Application of NiCo2S4@N,S-rGO nanomaterials in visual detection of glucose

Abstract

The present invention provides the use of a NiCo2S4@N,S-rGO nanomaterial for visual glucose detection. The NiCo2S4@N,S-rGO nanomaterial used in the present invention can catalyze the oxidation of glucose to produce hydrogen peroxide and simultaneously catalyze the reaction between hydrogen peroxide and a chromogenic substrate. The glucose colorimetric assay developed based on this nanocomposite material yields results consistent with those of conventional blood glucose meters, providing satisfactory experimental accuracy and precision for glucose determination in human serum samples. This opens up more options and strategies for the development of rapid, accurate, and low-cost detection of hydrogen peroxide and glucose in complex human fluid samples.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to the application of NiCo2S4@N,S-rGO nanomaterials in glucose visualization detection. Background Art

[0002] Diabetes mellitus is a chronic disease caused by insulin secretion defects or insulin dysfunction, characterized by hyperglycemia. Severe diabetes can lead to various acute and chronic complications, such as cardiovascular disease, diabetic ketoacidosis, renal insufficiency, and insomnia. The International Diabetes Federation predicts that by 2035, nearly 592 million people worldwide will have diabetes. Therefore, timely monitoring of patients' blood glucose and urine glucose levels is extremely important for the prevention and control of diabetes. Hydrogen peroxide (H2O2) is one of the important signaling molecules that regulates fundamental biological processes. Elevated H2O2 levels can trigger irreversible oxidative damage to lipids, proteins, and DNA, as well as several diseases such as neurodegeneration, diabetes, Alzheimer's disease, and even cancer. Therefore, the development of a rapid, sensitive, efficient, and inexpensive method for the determination of hydrogen peroxide and glucose is of practical significance.

[0003] Currently, various methods have been established for glucose detection, including enzymatic methods, glucose meters, electrochemical non-enzymatic methods, high-performance liquid chromatography (HPLC), capillary zone electrophoresis, and Fourier transform infrared spectroscopy (FT-IR). These methods suffer from disadvantages such as the difficulty of storing natural enzymes, complex pretreatment procedures for chromatography, spectroscopy, and electrochemical detection, and low sensitivity of glucose meters. In contrast, the colorimetric method based on horseradish peroxidase (HRP) has attracted widespread attention due to its simplicity, rapidity, minimal background interference, and high sensitivity. This method first oxidizes glucose molecules with oxygen in the presence of glucose oxidase to produce gluconic acid and hydrogen peroxide (H2O2). The peroxidase then decomposes the H2O2 into reactive oxygen species (ROS) such as hydroxyl radicals (·OH). These ROS oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to 3,3',5,5'-tetramethylbenzidinequinone (TMBox). The concentrations of glucose, H2O2, and TMBox exhibit a linear relationship. Therefore, the glucose concentration can be obtained by measuring the absorbance of TMBox at 652nm.

[0004] However, the natural peroxidase is horseradish peroxidase (HRP), an important natural enzyme that is expensive to prepare and difficult to store after denaturation, heating, or chemical changes. In recent years, novel nanozymes with peroxidase-like activity have been developed as alternatives to address the high cost and storage difficulties of these natural enzymes. Notably, their application in glucose and hydrogen peroxide detection has become a research hotspot.

[0005] Since the first introduction of Fe3O4 with peroxidase activity, various nanomaterials have been widely developed as various colorimetric sensing platforms, including metal oxides, bio-carbon-based materials, metal-organic frameworks with porous structures, and various nanohybrids and their derivatives.

[0006] Compared to corresponding metal compounds or metal oxides, transition metal sulfides exhibit superior redox properties and are considered promising nanozymes. Borthakur et al. fabricated transition metal sulfide / porous reduced graphene oxide-based nanohybrids with intrinsic peroxidase-like activity and developed a simple colorimetric sensor utilizing the inhibitory properties of Hg(II) nanozymes. Bimetallic sulfides, particularly NiCo2S4, offer a viable platform for constructing colorimetric sensing due to their complex crystal structure and high peroxidase activity in multiple valence states. However, SSA and poor electrical conductivity compromise the catalytic activity and stability of NiCo2S4. Reported peroxidase-like activities of Cu-Ag / rGO and WS2 / rGO, among others, need further improvement. However, the sensitivity and accuracy of glucose detection in complex human fluid samples are not very good, making them unsuitable for glucose detection in complex human fluid samples. Summary of the Invention

[0007] The purpose of the present invention is to provide an application of NiCo2S4@N,S-rGO nanomaterials containing sulfur vacancies in the visualization detection of glucose.

[0008] Another object of the present invention is to provide a method for visually detecting glucose.

[0009] Another object of the present invention is to provide a glucose visualization detection kit, which includes NiCo2S4@N,S-rGO nanomaterials and a chromogenic substrate.

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

[0011] Application of NiCo2S4@N,S-rGO nanomaterials in visual detection of glucose, wherein the NiCo2S4@N,S-rGO nanomaterials have peroxidase-like activity.

