Cobalt monatomic nano-enzyme and application thereof in construction of glutathione colorimetric sensor

Abstract

The invention discloses a cobalt monatomic nano-enzyme and application thereof in construction of a glutathione colorimetric sensor, a preparation method of the cobalt monatomic nano-enzyme comprises the following steps: dispersing CaC2, halogenated benzene, a cobalt source and a nitrogen source in an aqueous solution of ethanol, carrying out ball milling under the protection of an inert atmosphere, and carrying out vacuum drying to obtain the cobalt monatomic nano-enzyme. And carrying out vacuum drying, heat treatment, washing, stripping, centrifugation and vacuum drying again to prepare the Co-2N-GY. Wherein 2N-GY and transition metal Co atoms form a new coordination form, and have a strong catalysis effect on adsorbed oxygen, so that superoxide free radicals are formed, and oxidation color development of the color developing agent is further caused. Meanwhile, the in-situ growth Co monatomic coordinated 2N-GY nano-enzyme has a high specific surface area and abundant active sites, so that the activity of the oxide-like enzyme is greatly enhanced. The colorimetric sensor constructed based on Co-2N-GY and used for detecting GSH has the advantages of being high in sensitivity, good in selectivity and wide in linear range.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials and biological detection, and in particular relates to a cobalt single-atom nanozyme and its application in constructing a glutathione colorimetric sensor, and specifically relates to a dinitrogen graphene-based cobalt single-atom nanozyme and its preparation method, and a method for detecting glutathione using a colorimetric sensor constructed using the same. Background Art

[0002] As a new generation of artificial enzymes, nanozymes have shown great application potential in many fields such as biomedicine, environmental monitoring, and food safety. However, traditional nanozymes often have problems such as unclear catalytic mechanisms, and the need to improve activity and specificity, which limits their further development and wide application. The emergence of single-atom nanozymes has brought new hope for solving these problems. Single-atom nanozymes have become a research hotspot in the current field of nanozymes due to their unique atomically dispersed active site structure, high atomic utilization, clear and uniform active sites, and easy regulation of catalytic performance.

[0003] In the prior art, single-atom nanozymes are generally made of carbon materials and metal elements such as iron, molybdenum, and manganese. Cobalt has broad application prospects in the fields of catalysis and biomedicine. In terms of catalysis, Co-based alloys exhibit unique catalytic ability, enhance mobility-induced activity, and thus effectively improve the efficiency of catalytic reactions; at the same time, Co 2+ with Fe 3+ It has similarity and can be simulated by Fe 3+ It can enter the bacteria to achieve antibacterial treatment without causing cross-resistance of bacteria. However, traditional cobalt catalysts are generally in block form, and their catalytic activity still needs to be further improved.

[0004] Glutathione (GSH) is widely present in animals and plants, and plays an important role in organisms. For example, as an important antioxidant in the body, glutathione can remove free radicals in the human body; because glutathione itself is susceptible to oxidation by certain substances, it can protect the sulfhydryl groups in many proteins and enzymes from being oxidized by harmful substances in the body, thereby ensuring the normal physiological functions of proteins and enzymes; the content of glutathione in human red blood cells is high, which is of great significance for protecting the sulfhydryl groups of proteins on the red blood cell membrane in a reduced state and preventing hemolysis. Therefore, it is of great practical significance to construct a sensor for the detection of glutathione.

[0005] Currently, the detection methods of glutathione still have technical problems such as low sensitivity, time-consuming detection, high cost and complicated steps. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. For this purpose, the present invention provides a cobalt single-atom nanozyme and its application in constructing a glutathione colorimetric sensor. The cobalt single-atom nanozyme is a novel nanozyme with high efficiency and stability and single-atom-like peroxidase activity. The colorimetric sensing system constructed using this cobalt single-atom nanozyme for detecting glutathione can achieve high sensitivity and high selectivity in glutathione detection.

[0007] The inventive concept of the present invention is as follows: Traditional nitrogen doping can change the electronic configuration of graphdiyne, and cooperate with metals to further optimize the electron arrangement, which is more conducive to the adsorption and desorption of metal atoms, and ultimately significantly improves the reaction performance. However, the nitrogen-doped carbon obtained by such methods lacks uniformity, so the so-called M-N-C active centers constructed are randomly distributed, which greatly reduces their activity. In the present invention, a nitrogen source and a carbon source are subjected to high-energy ball milling in a fixed ratio to form a nitrogen-carbon compound with a special structure, achieving a structure with uniform distribution of nitrogen and carbon at the atomic level, and then forming dinitrogen graphdiyne (2N-GY). The interaction between nitrogen and carbon in 2N-GY is quite different from that of C-C, enabling it to coordinate with metal ions at the atomic level to form a new type of M-N-C structure. Due to the anisotropy of this structure, the metal active centers are more active and suitable for applications in the sensing field.

