Colorimetric glucose analysis method based on triangular gold nanosheets and glucose oxidase

Abstract

The present invention provides a colorimetric glucose analysis method based on triangular gold nanosheets and glucose oxidase. The method comprises: 1) preparing triangular gold nanosheets using an oxidative etching method; 2) functionalizing the triangular gold nanosheets with 11-mercaptoundecanoic acid; and 3) activating the gold nanosheets with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to immobilize glucose oxidase on their surfaces. The triangular gold nanosheets with immobilized glucose oxidase are then mixed with a glucose sample to be tested to obtain a test reagent. The glucose concentration in the test glucose solution is determined based on the color changes and ultraviolet absorption spectrum of the test reagent. This method has a wide detection range, is easy to use, and enables visual detection of glucose.

Description

Technical Field

[0001] The present invention relates to a method for detecting glucose, in particular to a colorimetric glucose analysis method based on triangular gold nanosheets and glucose oxidase. Background Art

[0002] Diabetes is a major health threat to human health. Measuring glucose in blood and urine can be used to diagnose diabetes. Urine glucose testing is convenient, but its accuracy needs to be improved. Compared to urine glucose testing, blood glucose testing is more accurate, and blood glucose levels are often used as a clinical indicator of diabetes.

[0003] Various methods for glucose detection have been reported both domestically and internationally, primarily categorized into two main categories: enzymatic colorimetry and electrochemical methods. Electrochemical methods are complex to operate and generally require specialized testing instruments and electrochemical workstations. Compared to electrochemical methods, enzymatic colorimetric analysis offers advantages such as ease of operation, low cost, rapid speed, and high practicality. Furthermore, it can be detected with the naked eye and does not rely on complex instrumentation.

[0004] Currently, metal nanomaterial-based sensors have become an important glucose colorimetric tool for detecting biomolecules associated with disease pathogenesis, aiding early diagnosis. When gold and silver nanomaterials are used to detect glucose, the reaction product, hydrogen peroxide, causes a color change in the solution. This color change is due to the presence of a color developer. When natural enzymes catalyze the decomposition of glucose, hydrogen peroxide is produced. The reaction between hydrogen peroxide and the color developer produces a color change, which can be used to measure glucose concentration. However, natural peroxidases (such as HRP) are easily affected by external conditions such as pH and temperature, and can lose stability due to drastic changes in these conditions. Furthermore, they are expensive.

[0005] Furthermore, when round nanoparticles form or aggregate, they can also be accompanied by a noticeable color change, without the need for a color developer. As the particle size increases, the overall absorption peak red-shifts. This principle can be used for glucose detection, but the aggregation of round nanoparticles themselves can interfere with the experiment and affect the accuracy of glucose measurements.

[0006] CN 105424690 A discloses a colorimetric detection method for glucose, which uses a mixture of a triangular silver nanoparticle solution and a glucose oxidase solution to detect glucose concentration. Silver nanoparticles have a higher extinction coefficient than gold nanoparticles of the same size, but they are easily oxidized, which makes them unstable and limits their analytical applications. Summary of the Invention

[0007] The invention provides a colorimetric glucose analysis method based on triangular gold nanosheets and glucose oxidase, which has a wide test range, is easy to use, and can be visually detected.

[0008] The technical solution of the present invention is a colorimetric glucose analysis method based on triangular gold nanosheets and glucose oxidase, comprising the following steps:

[0009] S1, mixing a dispersion containing triangular gold nanosheets (AuTNPs) and a phosphate buffer solution (PBS), then adding a 11-mercaptoundecanoic acid solution (MUA) to carry out a mixing reaction, then separating the triangular gold nanosheets, and re-dispersing the obtained Au-MUA in the phosphate buffer solution;

[0010] S2, first adding crosslinking agents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the solution obtained in S1, and then adding glucose oxidase solution (GOD) for crosslinking; finally, separating the triangular gold nanosheets to obtain the Au-GOD conjugate detection system, and dispersing it in phosphate buffer solution;

[0011] S3. Mix the detection system with a series of prepared glucose samples to obtain a test reagent, and determine the glucose concentration in the test glucose solution based on the color change and ultraviolet absorption spectrum change of the test reagent.