[0012] Specifically, the NiCo2S4@N,S-rGO nanomaterial is a composite nanomaterial in which NiCo2S4 nanoparticles are uniformly dispersed on N, S co-doped graphene sheets.

[0013] Specifically, the specific surface area (SAA) of the NiCo2S4@N,S-rGO is 120-200m 2 / g, more preferably 130 to 180 m 2 / g, more preferably 140 to 160m 2 / g.

[0014] Specifically, the NiCo2S4@N,S-rGO nanomaterial has a porous structure with a pore diameter of 5 to 10 nm, more preferably 7 to 9 nm.

[0015] According to some embodiments, the NiCo2S4@N,S-rGO nanomaterial is prepared by calcining Ni(CH3COO)2·4H2O, CoCl2·6H2O, TAA and graphene oxide.

[0016] Preferably, the calcination temperature is 280-320°C, such as 280°C, 290°C, 300°C, 310°C, 320°C.

[0017] Preferably, the calcination is carried out under N2 atmosphere.

[0018] Preferably, the calcination is carried out in the presence of ethylene glycol.

[0019] Preferably, the calcination is carried out at a heating rate of 3-8°C / min to the desired temperature, for example, 4°C / min, 6°C / min, 7°C / min, or 8°C / min.

[0020] Preferably, the calcination time is 1 to 2 hours.

[0021] Preferably, the feeding mass ratio of Ni(CH3COO)2·4H2O, CoCl2·6H2O, thioacetamide (TAA) and graphene oxide (Go) is (2-5):(3-8):(15-20):1.

[0022] Furthermore, the feed mass ratio of Ni(CH3COO)2·4H2O, CoCl2·6H2O, TAA and graphene oxide is (2.5-3.5):(5-8):(17-19):1.

[0023] According to some embodiments, the preparation method of the NiCo2S4@N,S-rGO nanomaterial is:

[0024] Ni(CH3COO)2·4H2O, CoCl2·6H2O, and TAA were dissolved in ethylene glycol and mixed at room temperature to obtain Solution A. Graphene oxide was then mixed with ethylene glycol to obtain a suspension, which was then slowly added dropwise to Solution A. The resulting mixed solution was heated to 280-320°C at a heating rate of 3-8°C / min and calcined in an N2 atmosphere for 50-80 minutes. The resulting material was washed alternately with deionized water and ethanol one to five times and oven-dried at 50-70°C to obtain dark gray NiCo2S4@N,S-rGO.

[0025] The present invention also provides a method for visually detecting glucose, which is based on the colorimetric method for testing glucose. NiCo2S4@N,S-rGO nanomaterials are used to catalyze the oxidation of glucose to produce hydrogen peroxide, and simultaneously catalyze the reaction between hydrogen peroxide and a chromogenic substrate, wherein the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine.

[0026] Preferably, the temperature of the catalytic reaction of the NiCo2S4@N,S-rGO nanomaterial is 35-50°C, more preferably 38-45°C, more preferably 38-42°C, and even more preferably 39-41°C.

[0027] Preferably, the concentration of the NiCo2S4@N,S-rGO nanomaterial in the reaction system is 0.5-2.0 mg / mL, more preferably 0.5-1.75 mg / mL, more preferably 0.5-1.5 mg / mL, and even more preferably 0.8-1.2 mg / mL.

[0028] Preferably, the initial pH value of the reaction system is 3-4.

[0029] Preferably, after the reaction is completed, the absorbance of the reaction system is tested, and the absorbance range is 625-675 nm, more preferably 630-660 nm, more preferably 640-660 nm, and even more preferably 650-658 nm.

[0030] Preferably, the test sample is one or more of blood, serum or urine.

[0031] The present invention also provides a glucose visualization detection kit, which includes a NiCo2S4@N,S-rGO nanomaterial and a chromogenic substrate. The NiCo2S4@N,S-rGO nanomaterial is the NiCo2S4@N,S-rGO nanomaterial in the application or the detection method, and the chromogenic substrate is one or more of 3,3',5,5'-tetramethylbenzidine, 1,2-phenylenediamine or 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] The nanocomposite material (NiCo2S4@N,S-rGO) used in the present invention has significantly higher peroxidase activity than that of peroxidase precursors (NiCo2S4 and N,S-rGO). The glucose colorimetric detection results established based on the nanocomposite material are consistent with those of traditional blood glucose meters, providing satisfactory experimental accuracy and precision for glucose determination in human serum samples, and offering more options and strategies for the development of rapid, accurate, and low-cost detection of hydrogen peroxide and glucose in complex human fluid samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the synthesis process of NiCo2S4@N,S-rGO, NiCo2S4 and N S-rGO;

[0035] Figure 2 SEM and HRTEM images of NiCo2S4@N,S-rGO, NiCo2S4 and N,S-rGO, including (a) SEM image of NiCo2S4; (b) SEM image of N,S-rGO; (c) SEM image of NiCo2S4@N,S-rGO; (d) TEM image of NiCo2S4@N,S-rGO; (e) HRTEM image of NiCo2S4@N,S-rGO; (f) selected area electron diffraction (SAED) of NiCo2S4@N,S-rGO;