[0008] At the same time, 2N-GY is a two-dimensional carbon-based material with sp and sp 2 two hybrid carbons, so it has characteristics such as uniformly distributed pores, high carrier mobility, excellent stability, large specific surface area, and superior conductivity. These characteristics make it an ideal single-atom carrier material. Moreover, 2N-GY has complex C-N, -N=N- functional groups, which form a new coordination form with transition metal Co atoms and have a strong catalytic effect on the adsorption of oxygen to form superoxide radicals, thereby causing the oxidation and color development of the color reagent.

[0009] The present invention synthesizes dinitrogen graphdiyne-based cobalt single-atom nanozyme (Co-2N-GY) by a one-pot method. Through the ball milling of a nitrogen source (such as polydopamine, PDA) and transition metal cobalt, the 2N-GY nanozyme coordinated with in-situ grown Co single atoms has a high specific surface area and abundant active sites. Different from the traditional coordination form of single atoms, the cobalt single-atom nanozyme of the present invention has peroxidase-like activity and can catalyze free O 2 to form superoxide radicals. Based on the peroxidase-like activity of Co-2N-GY, a colorimetric sensor for quantitative determination of GSH is established. In the absence of GSH, the peroxidase-like activity of Co-2N-GY catalyzes free O 2Superoxide radicals are formed to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) into oxidized TMB (ox-TMB), and ox-TMB has strong ultraviolet absorption at 652 nm. When GSH is present, the generated ox-TMB in the system decreases, and the absorbance at 652 nm decreases significantly, and it has excellent stability and reusability. This not only helps to promote the development of single-atom nanozyme technology, but also lays a foundation for the development of other cobalt-based single-atom biosensors and bioanalysis methods, and has broad application prospects in the fields of biomedical research and clinical diagnosis.

[0010] To solve the above technical problems, the first aspect of the present invention provides a preparation method of a cobalt single-atom nanozyme, comprising the following steps:

[0011] Disperse CaC 2 , halogenated benzene, cobalt source, and nitrogen source in an aqueous solution of ethanol, carry out ball milling under the protection of an inert atmosphere, and obtain cobalt single-atom nanozyme with two-nitrogen graphdiyne groups, denoted as Co-2N-GY, after vacuum drying, heat treatment, washing, exfoliation, centrifugation, and then vacuum drying.

[0012] In some embodiments of the present invention, the halogenated benzene is selected from at least one of fluorobenzene, chlorobenzene, and bromobenzene.

[0013] In some embodiments of the present invention, the cobalt source includes cobalt acetylacetonate [Co(acca) 3 .

[0014] In some embodiments of the present invention, the nitrogen source includes polydopamine.

[0015] In some embodiments of the present invention, the mass-volume ratio of the CaC 2 and the halogenated benzene is (1-50) g:(0.1-20) mL.

[0016] In some embodiments of the present invention, the mass ratio of the nitrogen source to the cobalt source is 1:(0.1-0.5).

[0017] In some embodiments of the present invention, the mass ratio of the CaC 2 and the nitrogen source is 1:(0.1-5).

[0018] In some embodiments of the present invention, the inert atmosphere is a nitrogen atmosphere or an argon atmosphere.

[0019] In some embodiments of the present invention, the process conditions for ball milling are as follows: using zirconia as the ball milling beads, first ball milling at a speed of 200 - 800 rpm for 6 - 10 hours, with a pause of 10 - 20 minutes for cooling every 20 - 40 minutes during this period; then ball milling at a speed of 200 - 600 rpm for 6 - 10 hours, with a pause of 10 - 20 minutes for cooling every 20 - 40 minutes during this period.

[0020] In some embodiments of the present invention, the vacuum drying is carried out overnight at a temperature of 50 - 70 °C to remove the solvent.

[0021] In some embodiments of the present invention, the temperature regime for heat treatment is as follows: first heating at a rate of 3 - 8 °C / min to 300 - 500 °C; then heating at a rate of 1 - 3 °C / min to 700 - 900 °C, holding for 100 - 150 minutes, and naturally cooling to room temperature.