[0012] Furthermore, the side length of the triangular gold nanosheet is between 40 nm and 120 nm.

[0013] Furthermore, when preparing a dispersion containing triangular gold nanosheets, hexadecyltrimethylammonium chloride is first diluted with ultrapure water, and then potassium iodide solution, chloroauric acid solution, sodium hydroxide solution, and L-ascorbic acid solution are added to react to obtain the dispersion.

[0014] Furthermore, the pH of the phosphate buffer solution added to S1 and S2 is 5.0-7.0; and the addition ratio of the phosphate buffer solution to the triangular gold nanosheet dispersion is 1:1-3:1.

[0015] Furthermore, in S1, the addition ratio of the dispersion containing triangular gold nanosheets to the 11-mercaptoundecanoic acid solution is 1:1 to 3:1.

[0016] Furthermore, in S2, the ratio of the cross-linking agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide (10 mM) is 1:1 to 5:1; the concentration of glucose oxidase is 1 to 5 mg / mL, and the addition ratio of the dispersion containing triangular gold nanosheets to the glucose oxidation solution is 1:1 to 5:1.

[0017] Furthermore, the cross-linking reaction temperature in S2 is 4° C. to 8° C., and the time is more than 12 hours.

[0018] Furthermore, the reaction time between the detection system and the glucose solution to be tested is more than 4 hours.

[0019] Furthermore, the concentration of the glucose solution to be tested is 0-12 mM.

[0020] Furthermore, an ultraviolet spectrophotometer is used to test the ultraviolet absorption spectrum of the reagent to be detected, and the measuring wavelength range of the instrument is ultraviolet-visible light of 190nm to 900nm.

[0021] The present invention has the following beneficial effects:

[0022] 1. Triangular gold nanosheets are subject to increasing attention due to their extremely high anisotropy and excellent LSPR performance. The present invention utilizes chemical crosslinking to fix glucose oxidase on the surface of triangular gold nanosheets. When there are glucose molecules in the system, glucose oxidase can catalyze glucose and oxygen to react, producing hydrogen peroxide (H2O2), and then the triangular gold nanosheets are etched, gradually losing their edges and corners, and turning to a circle. The solution color changes from blue to lavender, and both enzymatic reaction and etching can cause the absorption peak of the gold nanosheet to change. This method carries out the mensuration of glucose according to the color change of reagent to be detected and the absorption peak shift change situation, and compared with the change of detection absorption peak absorbance, greatly improves the accuracy of detection. In addition, the glucose oxidase in the present invention is fixed on the surface of triangular gold nanosheets by chemical crosslinking, and compared with adding free enzyme, improves stability.

[0023] 2. The present invention uses an oxidative etching method to produce triangular gold nanosheets with high purity and uniform size. The preparation method is simple, can be completed at room temperature, and has high reproducibility. The edges of the triangular gold nanosheets can be adjusted during preparation. Specifically, by adjusting the amount of L-ascorbic acid added, triangular gold nanosheets with side lengths of 40 nm to 120 nm can be produced. The triangular gold nanosheets can be grown at room temperature in less than 10 minutes, resulting in a high yield.

[0024] 3. The present invention is easy to operate and can realize visual detection.

[0025] The method of the present invention uses a ligand and a cross-linking agent to bind glucose oxidase to the surface of a gold nanosheet to prepare an Au-GOD conjugate for biological detection. When used for detection, the conjugate responds only to glucose. Due to the enzymatic and etching detection mechanism, the absorption peak is blue-shifted, allowing for visual detection of glucose. The method is easy to operate and has a wide detection range. The sensitivity of the glucose detection method can reach 0 to 12 mM, making it suitable for detecting glucose in real samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Testing the glucose process for triangular gold nanosheets and enzymes.

