Preparation method and application of gold single-atom glucose oxidizing nanozyme

Abstract

The present invention discloses a preparation method and application of a gold single-atom glucose oxidizing nanozyme, which belongs to the field of bioanalysis and detection. The gold single-atom glucose oxidizing nanozyme is a single-atom gold loaded on a graphene carrier. The prepared gold single-atom glucose oxidizing nanozyme itself has glucose oxidase activity and can directly replace natural glucose oxidase for application in devices. The gold single-atom glucose oxidizing nanozyme prepared by the present invention can replace natural glucose oxidase to achieve glucose detection, and compared with glucose sensors based on natural glucose oxidase, it improves detection stability and service life.

Description

Technical Field

[0001] The present invention belongs to the field of biological analysis and detection, and specifically relates to a preparation method and application of a gold single-atom glucose oxidizing nanozyme. Background Art

[0002] Diabetes and other blood sugar-related diseases have become a serious threat to human health. Patients with diabetes need to continuously monitor their blood sugar levels in real time, receive early warnings for abnormal blood sugar levels, and monitor changes in blood sugar levels over several hours or days. Real-time monitoring of blood sugar levels can help patients make more informed decisions about how to balance food, physical activity, and medication throughout the day. Common blood sugar monitoring technologies are based on natural glucose oxidase sensors, which use glucose oxidase to recognize glucose and transmit signals for sensitive detection. However, a major challenge facing continuous monitoring technology is that natural glucose oxidase has a short lifespan. After repeated use, the detection sensitivity significantly decreases, greatly shortening the lifespan of the detection instrument.

[0003] A promising solution is to replace traditional glucose oxidase with nanozymes. Nanozymes are a new generation of artificial enzyme mimics, a class of nanomaterials with catalytic efficiency and enzymatic reaction kinetics similar to natural enzymes. Compared to traditional natural enzymes (primarily proteins), they are more stable and can maintain high activity under extreme conditions. Therefore, using nanozyme materials with glucose oxidase activity to replace natural enzymes and applying them to blood glucose monitors can greatly improve the lifespan of the monitoring equipment.

[0004] Based on the above analysis, the preparation method and application of a gold single-atom glucose oxidase nanozyme that has the characteristics of simulating glucose oxidase, can efficiently catalyze glucose oxidation, realize electrochemical detection and colorimetric detection of glucose, and improve the life of glucose oxidase sensors are urgently needed in the industry. Summary of the Invention

[0005] In view of the above-mentioned shortcomings, the present invention provides a preparation method and application of a gold single-atom glucose oxidase nanozyme. The gold single-atom nanozyme has the characteristics of mimicking glucose oxidase, can efficiently catalyze glucose oxidation, realize electrochemical detection and colorimetric detection of glucose, and improve the life of the glucose oxidase sensor.

[0006] The present invention is achieved by the following means:

[0007] A method for preparing a gold single-atom glucose oxidizing nanozyme, comprising:

[0008] Gold single-atom nanozymes are prepared by loading gold in the form of single atoms on a graphene carrier. The specific preparation process is as follows:

[0009] (1) 30 mL of a graphene oxide suspension having a concentration of 5-20 mg / mL prepared by the Hummers method was taken for later use, 15 mL of a 0.5-2 mol / L ammonium carbonate solution was added thereto, and the mixture was thoroughly mixed by magnetic stirring for 20-60 min to obtain a first mixed solution;

[0010] (2) slowly adding 15 mL of a 0.5-2 mmol / L aqueous solution of chloroauric acid to the first mixed solution, continuing stirring for 2-4 h and mixing evenly to obtain a second mixed solution;

[0011] (3) filtering the second mixed solution, collecting a solid sample, repeatedly rinsing with deionized water, and then drying at 70° C. for 2 h to obtain a solid sample;

[0012] (4) The solid sample is placed in a tube furnace and sintered at 400-550°C for 1-4 hours in an air atmosphere to obtain a gold single-atom glucose oxidizing nanozyme.

[0013] The present invention also discloses a gold single-atom glucose oxidizing nanozyme prepared according to the above preparation method.

[0014] The present invention also discloses an application of the gold single-atom glucose oxidizing nanozyme in glucose activity detection.

[0015] Furthermore, the glucose activity detection includes:

[0016] Colorimetric detection; and

[0017] Electrochemical detection.