[0022] In some embodiments of the present invention, the washing is first carried out by washing 1 - 5 times with 1 - 3 mol / L HNO 3 and 0.1 - 2 mol / L HAc respectively, and then repeatedly washing with deionized water until neutral.

[0023] In some embodiments of the present invention, the exfoliation is first to disperse the washed product in ethanol, and then use a high - energy ultrasonic rod to carry out exfoliation at a power of 2500 - 3500 W for 2 - 12 hours.

[0024] The second aspect of the present invention provides a cobalt single - atom nanozyme, which is prepared by the above - mentioned preparation method. The cobalt single - atom nanozyme includes a diazographene substrate and cobalt single atoms, and the cobalt single atoms are uniformly distributed on the surface of the diazographene substrate.

[0025] The third aspect of the present invention provides a method for detecting glutathione based on the above - mentioned cobalt single - atom nanozyme - constructed colorimetric sensor, including the following steps:

[0026] (1) Constructing a glutathione colorimetric sensor: adding different concentrations of glutathione, 3,3',5,5' - tetramethylbenzidine, H 2 O 2 and the above - mentioned Co - 2N - GY into a buffer solution, heating, and incubating to obtain a detection system; then measuring the absorbance value of the detection system at 652 nm by ultraviolet - visible spectrophotometry. Taking the concentration of glutathione as the abscissa and the absorbance value at 652 nm as the ordinate, establish a glutathione standard curve;

[0027] (2) Colorimetric sensor sample detection: Measure the absorbance value of the sample to be tested containing glutathione at 652 nm according to step (1), and substitute the absorbance value at 652 nm into the glutathione standard curve of step (1) to obtain the concentration of glutathione in the sample to be tested.

[0028] In some embodiments of the present invention, in step (1), the buffer solution is an acetic acid-sodium acetate (HAc-NaAc) buffer solution with a pH value of 3-5.

[0029] In some embodiments of the present invention, in step (1), the incubation temperature is 40-50 °C; and / or, the incubation time is 1-30 min.

[0030] In some embodiments of the present invention, in step (1), in the detection system, the final concentration of Co-2N-GY is 0.05-0.25 μg / mL, the concentration of 3,3',5,5'-tetramethylbenzidine is 0.5-1.0 mmol / L, and the concentration of glutathione is 0-600 μmol / L.

[0031] In some embodiments of the present invention, in step (1), the H 2 O 2 addition amount is quantitative; preferably 5 wt% H 2 O 2 60 μL.

[0032] The above technical solutions of the present invention have at least the following technical effects or advantages compared with the prior art:

[0033] (1) The present invention synthesizes cobalt single-atom nanozyme (Co-2N-GY) based on two-dimensional nitrogenated graphdiyne by a one-pot method. Among them, 2N-GY has complex C-N and -N=N- functional groups, which form a new coordination form with transition metal Co atoms, and has a strong catalytic effect on adsorbed oxygen, thus forming superoxide radicals, which in turn cause the oxidation and color development of the color developer. At the same time, the present invention uses PDA and transition metal cobalt for ball milling, and the in-situ growth of 2N-GY nanozyme coordinated with Co single atoms has a high specific surface area and rich active sites, different from the coordination forms of traditional single atoms, which greatly enhances the peroxidase-like activity.

[0034] (2) The preparation method of the present invention has strong controllability. The performance of the nanozyme can be regulated by changing the heat treatment temperature, which provides possibilities for its diverse applications. The prepared Co-2N-GY has high pseudo-oxidase activity, as well as excellent stability and reusability. Therefore, the colorimetric sensor for detecting GSH constructed based on Co-2N-GY has the advantages of high sensitivity, good selectivity, and a wide linear range. The linear range of GSH is 0 - 600 μmol / L; the detection is linearly good in the range of 0 - 30 μmol / L, and the lowest detection limit is 0.55 μmol / L. Description of the Drawings

[0035] Figure 1 It is the route map for synthesizing Co-2N-GY in Example 1;

[0036] Figure 2 It is the structural schematic diagram of Co-2N-GY synthesized in Example 1;

[0037] Figure 3 It is the XRD pattern of GY in Comparative Example 2, 2N-GY and Co-2N-GY synthesized in Comparative Example 1 and Example 1;