[0027] Figure 2 A is a photo of the Au-GOD solution, and B is the mixed solution after adding glucose.

[0028] Figure 3 The UV absorption spectra of Example 1 are obtained when different concentrations of glucose are added.

[0029] Figure 4 for Figure 3 The change in absorption peak Δλ after adding different concentrations of glucose is linearly related to the glucose concentration.

[0030] Figure 5 A is the UV-visible spectrum of the AuTNPs, Au-MUA and Au-GOD conjugate prepared in Example 2; B is the FTIR spectrum of the AuTNPs and Au-GOD conjugate prepared in Example 2.

[0031] Figure 6 This is a transmission electron microscopy image in Example 2, where A is uniformly dispersed AuTNPs and B is the Au-GOD conjugate.

[0032] Figure 7 3 are related figures in Example 3, wherein A is the UV-visible spectrum of Au-GOD and Au-Glu; B and C are electron microscope images of AuTNPs changing from triangle to circle.

[0033] Figure 8 This is the ultraviolet absorption spectrum of AuTNPs at different glucose concentrations of 0 to 12 mM in Example 3.

[0034] Figure 9 for Figure 8 The change in absorption peak Δλ after adding different concentrations of glucose from 0 to 12 mM is linearly related to the glucose concentration.

[0035] Figure 10 This is a relationship diagram of the absorption peak change Δλ when Au-GOD detects different analytes in Example 4.

[0036] Figure 11 This is a relationship diagram of the absorption peak change Δλ when Au-GOD detects glucose in the presence of different interferents in Example 5.

[0037] Figure 12 A is a graph showing the relationship between the absorption peak change Δλ and the glucose detection under different cross-linking agent ratios in Example 7. B is a graph showing the relationship between the absorption peak change Δλ and the glucose detection under different cross-linking agent concentrations in Example 7.

[0038] Figure 13 This is a relationship diagram of the absorption peak change Δλ when glucose is detected at different system pH (the final detection system pH = 6) in Example 8.

[0039] Figure 14 This is a graph showing the relationship between the absorption peak change Δλ when glucose is detected at different glucose oxidase concentrations in Example 9. DETAILED DESCRIPTION

[0040] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0041] The present invention provides a novel colorimetric glucose analysis method based on triangular gold nanomaterials and glucose oxidase, the steps of which are as follows:

[0042] 1) triangular gold nanosheets (AuTNPs) were prepared by oxidation etching, and a triangular gold nanosheet dispersion was uniformly mixed with an 11-mercaptoundecanoic acid (MUA) solution to functionalize the gold nanosheets and obtain an Au-MUA dispersion;

[0043] 2) Glucose oxidase (GOD) was immobilized on the functionalized gold nanoparticle surface using crosslinking agents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), so that the gold nanoparticles and the enzyme were combined to obtain an Au-GOD conjugate detection system;

[0044] 3) The detection system is mixed with the glucose solution to be detected to obtain a reagent to be detected, and glucose is measured based on the color change and absorption peak change of the reagent to be detected.

[0045] Example 1:

[0046] 1. The preparation method of triangular gold nanosheets is as follows:

[0047] Hexadecyltrimethylammonium chloride (6.4 mL, 0.1 M) was placed in a two-necked flask and diluted with 32 mL of ultrapure water. Potassium iodide solution (300 μL, 0.01 M), chloroauric acid solution (320 μL, 25.4 mM), sodium hydroxide solution (81.2 μL, 0.1 M), L-ascorbic acid solution (320 μL, 0.064 M), and sodium hydroxide solution (40 μL, 0.1 M) were then added to the flask. The flask was shaken rapidly at room temperature for 1-2 seconds. The mixed solution turned from colorless to red, purple, and finally blue. The growth of triangular gold nanosheets (AuTNPs) was completed in approximately 10 minutes.