[0018] Furthermore, the color reaction detection includes:

[0019] Colorimetric detection includes:

[0020] (1) In 60 μL of 5-20 mg / mL gold single-atom glucose oxidizing nanozyme suspension, 72 μL of 15-60 mM chromogenic substrate 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 30 μL of 2-10 mg / mL ferrosoferric oxide suspension were mixed to obtain a first mixed solution;

[0021] (2) Add 2.4 mL of HAc-NaAc buffer solution with a pH of 3.6 to the first mixed solution, place the solution in a quartz dish of a UV spectrophotometer, preheat to 37°C, and measure the absorption spectrum in the wavelength range of 400 nm to 900 nm. The wavelength for measuring absorbance is 419 nm.

[0022] (3) Measure the absorbance of the solution at a wavelength of 419 nm. After adding 600 μL of a glucose solution of a certain concentration ranging from 1 mM to 20 mM, monitor the absorbance change in real time.

[0023] (4) Establish the relationship between absorbance and glucose concentration to achieve sensitive detection of glucose.

[0024] Furthermore, the gold single-atom glucose oxidizing nanozyme suspension is prepared by the following method:

[0025] 5-20 mg of gold single-atom glucose oxidizing nanozyme was added to 1 mL of water and ultrasonically shaken for 15 minutes to obtain a gold single-atom glucose oxidizing nanozyme suspension.

[0026] Furthermore, the electrochemical detection includes:

[0027] (1) Electrode coating: Grind and mix 10-50 mg of gold single-atom glucose oxidizing nanozyme and 1-5 mg of PVDF, add NMP and continue grinding until it becomes a slurry, then evenly apply the slurry on the surface of the current collector and dry it at 60°C for 10-15 hours;

[0028] (2) Establishment of a three-electrode reaction cell: the working electrode is a current collector loaded with gold single-atom glucose oxidizing nanozyme, the counter electrode is Pt, the reference electrode is saturated AgCl, and the electrolyte is PBS buffer with pH 7-7.5 or 0.05-0.1 mM NaOH solution;

[0029] (3) Cyclic voltammetry for glucose detection: Select an appropriate voltage scanning range based on the electrolyte and electrode. Taking PBS electrolyte as an example, the scanning voltage is -0.6V to 0V. Glucose solutions of different concentrations are added to the electrolyte, and the response cyclic voltammetry curve is collected. For the conductive carbon cloth current collector, read the current response value corresponding to the boundary voltage, establish the relationship between this value and the glucose concentration, and achieve the purpose of glucose detection. For the copper foil current collector, read the response current intensity at the redox peak (-0.3V to -0.4V), establish the relationship between this intensity and the glucose concentration, and achieve the purpose of glucose detection.

[0030] (4) Chronoamperometry for glucose detection: Under a constant voltage of -0.35 V, the instantaneous current response was monitored in real time. After voltage loading for 1 min, the initial current response stabilized. Thereafter, 1 mM to 10 mM glucose was added to the electrolyte every 2 min, and the changes in the instantaneous current response were observed.

[0031] The beneficial effects of the present invention are:

[0032] The gold single-atom glucose oxidizing nanozyme prepared by the present invention can replace natural glucose oxidase and be used in devices to realize glucose detection. Compared with glucose sensors based on natural glucose oxidase, it has improved stability and service life. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the color reaction result of gold single-atom glucose oxidation nanozyme;

[0034] Figure 2 To characterize the glucose oxidase activity of gold single-atom glucose oxidizing nanozyme;

[0035] Figure 3 is the absorbance of the solution at a wavelength of 419 nm, and the absorbance change after adding glucose solution;

[0036] Figure 4 The results show the effect of glucose solution concentration on the slope of the cyclic voltammetry curve of the conductive carbon cloth current collector;

[0037] Figure 5 The results show the effect of glucose solution concentration on the shape of cyclic voltammetry curve and redox peak intensity of copper foil current collector;

[0038] Figure 6 In order to use copper foil as the current collector, the results of the instantaneous current response were monitored in real time at a constant pressure of -0.35V. DETAILED DESCRIPTION

[0039] The present invention is further illustrated by the following examples, but is not limited to the following examples. Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0040] Example 1

[0041] A method for preparing a gold single-atom glucose oxidizing nanozyme, comprising:

[0042] Gold single-atom nanozymes are prepared by loading gold in the form of single atoms on a graphene carrier. The specific preparation process is as follows:

[0043] (1) Take 30 mL of a 20 mg / mL graphene oxide suspension prepared by the Hummers method, add 15 mL of a 1 mol / L ammonium carbonate solution, and stir magnetically for 30 min to mix thoroughly.