[0038] Figure 4 It is the SEM image of Co-2N-GY and 2N-GY synthesized in Example 1 and Comparative Example 1;

[0039] Figure 5 It is the AC-HAADF-STEM image of Co-2N-GY synthesized in Example 1;

[0040] Figure 6 It is the test result graph for optimizing the optimal wavelength for TMB detection;

[0041] Figure 7 It is the test result graph for optimizing the pH value range for TMB color development;

[0042] Figure 8 It is the test result graph for optimizing the concentration range for TMB detection;

[0043] Figure 9 It is the test result graph of the standard curve of GSH. Detailed Description of the Invention

[0044] The present invention will be specifically described below in conjunction with embodiments to facilitate the understanding of those skilled in the art. It is necessary to specifically point out here that the embodiments are only used to further illustrate the present invention and should not be construed as limiting the protection scope of the present invention. Those skilled in the art, based on the above-mentioned invention content, make non-essential improvements and adjustments to the present invention, which should still fall within the protection scope of the present invention. At the same time, for the raw materials not specifically described below, they are all commercially available products; for the process steps or preparation methods not specifically mentioned, they are all process steps or preparation methods known to those skilled in the art.

[0045] Example 1: One-pot synthesis of cobalt single-atom nanozyme Co-2N-GY based on diazographene

[0046] The one-pot synthesis of cobalt single-atom nanozyme based on diazographene has a synthesis route as Figure 1 shown, and specifically includes the following steps:

[0047] Take 10 g of CaC 2 , 2 mL of hexabromobenzene, 3 mmol of Co(acca) 3 , 3 g of polydopamine and 20 g of zirconia ball milling beads with a diameter of 5 mm and place them in a 45 mL ball milling jar lined with ZrO 2 . Add 30 mL of absolute ethanol as a solvent. Under the protection of a nitrogen atmosphere (ensuring isolation of O 2 and water vapor), first ball mill at a speed of 800 rpm for 8 hours (pause for 15 minutes every 30 minutes for cooling), and then ball mill at a speed of 400 rpm for 8 hours (pause for 15 minutes every 30 minutes for cooling). The obtained intermediate product is vacuum dried at 60 °C overnight to remove the solvent, and then heat treated under the protection of a nitrogen atmosphere. First, heat up from room temperature to 400 °C at a rate of 5 °C / min, and then heat up to 800 °C at a rate of 1 °C / min, hold for 120 minutes, and naturally cool to room temperature. The heat-treated product is washed 5 times with 1 - 3 mol / L of HNO 3 and 0.1 - 2 mol / L of HAc respectively, and then repeatedly washed with deionized water until neutral; then it is dispersed in ethanol and ultrasonically peeled (high-energy ultrasonic rod) for 6 hours, centrifuged and washed, and then vacuum dried to obtain cobalt single-atom nanozyme based on diazographene, denoted as Co-2N-GY, and its structural schematic diagram is as Figure 2 shown.

[0048] Comparative Example 1: Synthesis of diazographene 2N-GY

[0049] The synthesis of diazographene by ball milling method includes the following steps:

[0050] Take 5 g of CaC 2, 1 mL of hexabromobenzene, 50 mg of polydopamine, and 80 g of zirconia milling beads with a diameter of 5 mm were placed in a 45 mL ball milling jar lined with ZrO 2 . 17 mL of absolute ethanol was poured into the jar as a solvent. Under the protection of a nitrogen atmosphere (ensuring isolation from O 2 and water vapor), first, ball milling was carried out at a speed of 600 rpm for 8 hours (pausing for 15 minutes of cooling every 30 minutes), and then ball milling was carried out at a speed of 450 rpm for 8 hours (pausing for 15 minutes of cooling every 30 minutes). After ball milling, the obtained product was pickled with 1 mol / L HNO 3 and 2 mol / L HAc, and then dried overnight in a vacuum environment at 60 °C. Then, the dried solid sample was dispersed in ethanol and ultrasonically peeled (using a high-energy ultrasonic rod) for 6 hours, centrifuged and washed 5 times, and dried again in a vacuum environment to obtain dinitrogen graphdiyne, denoted as 2N-GY.