[0048] It is worth noting that the concentration ratio of chloroauric acid solution to ultrapure water, potassium iodide, sodium hydroxide, and hexadecyltrimethylammonium chloride solution is fixed. By adjusting the amount of L-ascorbic acid added (from 0.01536mM to 0.0256mM), triangular gold nanosheets with a side length of 40nm to 120nm can be prepared.

[0049] 2. The gold nanoparticles are functionalized as follows:

[0050] A triangular gold nanoparticle (5 mL) was placed in a two-necked flask and mixed with phosphate buffer (5 mL, pH 6.8, 10 mM) for 20 minutes. 11-Mercaptoundecanoic acid (5 mL, 0.5 mM) was added to the mixture and stirred at room temperature for 4 hours at 30 rpm. The mixture was then centrifuged (13,000 rpm for 15 minutes) and the resulting Au-MUA was redispersed in phosphate buffer.

[0051] 3. The method for immobilizing glucose oxidase on the surface of triangular gold nanosheets is as follows:

[0052] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (5 mL, 10 mM each) were added to the functionalized gold nanoparticle solution and incubated at room temperature for 30 minutes. Glucose oxidase (GOD) solution (2 mL, 1 mg / mL in phosphate buffer) was then added to the mixture, which was then incubated at 4°C overnight. The mixture was centrifuged at 13,000 rpm for 15 minutes, and the resulting Au-GOD conjugate solution was redispersed in 3 mL of phosphate buffer.

[0053] 4. Detection of glucose:

[0054] Prepare 10 mL of glucose at three different concentrations in PBS (pH 7). Then, incubate 3 mL of the Au-GOD conjugate solution with 3 mL of glucose solution at 30°C for 4 hours. After 4 hours, observe the color change of the mixed solution and measure the UV absorption spectrum to observe the absorption peak.

[0055] This test process is simple and easy to operate. Figure 2 and 3 As shown in Figure 3, after adding glucose, the color of the reagent changes from blue to lavender, and its absorption peak shifts to the blue as a whole.

[0056] Example 2:

[0057] In this example, samples AuTNPs and Au-MUA were prepared according to steps 1 and 2 of Example 1 above, respectively. The EDC:NHS concentration ratio in step 3 was changed to (120 mM:60 mM), and the GOD concentration was adjusted to 4 mg / mL to obtain an improved Au-GOD conjugate.

[0058] The present invention uses an ultraviolet spectrophotometer to characterize the formation and properties of a combination of triangular gold nanosheets (AuTNPs) and glucose oxidase triangular gold nanosolution (Au-GOD). Figure 5 A shows the UV-visible spectra of the prepared AuTNPs, Au-MUA and Au-GOD conjugates. It can be clearly seen from the figure that the LSPR peak of AuTNPs is 626nm, and the LSPR peak of Au-MUA is about 634nm. In comparison, the overall absorption peak is red-shifted by 8nm. This is because the alkanethiol molecules are bound to the surface of the gold nanosheets through the Au-S interaction, and the dielectric layer around the gold nanoparticles causes the LSPR peak of AuTNPs to red-shift. The red shift of the absorption peak (642nm) of the Au-GOD conjugate is due to the binding of glucose oxidase to the surface of the gold nanosheets. It can be clearly seen that the LSPR spectrum of the functionalized gold nanoparticles is not broadened, and the structure of the particles remains intact. In addition, the FTIR spectra of the AuTNPs and Au-GOD conjugates are shown in Figure 2. Figure 5 As shown in B, AuTNPs showed infrared absorption peaks at 1470 and 2849 cm-1, which are the characteristics of surfactants. GOD-bound AuTNPs showed infrared absorption peaks at 3380 and 3430 cm-1. -1 The peak is shown at , which is a typical feature of hydroxyl NH / OH in protein GOD.

[0059] To further verify the mechanism of gold surface modification, the structures of AuTNPs and Au-GOD conjugates were characterized by transmission electron microscopy. Figure 6 A shows that uniformly dispersed AuTNPs were synthesized. Figure 6 B shows the Au-GOD conjugate. It is obvious that AuTNPs are well dispersed after conjugation with GOD, and TEM images confirm that GOD is successfully attached to the gold surface.