[0044] (2) Slowly add 15 mL of 1 mmol / L aqueous solution of gold chloride to the mixed solution and continue stirring for 3 h to mix evenly;

[0045] (3) Filter the resulting mixed solution, collect the solid sample, rinse it repeatedly with deionized water, and then dry it at 70°C for 2 h;

[0046] (4) The solid sample was placed in a tube furnace and sintered at 450 °C for 2 h in an air atmosphere to obtain a gold single-atom glucose oxidizing nanozyme.

[0047] Example 2

[0048] A method for preparing a gold single-atom glucose oxidizing nanozyme, comprising:

[0049] Gold single-atom nanozymes are prepared by loading gold in the form of single atoms on a graphene carrier. The specific preparation process is as follows:

[0050] (1) Take 30 mL of a 5 mg / mL graphene oxide suspension prepared by the Hummers method, add 15 mL of a 0.5 mol / L ammonium carbonate solution, and stir magnetically for 30 min to mix thoroughly.

[0051] (2) Slowly add 15 mL of 0.5 mmol / L aqueous solution of gold chloride to the mixed solution and continue stirring for 2 h to mix evenly;

[0052] (3) Filter the resulting mixed solution, collect the solid sample, rinse it repeatedly with deionized water, and then dry it at 60°C for 3 h;

[0053] (4) The solid sample was placed in a tube furnace and sintered at 400 °C for 3 h in an air atmosphere to obtain a gold single-atom glucose oxidizing nanozyme.

[0054] Example 3

[0055] A method for preparing a gold single-atom glucose oxidizing nanozyme, comprising:

[0056] Gold single-atom nanozymes are prepared by loading gold in the form of single atoms on a graphene carrier. The specific preparation process is as follows:

[0057] (1) Take 30 mL of a 10 mg / mL graphene oxide suspension prepared by the Hummers method, add 15 mL of a 2 mol / L ammonium carbonate solution, and stir magnetically for 30 min to mix thoroughly.

[0058] (2) Slowly add 15 mL of 2 mmol / L aqueous solution of gold chloride to the mixed solution and continue stirring for 4 h to mix evenly;

[0059] (3) Filter the resulting mixed solution, collect the solid sample, rinse it repeatedly with deionized water, and then dry it at 80°C for 1 h;

[0060] (4) The solid sample was placed in a tube furnace and sintered at 550 °C for 1 h in an air atmosphere to obtain a gold single-atom glucose oxidizing nanozyme.

[0061] Test Example 1

[0062] Gold single-atom glucose oxidase nanoenzyme material exhibits glucose oxidase activity

[0063] The gold single-atom glucose oxidizing nanozyme prepared in Example 1 has glucose oxidase activity itself and can directly replace natural glucose oxidase and be used in devices.

[0064] The glucose oxidase activity of the gold single-atom glucose oxidizing nanozyme was calibrated using a colorimetric reaction. The specific steps are as follows:

[0065] (1) Mix 20 μL of 20 mg / mL gold single-atom glucose oxidizing nanozyme suspension with 200 μL of 80 mM glucose solution and incubate at 37°C for 10-60 min.

[0066] (2) 24 μL of 60 mM chromogenic substrate 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 10 μL of 10 mg / mL peroxidase-active ferroferric oxide suspension, and 800 μL of pH 3.6 HAc-NaAc buffer were added to the above solution in sequence and mixed well.

[0067] (3) Observe the color development process of the solution (such as Figure 1 As shown), in the control sample, no gold single atom material was added, and the solution did not change color; after 10 minutes, the color of the solution with gold single atom glucose oxidizing nanozyme changed from colorless to green, and the color became darker after 5 hours, which was close to the effect of natural enzyme (the first sample on the right); this shows that gold single atom has catalytic activity similar to glucose oxidase, which can catalyze the oxidation reaction of glucose to produce hydrogen peroxide, thereby causing ABTS to change color; the glucose oxidase activity of gold single atom glucose oxidizing nanozyme was characterized by measuring the absorbance of the mixed solution at a wavelength of 419nm (as shown Figure 2 As shown in the figure, it can be seen that the absorbance at 419 nm wavelength increases significantly after the addition of glucose. The higher the absorbance intensity, the darker the solution color, indicating a higher glucose oxidase activity.