[0051] Comparative Example 2: Synthesis of graphdiyne GY

[0052] The graphdiyne was synthesized by ball milling, including the following steps:

[0053] Take 5 g of CaC 2 , 1 mL of benzene, and 20 g of zirconia milling beads with a diameter of 5 mm, and place them in a 45 mL ball milling jar lined with ZrO 2 . 15 mL of absolute ethanol was poured into the jar as a solvent. Under the protection of a nitrogen atmosphere (ensuring isolation from O 2 and water vapor), first, ball milling was carried out at a speed of 600 rpm for 8 hours (pausing for 15 minutes of cooling every 30 minutes), and then ball milling was carried out at a speed of 450 rpm for 8 hours (pausing for 15 minutes of cooling every 30 minutes). After ball milling, the obtained product was pickled with 1 mol / L HNO 3 and 2 mol / L HAc, and then dried overnight in a vacuum environment at 60 °C. Then, the dried solid sample was dispersed in ethanol and ultrasonically peeled (using a high-energy ultrasonic rod) for 5 hours, centrifuged and washed, and then dried again in a vacuum environment to obtain graphdiyne, denoted as GY.

[0054] Performance test:

[0055] 1. XRD analysis

[0056] XRD was used to analyze the crystal structures of GY synthesized in Comparative Example 2, and Co-2N-GY and 2N-GY synthesized in Example 1 and Comparative Example 1, respectively. The results are as Figure 3 shown, Figure 3 where the abscissa 2-Theta represents the diffraction angle 2θ, and the ordinate Intensity represents the intensity of the diffraction peak. From Figure 3It can be seen that GY, 2N-GY, and Co-2N-GY all have a broad diffraction peak in the range of 22°-25°, which is generally considered to be a diffraction peak related to the graphite structure, and the absence of any other diffraction peaks indicates that cobalt in Co-2N-GY exists in the form of single atoms. The EDS spectrum of Co-2N-GY also confirms the uniform distribution of Co, N, and C, further indicating that Co exists in the form of single atoms, and verifying that the one-pot synthesis route can obtain highly dispersed single-atom metal nanozymes.

[0057] 2. Microscopic morphology

[0058] The SEM images of Co-2N-GY and 2N-GY synthesized in Example 1 and Comparative Example 1 are as Figure 4 shown. It can be seen from Figure 4 that 2N-GY has a lamellar structure, while there is no obvious phase separation in Co-2N-GY. Further, the AC-HAADF-STEM technique was used to observe the distribution of Co atoms in Co-2N-GY, as Figure 5 shown. It can be clearly observed from Figure 5 that single cobalt atoms are uniformly dispersed on the graphdiyne substrate, and there is no agglomeration of cobalt atoms, providing intuitive and powerful evidence for the structural characteristics of Co-2N-GY as a single-atom nanozyme.

[0059] Example 3: Construction of a GSH colorimetric sensor based on Co-2N-GY

[0060] 1. Comparison of peroxidase-like activities

[0061] The catalytic activities of 2N-GY and Co-2N-GY synthesized in Comparative Example 1 and Example 1 were compared respectively. The experimental process of the catalytic activity is as follows:

[0062] In a saturated HAc-NaAc buffer solution with pH = 4.02 of N 2 , 60 μL of 1 mg / mL Co-2N-GY synthesized in Example 1 and 60 μL of a GSH solution with a concentration of 100 μmol / L were added. Then, 60 μL of 5 wt% H 2 O 2 was added. Subsequently, 30 μL of 0.2 mmol / L TMB was quickly added. It was placed in an oven at 40 °C and incubated for 1-5 min to obtain a detection system. The ultraviolet absorption curve of the detection system in the range of 400-800 nm was measured by ultraviolet-visible (UV-vis) spectrophotometry (as Figure 6 shown), and the absorbance value of the detection system at 652 nm was obtained. The concentration of TMB was detected according to the change in absorbance.

[0063] The results showed that 2N-GY had a lower POD-like activity. Co-2N-GY could catalyze the oxidation of TMB more efficiently compared to 2N-GY, making the absorbance change of the oxidation product of TMB at a specific wavelength (652 nm) more obvious.