[0060] Example 3. Etching mechanism verification experiment:

[0061] The present invention also relates to verification of the glucose oxidase etching mechanism: a sample Au-GOD conjugate was prepared according to the above Example 2, and a series of glucose solutions (PBS, pH = 7) with different concentrations of 0 to 12 mM were prepared. 1 ml of the above Au-GOD conjugate solution and 1 ml of glucose solution were respectively taken and reacted at 30°C for 4 hours. After 4 hours, the color change of the mixed solution was observed, the change of the ultraviolet absorption spectrum was tested, and the absorption peak was observed.

[0062] When glucose was introduced and reacted for 4 hours, the results were as follows Figure 7 As shown in A, the absorption peak of the nanosheets gradually shifts from 642 nm to 600 nm due to the enzymatic and etching reactions. The blue shift of the absorption peak is usually caused by the change in the shape and size of the nanosheets. TEM tests confirmed that the AuTNPs transformed from triangles to round shapes ( Figure 7 B and Figure 7 C).

[0063] In addition, with the increase of glucose concentration, the absorption peak of AuTNPs gradually shifted to the blue. Figure 8 This is because glucose oxidase reacts with glucose, and the enzymatic reaction causes the LSPR peak to blue-shift. The product of the reaction, hydrogen peroxide, etches the AuTNPs, causing the edge length of the nanosheets to continuously decrease, which will also cause the absorption peak to blue-shift. In addition, the color of the mixed solution gradually changes from blue to lavender. Figure 9 As shown, the absorption peak change Δλ is linearly related to the glucose concentration in the two linear detection ranges of 0.2-1mM and 1-12mM. When the glucose concentration is in the range of 0.2-1mM, the sensor shows higher sensitivity. This is because the GOD adsorbed on the surface of the nanosheet reacts with glucose, and the enzymatic reaction causes the LSPR peak to blue-shift. The product H2O2 generated by the reaction also begins to etch the nanosheet, causing the size of the nanosheet to change, and the LSPR peak will also change further. The fitting line of the absorption peak change Δλ and glucose concentration can be described by the equation: Δλ = 17.887*X(mM)-2.229(R 2 =0.983). However, when the glucose concentration exceeds 1 mM, the enzyme adsorbed on the gold nanosheet surface has completely reacted, and H2O2 etches the AuTNPs, resulting in changes in color and Δλ. The entire etching process is slow. Therefore, the detectable glucose range is 1 to 12 mM, and the fitted line can be expressed as the equation: Δλ = 2.226*X(mM) + 14.828(R 2 =0.946). Its detection limit was 0.1 mM.

[0064] Example 4. Selectivity test:

[0065] According to the above Example 2, Au-GOD conjugate samples were prepared. A series of reactants, glucose, fructose, lactose, Fe 2+ 、Na + 、Cl - (Concentration of each is 10 mM) were mixed with Au-GOD conjugate, 1 mL of each was taken, and reacted at 30°C for 4 h. After 4 h, the color change of the mixed solution was observed, and the changes in the UV absorption spectrum were tested to observe the absorption peak.

[0066] The relevant drawings are shown in Figure 10The change in the absorption peak after adding glucose was Δλ=26 nm, while the change in the absorption peak of the reactants in the other experimental groups was Δλ<3 nm. This shows that the Au-GOD conjugate prepared by the present invention has a specific response to glucose.

[0067] Example 5. Anti-interference test:

[0068] The Au-GOD conjugate samples were prepared according to the above Example 2. A series of interfering substances, Mn 2+ , K + 、Fe 2+ 、Na + , mixed with Au-GOD conjugate and 2mM glucose solution, 1mL each, reacted at 30℃ for 4h. After 4h, observed the color change of the mixed solution, tested the change of UV absorption spectrum, and observed the absorption peak.

