration of the insulin standard sample solution are counted to obtain the relationship curve y = ax + b between the concentration of the insulin standard sample and its corresponding absorbance ratio, where x is the concentration of the insulin standard sample solution, y is, and a and b are constants.

[0044] Figure 4 The results are as follows: a. Insulin detemir injection, y = 0.88x - 2.67, R 2=0.9966; b. Protamine human insulin mixed injection 30R, y = 1.11x-2.23, R 2 =0.9912; c. Recombinant human insulin injection, y = 0.24x + 0.93, R 2 =0.9956; d. Insulin aspart injection, y = 0.08x + 1.2, R 2 =0.9775; e. Protamine human insulin injection, y = x-1.48, R 2 =0.9974; f. Neutral insulin injection, y = 0.12x + 0.66, R 2 =0.9879. Figure 4 It can be seen that the graphene oxide / nanogold composite sensing platform for rapid insulin detection of the present invention can quickly detect different concentrations of insulin by oscillation after sample addition, and the results are stable and reproducible.

[0045] 4. Rapid optical detection of commercial insulin concentration based on graphene oxide / nano-gold composites

[0046] Take 0.1mL of the commercial insulin solution to be tested and mix it with 1mL of graphene oxide / nano-gold composite colloid solution. Repeat the measurement steps in step 3 for 3-5 times to obtain the stable characteristic spectrum of the solution and the average displacement of its absorbance peak point. When the type of the test solution is known but the concentration is unknown, substitute the average displacement into the standard concentration dependence curve of the corresponding type of insulin obtained in step 3 to obtain the corresponding concentration. This completes the concentration detection of the test solution. Figure 5 shown.

[0047] In summary, the rapid optical sensing detection method for insulin based on graphene oxide / nanogold composite nanomaterials provided by the present invention can, on the one hand, explore the biosensing properties of the complex and provide guidance for the research of the complex; on the other hand, it can assist existing insulin injection devices, such as insulin pumps and insulin patches, to provide drug remaining indications, further improving the quality of chronic disease management for diabetic patients.

[0048] The embodiment described above is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent substitution or equivalent transformation falls within the scope of protection of the present invention.

Claims

1. A method for optical detection of insulin based on graphene oxide / nano-gold composite material, characterized in that: The details are as follows: S1: Using different commercial insulins, several groups of insulin standard sample solutions with different concentrations ranging from 5 μmol / L to 20 μmol / L were prepared; the insulin standard sample solutions were then mixed with graphene oxide / nano-gold composite solutions and tested for visible light absorbance; a control group was prepared by mixing the graphene oxide / nano-gold composite solution with water and also testing for visible light absorbance; S2: Using the average displacement of the absorbance peak of the insulin standard sample solution compared to the absorbance peak of the control group as the ordinate and the concentration of the insulin standard sample solution as the abscissa, a standard concentration dependence curve is drawn, which serves as the calibration curve for subsequent insulin detection; S3: Mixing the commercial insulin solution to be tested with the graphene oxide / nanogold composite material solution and performing visible light absorbance detection to obtain an average displacement of the absorbance peak of the solution compared to the absorbance peak of the control group, substituting the average displacement into the corresponding commercial insulin calibration curve obtained in step S2 to obtain the corresponding concentration, thereby achieving optical detection of insulin in the commercial insulin solution to be tested; The preparation method of the graphene oxide / nano-gold composite material solution is as follows: 1.225 mL of 1% chloroauric acid solution and 3 mL of graphene oxide dispersion were mixed, 150 mL of deionized water was added, and the mixture was allowed to stand in the dark for 30 minutes, then stirred continuously and heated to 80°C; then 1.0-2.5 mL of 1% sodium citrate solution was added to the mixture at one time, and stirring and heating at 80°C was continued for 1 hour; then heating was stopped, stirring was continued for 15 minutes, and the obtained solution was allowed to stand in the dark to room temperature to obtain a graphene oxide / nano-gold composite material solution.

2. The method for optical detection of insulin based on graphene oxide / nano-gold composite material according to claim 1, characterized in that: The commercial insulins include detemir insulin, protamine human insulin mixed 30R, recombinant human insulin, aspart insulin, protamine human insulin and neutral insulin.

3. The method for optical detection of insulin based on graphene oxide / nano-gold composite material according to claim 1, characterized in that: In step S1, four insulin standard sample solutions of different concentrations are prepared.

4. The method for optical detection of insulin based on graphene oxide / nano-gold composite material according to claim 1, wherein: In step S1, 0.1 mL of insulin standard sample solutions of different concentrations are mixed with 1.0 mL of graphene oxide / nano-gold composite material solution respectively; 0.1 mL of deionized water is mixed with 1.0 mL of graphene oxide / nano-gold composite material solution.

5. The method for optical detection of insulin based on graphene oxide / nano-gold composite material according to claim 1, wherein: During the visible light absorbance detection process, the spectrum detection parameters were set as an integration time of 100 ms, an averaging number of 10 times, a scanning range of 180 nm to 800 nm, and a smoothness of 5.

6. The method for optical detection of insulin based on graphene oxide / nano-gold composite material according to claim 1, characterized in that: In the step S1, the mixed solution is shaken for 1 minute before the visible light absorbance is detected.

7. The method for optical detection of insulin based on graphene oxide / nano-gold composite material according to claim 1, characterized in that: The graphene oxide / nano-gold composite material solution was placed in a 4° C. refrigerator away from light and refrigerated for later use.

8. The method for optical detection of insulin based on graphene oxide / nano-gold composite material according to claim 1, wherein: The amount of sodium citrate solution added was 1.8 mL.

9. The method for optical detection of insulin based on graphene oxide / nano-gold composite material according to claim 1, wherein: The concentration of the graphene oxide dispersion is 1 mg / mL.

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