[0064] 2. Optimization of pH test range

[0065] The catalytic activity of Co-2N-GY synthesized in Example 1 towards TMB in different pH test ranges was investigated. The experimental procedure was as follows:

[0066] Using TMB as the chromogenic agent, following the same method as for the comparison of POD-like activity, at a fixed wavelength of 652 nm, tests were carried out using buffer solutions with different pH ranges. When pH was between 2 - 6, different ratios of HAc-NaAc buffer solutions were selected. When pH was 7 - 8, different ratios of potassium dihydrogen phosphate - dipotassium hydrogen phosphate buffer systems were used. At N 2 saturation, 60 μL of 1 mg / mL Co-2N-GY synthesized in Example 1 was added, then 60 μL of a 100 μmol / L GSH solution was added, and then 60 μL of 5 wt% H 2 O 2 was added. Subsequently, 30 μL of 0.2 mmol / L TMB was quickly added. It was placed in an oven at 40 °C and incubated for 1 - 5 min to obtain the detection system. The absorbance concentration of TMB was measured by ultraviolet-visible (UV-vis) spectrophotometry.

[0067] The results were as Figure 7 shown, Figure 7 where the abscissa pH value represents the pH value and the ordinate Absorbance represents the absorbance. As Figure 7 can be seen, a maximum value was shown at pH = 4.02 within 5 minutes, demonstrating that under the same temperature and time conditions, Co-2N-GY could catalyze the oxidation of TMB more efficiently at around pH = 4, making the absorbance change of the oxidation product of TMB at a specific wavelength (such as 652 nm) more obvious.

[0068] 3. Optimization of TMB detection concentration range

[0069] The V 0 rates of 2N-GY synthesized in Comparative Example 1 and Co-2N-GY synthesized in Example 1 towards different concentration ranges of TMB were tested. The experimental procedure was as follows:

[0070] On the basis of determining the pH value of the buffer solution to be 4.02, at a fixed wavelength of 652 nm, at N 2Under saturation conditions, 60 μL of 1 mg / mL Co-2N-GY synthesized in Example 1 and 2N-GY synthesized in Comparative Example 1 were respectively added, then 60 μL of a GSH solution with a concentration of 100 μmol / L was added, and then 60 μL of 5 wt% H 2 O 2 was added. Subsequently, 0 - 1 mmol / L of TMB was quickly added. It was placed in an oven at 40 °C and incubated for 1 - 5 min to obtain a detection system. The absorbance of different concentrations of TMB was measured by ultraviolet-visible (UV-vis) spectrophotometry.

[0071] The results are as Figure 8 shown. Figure 8 In [the figure], the abscissa "TMB concentration" represents the concentration of TMB, and the ordinate "V 0 Rate" represents the V 0 rate. As can be seen from Figure 8 it, the Co-2N-GY sample has a high V 0 rate, indicating that its response range to TMB is wider, mainly due to the high dispersion and high activity of Co single atoms, and the highly uniform coordination of Co with the N-C structure is the main catalytic active center.

[0072] 4. Testing of the standard curve

[0073] In 5 mL of N 2 -saturated HAc-NaAc buffer solution with a pH of 4.02, 60 μL of 1 mg / mL Co-2N-GY synthesized in Example 1 and 60 μL of GSH solutions with different concentrations (0, 50 μmol / L, 100 μmol / L, 200 μmol / L, 300 μmol / L, 400 μmol / L, 500 μmol / L, 600 μmol / L) were added. Then 60 μL of 5 wt% H 2 O 2 was added. Subsequently, 30 μL of 0.2 mmol / L of TMB was quickly added. After incubating it in an oven at 30 - 40 °C for 30 min, the absorbance at 652 nm was recorded with a UV-vis spectrophotometer. According to the standard curve of absorbance versus GSH concentration, the quantitative detection of the GSH concentration in an unknown sample was achieved. A GSH standard curve was established with the concentration of GSH as the abscissa and the absorbance value at 652 nm as the ordinate, as Figure 9 shown: Y = 0.3432 - 0.0117x, R 2 = 0.959, and the detection limit LOD was 0.55 μmol / L.

[0074] Example 4: Sample detection

[0075] Dissolve two commercial glutathione supplements in absolute ethanol, let it stand, and take the supernatant to prepare the solution to be measured. Add glutathione standard solutions at three different concentration levels of low, medium, and high (100 μmol / L, 300 μmol / L, and 500 μmol / L respectively) to the solution to be measured so that the concentration is within the linear range (0 - 600 μmol / L), and repeat the measurement 3 times for each concentration level. Measure the absorbance at 652 nm according to the above sensor detection method, and calculate the sample addition recovery rate. The calculation formula is:

[0076] Sample addition recovery rate = (measured value - original value of the sample) / added standard value × 100%.