[0068] (4) Comparative Sample Preparation and Characterization The above process was repeated, replacing the glucose solution with an equal volume of deionized water.

[0069] Test Example 2

[0070] Establishment of a glucose detection method based on the glucose oxidase activity of gold single-atom glucose oxidizing nanozymes

[0071] 1. Color reaction method for detecting glucose

[0072] Gold single-atom glucose oxidizing nanozymes catalyze the reaction of glucose to produce H2O2. Based on the peroxidase activity of the H2O2 product and ferroferric oxide, the chromogenic substrate undergoes a color reaction. The presence of glucose is detected by measuring the change in absorbance of the mixed solution at a wavelength of 419nm after the color reaction. The specific steps are as follows:

[0073] (1) In 60 μL of a 5-20 mg / mL gold single-atom glucose oxidizing nanozyme suspension (5-20 mg of gold single-atom glucose oxidizing nanozyme was added to 1 mL of water and ultrasonicated for 15 min to obtain a gold single-atom glucose oxidizing nanozyme suspension), 72 μL of a 15-60 mM chromogenic substrate 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 30 μL of a 2-10 mg / mL peroxidase-active ferroferric oxide suspension were mixed evenly;

[0074] (2) Add 2.4 mL of HAc-NaAc buffer solution of pH 3.6 to the above mixed solution, place it in a quartz dish of UV spectrophotometer, preheat to 37°C, and measure the absorption spectrum in the wavelength range of 400 nm-900 nm (such as Figure 2 As shown), since the absorption spectrum has a characteristic absorption peak at 419 nm, the wavelength for testing absorbance is selected to be 419 nm;

[0075] (3) Measure the absorbance of the solution at a wavelength of 419 nm. After adding 600 μL of a glucose solution of a certain concentration, monitor the absorbance change in real time (e.g. Figure 3 As shown in the figure, it can be seen that after adding glucose solution, the absorbance increases instantaneously, indicating that the gold single-atom nanozyme catalyzes the reaction of glucose, thereby causing ABTS to react with color;

[0076] (4) Establish the relationship between absorbance and glucose concentration to achieve sensitive detection of glucose.

[0077] 2. Electrochemical detection

[0078] Gold single-atom glucose oxidizing nanozymes catalyze glucose reactions, generating electron transfer. Electrochemical methods are then used to collect the electrochemical signals generated by the electron transfer, achieving the purpose of glucose detection. The specific steps are as follows:

[0079] (1) Electrode coating: Grind and mix 10-50 mg of gold single-atom glucose oxidizing nanozyme and 1-5 mg of PVDF, add NMP and continue grinding until it becomes a slurry, then evenly apply the slurry on the surface of the current collector (conductive carbon cloth or copper foil) and dry it at 60°C for 10-15 hours;

[0080] (2) Establishment of a three-electrode reaction cell: the working electrode is a current collector loaded with gold single-atom glucose oxidizing nanozyme, the counter electrode is Pt, the reference electrode is saturated AgCl, and the electrolyte is PBS buffer with pH 7-7.5 or 0.05-0.1 mM NaOH solution;

[0081] (3) Cyclic voltammetry for glucose detection: Select an appropriate voltage scanning range based on the electrolyte and electrode. Taking PBS electrolyte as an example, the scanning voltage is -0.6V to 0V. Add glucose solutions of different concentrations to the electrolyte and collect the response cyclic voltammetry curves. For the conductive carbon cloth current collector (such as Figure 4 As shown in the figure, the concentration of glucose solution affects the slope of the cyclic voltammetry curve. At a voltage of -0.6V, the response current decreases as the glucose concentration increases. Read the current response value corresponding to the boundary voltage and establish the relationship between this value and the glucose concentration to achieve the purpose of glucose detection. For the copper foil current collector (such as Figure 5 As shown in the figure, the concentration of glucose solution affects the shape of the cyclic voltammetry curve and the intensity of the redox peak. The response current intensity at the redox peak (-0.3V to -0.4V) increases with the increase of glucose concentration. The relationship between this intensity and glucose concentration is established to achieve the purpose of glucose detection.