[0069] The relevant drawings are shown in Figure 11 In the presence of interfering substances, the variation of the absorption peak Δλ is not much different (overall <3%). Therefore, it can be seen that the Au-GOD conjugate prepared by the present invention has good anti-interference performance when detecting glucose.

[0070] Example 6:

[0071] The present invention also relates to the detection of glucose in artificial body fluids: Au-GOD conjugate samples are prepared according to Example 2, a series of glucose solutions of standard concentrations are prepared using artificial body fluids, the solutions are mixed with the Au-GOD conjugates, and the mixtures are incubated at 30° C. for 4 hours. After 4 hours, the color change of the mixed solution is observed, the change in the ultraviolet absorption spectrum is tested, and the absorption peak is observed.

[0072] As shown in Table 1, the recovery rate of glucose tested in artificial body fluids is between 92.47% and 106.43%, indicating that the glucose sensor has good performance and can be effectively applied to the detection of glucose in actual samples.

[0073] Table 1 Glucose testing in artificial body fluids

[0074]

[0075] Example 7. Selection of cross-linking agent ratio and concentration:

[0076] Au-MUA samples were prepared according to the steps in Example 1 above. Five different EDC:NHS ratios were then prepared, ranging from 1:1 (10 mM:10 mM) to 5:1 (50 mM:10 mM), each in a 5 mL volume. Each of the five different cross-linking agents was added to the Au-MUA solution and incubated at room temperature for 30 minutes. Glucose oxidase solution (2 mL, 1 mg / mL in phosphate buffer) was then added to the mixture, which was then incubated at 4°C overnight. The mixture was centrifuged at 13,000 rpm for 15 minutes, and the resulting Au-GOD conjugate solution was redispersed in 3 mL of phosphate buffer.

[0077] Au-MUA samples were prepared according to the steps in Example 1 above. Five different EDC:NHS concentration ratios were then prepared, ranging from 40mM:20mM to 120mM:60mM, each containing 5mL. The five different cross-linking agent concentrations were added to the Au-MUA solution and incubated at room temperature for 30 minutes. Glucose oxidase solution (2mL, 1mg / ml in phosphate buffer) was then added to the mixture, which was then kept at 4°C overnight. The mixture was centrifuged at 13,000rpm (15min), and the resulting Au-GOD conjugate solution was redispersed in 3mL of phosphate buffer.

[0078] Prepare 10 mL of a 10 mM glucose solution in PBS (pH 7). Take 3 mL of the Au-GOD conjugate solution and 3 mL of the glucose solution and react at 30°C for 4 hours. After 4 hours, observe the color change of the mixed solution and test the UV absorption spectrum to observe the absorption peak.

[0079] The relevant drawings are shown in Figure 12 The optimal glucose detection condition was achieved when the EDC:NHS ratio was 2:1. Under the optimal ratio, the EDC / NHS concentrations were adjusted, and the optimal glucose absorption peak change Δλ was achieved when the EDC:NHS ratio was 120mM:60mM.

[0080] Example 8. Control of system pH:

[0081] Sample Au-GOD conjugates were prepared according to Example 2. 10 mL of 10 mM glucose solution was prepared using five PBS solutions with pH values ​​ranging from 4 to 9. Three mL of the Au-GOD conjugate solution and three mL of the glucose solution were reacted at 30°C for 4 hours. After 4 hours, the mixed solutions were observed for color changes, UV absorption spectra, and absorbance values.

[0082] The relevant drawings are shown in Figure 13Because enzymes are sensitive to the pH of a solution, excessively alkaline or acidic conditions can disrupt the structure or spatial conformation of the enzyme molecule, affecting the binding and dissociation between the enzyme and the substrate glucose, thereby reducing the enzyme's catalytic efficiency. The Au-GOD conjugate achieved the optimal absorption peak change, Δλ, at a pH of 6 in the detection system.