[0077] Calculate the average value and relative standard deviation (RSD) of the three measurements to evaluate the accuracy and precision of the detection method of the present invention in the detection of actual samples. The results show that: for the sample addition recovery experiment of glutathione at a certain concentration level, the recovery rates of the three measurements are 98.5%, 100.2%, and 99.6% respectively, the average value is 99.4%, and the RSD is 0.8%, indicating that this method has good accuracy and precision.

[0078] For those of ordinary skill in the art to which the present invention pertains, several simple deductions or substitutions can be made without departing from the concept of the present invention, without the need for creative labor. Therefore, all simple improvements made by those skilled in the art based on the disclosure of the present invention should fall within the protection scope of the present invention. The above embodiments are the preferred embodiments of the present invention, and all processes similar to the present invention and equivalent changes made thereto shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a cobalt single-atom nanozyme, characterized in that: The following steps are involved: CaC2, halogenated benzene, cobalt source and nitrogen source are dispersed in an aqueous solution of ethanol, ball-milled under the protection of an inert atmosphere, and then vacuum-dried, heat-treated, washed, peeled, centrifuged and vacuum-dried again to obtain dinitrographene-based cobalt single-atom nanozyme, denoted as Co-2N-GY.

2. The method for preparing the cobalt single-atom nanozyme according to claim 1, characterized in that: The halogenated benzene is selected from at least one of fluorinated benzene, chlorobenzene and bromobenzene; and / or the cobalt source comprises cobalt acetylacetonate; and / or the nitrogen source comprises polydopamine.

3. The method for preparing the cobalt single-atom nanozyme according to claim 1 or 2, characterized in that: The mass volume ratio of the CaC2 and the halogenated benzene is (1-50) g: (0.1-20) mL; and / or, the mass ratio of the nitrogen source and the cobalt source is 1: (0.1-0.5); and / or, the mass ratio of the CaC2 and the nitrogen source is 1: (0.1-5).

4. The method for preparing the cobalt single-atom nanozyme according to claim 1, characterized in that: The inert atmosphere is a nitrogen atmosphere or an argon atmosphere.

5. The method for preparing the cobalt single-atom nanozyme according to claim 1, characterized in that: The temperature system of the heat treatment is: firstly heating to 300-500°C at a rate of 3-8°C / min; then heating to 700-900°C at a rate of 1-3°C / min, keeping the temperature for 100-150 minutes, and naturally cooling to room temperature.

6. A cobalt single-atom nanozyme, characterized in that: The cobalt single-atom nanozyme is prepared by the preparation method described in any one of claims 1 to 5, wherein the cobalt single-atom nanozyme comprises a dinitrogen graphyne substrate and a cobalt single atom, and the cobalt single atom is uniformly distributed on the surface of the dinitrogen graphyne substrate.

7. A method for detecting glutathione using a colorimetric sensor constructed based on cobalt single-atom nanozymes, characterized in that: The following steps are involved: (1) constructing a glutathione colorimetric sensor: adding glutathione, 3,3',5,5'-tetramethylbenzidine, H2O2 and the Co-2N-GY described in claim 6 to a buffer solution, heating, and incubating to obtain a detection system; then measuring the absorbance of the detection system at 652 nm by ultraviolet-visible spectrophotometry, using the concentration of glutathione as the horizontal axis and the absorbance at 652 nm as the vertical axis to establish a glutathione standard curve; (2) Colorimetric sensor sample detection: According to step (1), the absorbance value of the sample to be tested containing glutathione at 652 nm is measured, and the absorbance value at 652 nm is substituted into the glutathione standard curve of step (1) to obtain the concentration of glutathione in the sample to be tested.

8. The method for detecting glutathione using a colorimetric sensor constructed based on cobalt single-atom nanozyme according to claim 7, characterized in that: In step (1), the buffer solution is an acetic acid-sodium acetate buffer solution with a pH value of 3-5.

9. The method for detecting glutathione using a colorimetric sensor constructed based on cobalt single-atom nanozyme according to claim 7, characterized in that: In step (1), the incubation temperature is 40-50° C.; and / or the incubation time is 1-30 min.

10. The method for detecting glutathione using a colorimetric sensor constructed based on cobalt single-atom nanozyme according to claim 7, characterized in that: In step (1), in the detection system, the concentration of Co-2N-GY is 0.05-0.25 μg / mL, the concentration of 3,3',5,5'-tetramethylbenzidine is 0.5-1.0 mmol / L, and the concentration of glutathione is 0-600 μmol / L.

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