[0082] (4) Chronoamperometry for glucose detection: Using copper foil as the current collector, the instantaneous current response is monitored in real time at a constant pressure of -0.35 V. During this period, a certain concentration of glucose is added to the electrolyte to observe the changes in the instantaneous current response, such as Figure 6 As shown, after the concentration of glucose solution increased from 0 to 10 mM, the response current increased from -0.17 A to -0.11 A, indicating that this method can realize glucose detection.

[0083] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of a gold single-atom glucose oxidizing nanozyme in glucose activity detection, comprising: (1) Preparation of gold single-atom glucose oxidizing nanozymes; (2) Colorimetric detection or electrochemical detection; wherein: Preparation of gold single-atom glucose oxidizing nanozyme, including the following steps: ① Take 30 mL of 5-20 mg / mL graphene oxide suspension for later use, add 15 mL of 0.5-2 mol / L ammonium carbonate solution, and stir magnetically for 20-60 minutes to obtain a first mixed solution for later use; ② Slowly add 15 mL of 0.5-2 mmol / L aqueous solution of gold chloride to the first mixed solution and continue stirring for 2-4 hours to obtain a second mixed solution for use; ③ Filter the second mixed solution, collect the solid sample, rinse repeatedly with deionized water, and dry at 60-80°C for 1-4 hours to obtain a solid sample; ④ Place the solid sample in a tube furnace and sinter it at 400-550°C in an air atmosphere for 1-4 hours to obtain the gold single-atom glucose oxidizing nanozyme; The color reaction detection includes the following steps: (1.1) Add 5-20 mg of gold single-atom glucose oxidizing nanozyme to 1 mL of water and ultrasonically vibrate for 15 minutes to obtain a gold single-atom glucose oxidizing nanozyme suspension. Add 72 μL of 15-60 mM chromogenic substrate 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and 30 μL of 2-10 mg / mL ferrosoferric oxide suspension to 60 μL of 5-20 mg / mL gold single-atom glucose oxidizing nanozyme suspension and mix well to obtain a mixed solution; (1.2) Add 2.4 mL of HAc-NaAc buffer (pH 3.6) to the above mixture, place in a quartz cuvette of a UV spectrophotometer, preheat to 37°C, and measure the absorption spectrum in the wavelength range of 400 nm to 900 nm, with the wavelength of 419 nm selected for the absorbance measurement. (1.3) Measure the absorbance of the solution at 419 nm. After adding 600 μL of 1 mM to 20 mM glucose solution, monitor the absorbance change in real time. (1.4) Establish the relationship between absorbance and glucose concentration to achieve sensitive detection of glucose; Electrochemical detection includes the following steps: (2.1) Electrode coating: Grind and mix 10-50 mg of gold single-atom glucose oxidizing nanozyme and 1-5 mg of PVDF, add NMP and continue grinding until it becomes a slurry. Then, apply the slurry evenly on the surface of the current collector and dry it at 60°C for 10-15 hours. (2.2) Establishment of a three-electrode reaction cell: the working electrode is a current collector loaded with gold single-atom glucose oxidizing nanozyme, the counter electrode is Pt, the reference electrode is saturated AgCl, and the electrolyte is PBS buffer with pH 7-7.5 or 0.05-0.1 mM NaOH solution; (2.3) Cyclic voltammetry for glucose detection: Select an appropriate voltage scan range based on the electrolyte and electrodes, add glucose solutions of varying concentrations to the electrolyte, and collect response cyclic voltammetry curves. For the conductive carbon cloth current collector, read the current response value corresponding to the boundary voltage and establish a relationship between this value and glucose concentration to achieve glucose detection. For the copper foil current collector, read the response current intensity at the redox peak of -0.3V to -0.4V and establish a relationship between this intensity and glucose concentration to achieve glucose detection. (2.4) Chronoamperometry for glucose detection: At a constant voltage of -0.35 V, the instantaneous current response was monitored in real time. After 1 min of voltage loading, the initial current response stabilized. 1 mM to 10 mM glucose was then added to the electrolyte every 2 min, and the changes in the instantaneous current response were observed.

2. The use according to claim 1, wherein: The graphene oxide suspension in step ① is prepared by the Hummers method.

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