[0083] Example 9. Selection of glucose oxidase concentration:

[0084] Au-MUA samples were prepared according to the steps in Example 1. 5 mL each of EDC (120 mM) and NHS (60 mM) were added to the functionalized gold nanoparticle solution and incubated at room temperature for 30 minutes. Five different concentrations of glucose oxidase solution (2 mL, 1 mg / mL to 5 mg / mL) were then added to the mixture, which was then incubated at 4°C overnight. The mixture was centrifuged at 13,000 rpm for 15 minutes, and the resulting five Au-GOD conjugate solutions were redispersed in 3 mL of phosphate buffer.

[0085] Prepare 10 mL of 10 mM glucose in PBS (pH 6). Take 3 mL of the Au-GOD conjugate solution and 3 mL of the glucose solution and react at 30°C for 4 hours. After 4 hours, observe the color change of the mixed solution and test the UV absorption spectrum to observe the absorption peak.

[0086] The relevant drawings are shown in Figure 14 The reaction rate is related to the catalytic activity of the enzyme. The enzyme concentration was adjusted to achieve the optimal reaction conditions. When the added glucose oxidase concentration was 4 mg / mL, the absorption peak change Δλ reached its maximum when detecting glucose.

[0087] The above embodiments are merely preferred technical solutions of the present invention and should not be construed as limiting the present invention. The embodiments and features in the embodiments of this application may be arbitrarily combined with each other unless they conflict. The scope of protection of the present invention shall be the technical solutions described in the claims, including equivalent alternatives to the technical features of the technical solutions described in the claims. Equivalent alternatives and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. Application of a colorimetric glucose analysis method based on triangular gold nanosheets and glucose oxidase in glucose detection, characterized in that: The following steps are involved: S1. A dispersion containing triangular gold nanosheets (AuTNPs) is mixed with a phosphate buffer solution (PBS), and then an 11-mercaptoundecanoic acid solution (MUA) is added for a mixing reaction. The triangular gold nanosheets are then separated, and the obtained Au-MUA is redispersed in the phosphate buffer solution. The triangular gold nanosheets have a side length between 40 nm and 120 nm. When preparing the dispersion containing the triangular gold nanosheets, hexadecyltrimethylammonium chloride is first diluted with ultrapure water, and then potassium iodide solution, chloroauric acid solution, sodium hydroxide solution, and L-ascorbic acid solution are added for reaction to obtain the product. S2, first add crosslinking agents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to the solution obtained in S1, and then add glucose oxidase solution (GOD) for crosslinking; finally, separate the triangular gold nanosheets to obtain the Au-GOD conjugate detection system, and disperse it in phosphate buffer solution; S3. Mix the detection system with a series of prepared glucose samples to obtain a test reagent, and determine the glucose concentration in the test glucose solution based on the color change and ultraviolet absorption spectrum change of the test reagent.

2. The use according to claim 1, characterized in that: The pH of the phosphate buffer solution added to S1 and S2 is 5.0-7.0; the addition ratio of the phosphate buffer solution to the triangular gold nanosheet dispersion is 1:1-3:

1.

3. The use according to claim 1, characterized in that: In S1, the dispersion containing triangular gold nanosheets and the 11-mercaptoundecanoic acid solution are added in a ratio of 1:1 to 3:

1.

4. The use according to claim 1, characterized in that: In S2, the ratio of the crosslinking agents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1~5:1; the concentration of glucose oxidase is 1~5 mg / mL, and the addition ratio of the dispersion containing triangular gold nanosheets and the glucose oxidation solution is 1:1~5:

1.

5. The use according to claim 1, characterized in that: The cross-linking reaction temperature in S2 is 4°C~8°C, and the time is more than 12 hours.

6. The use according to claim 1, characterized in that: The reaction time between the detection system and the glucose solution to be tested is more than 4 hours.

7. The use according to any one of claims 1 to 6, characterized in that: The concentration of the glucose solution to be tested is 0~12mM.

8. The use according to claim 1, characterized in that: The ultraviolet absorption spectrum of the reagent to be tested is tested using an ultraviolet spectrophotometer, and the measuring wavelength range of the instrument is ultraviolet-visible light from 190nm to 900nm.

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