[0036] Figure 3 EDS spectrum of NiCo2S4@N,S-rGO;

[0037] Figure 4 XRD and Raman spectra of NiCo2S4@N,S-rGO, NiCo2S4 and N,S-rGO, where (a) XRD pattern; (b) Raman spectrum;

[0038] Figure 5 Infrared spectra of NiCo2S4@N,S-rGO and NiCo2S4;

[0039] Figure 6 XPS spectra of NiCo2S4@N,S-rGO, NiCo2S4 and N,S-rGO, including (a) full spectrum; (b) Ni 2p; (c) Co 2p; (d) C 1s; (e) N 1s; (f) S 2p;

[0040] Figure 7 ESR spectra of NiCo2S4@N,S-rGO and NiCo2S4;

[0041] Figure 8 N2 adsorption-desorption isotherms and pore size distribution diagrams of NiCo2S4@N,S-rGO, NiCo2S4 and N,S-rGO;

[0042] Figure 9 Figures showing the enzyme-like activities of NiCo2S4@N,S-rGO, NiCo2S4, and N,S-rGO, and the optimized conditions for the NiCo2S4@N,S-rGO colorimetric platform. (a) UV-visible absorption spectra of different reaction systems: 1, TMB; 2, H2O2; 3, TMB+H2O2; 4, r-GO+TMB+H2O2; 5, NiCo2S4+TMB+H2O2; 6, NiCo2S4@N,S-rGO+TMB+H2O2; 7, NiCo2S4@N,S-rGO+TMB; 8, NiCo2S4+TMB. The insets are photos of actual samples, showing (a) temperature optimization; (c) pH optimization; and (d) dosage optimization.

[0043] Figure 10 Steady-state kinetics of NiCo2S4@N,S-rGO, where (a) TMB concentration is in the range of 0.1-6.0 mM; (b) H2O2 concentration is in the range of 0.04-100 mM. The inset is the double reciprocal plot of the Michaelis-Menten equation versus TMB or H2O2 concentration.

[0044] Figure 11 This is an experiment to explore the mechanism of NiCo2S4@N,S-rGO catalytic oxidation of TMB, including (a) free radical scavenging experiment in NiCo2S4@N,S-rGO reaction system, the illustration is a photo of different free radical scavengers; (b) ESR spectrum of NiCo2S4@N,S-rGO color system of ·OH; (c) ·O2 - ESR spectrum of NiCo2S4@N,S-rGO color system; (d) ESR spectrum of NiCo2S4@N,S-rGO color system; (h + )’s ESR spectrum;

[0045] Figure 12 Schematic diagram of the mechanism of NiCo2S4@N,S-rGO for glucose detection;

[0046] Figure 13 NiCo2S4@N,S-rGO is used to detect glucose and H2O2, including (a) the calibration curve of absorbance versus H2O2; (b) the actual response curve of absorbance versus H2O2 concentration; (c) the calibration curve of absorbance versus glucose; (d) the actual response curve of absorbance versus glucose concentration.

[0047] Figure 14 This is the anti-interference and reusability test results of NiCo2S4@N,S-rGO nanozyme in detecting glucose. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific applications. The implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other. The implementation conditions used in the examples can be further adjusted according to specific requirements. The implementation conditions not specified are generally the conditions used in routine experiments.

[0049] Example 1

[0050] Synthesis of NiCo2S4@N,S-rGO Nanomaterials Containing Sulfur Vacancies

[0051] NiCo2S4@N,S-rGO nanocomposite materials were prepared by high temperature calcination method: 31.1mg Ni(CH3COO)2·4H2O, 59.48mg CoCl2·6H2O and 187.82mg TAA were dissolved in 5mL ethylene glycol and stirred at room temperature for 30min at a speed of 500rpm to obtain a uniform solution A. Subsequently, 10mg GO was added to 2mL ethylene glycol and dispersed into a homogeneous suspension under ultrasonic conditions, and then the resulting suspension was slowly added dropwise to the above solution A. The final mixed solution was heated to 300℃ at a heating rate of 5℃ / min and calcined in a N2 atmosphere for 60min. The material obtained after calcination was washed alternately with deionized water and ethanol 3 times and dried in an oven at 60℃ to obtain a dark gray material, NiCo2S4@N,S-rGO( Figure 1 -I).

[0052] In order to compare with NiCo2S4@N, S-rGO, NiCo2S4 and N, S-rGO were synthesized by similar methods. During the synthesis, NiCo2S4 ( Figure 1-Ⅱ ); During the preparation process, no nickel salt and cobalt salt were added, and N,S-rGO ( Figure 1-III ).

[0053] Characterization of NiCo2S4@N,S-rGO Nanomaterials Containing Sulfur Vacancies

[0054] The mixed solution of cobalt chloride, Ni(OCOCH3)2 and TAA was dropped into the graphene oxide suspension. 2+ and Co 2+ Dispersed and bonded to graphene oxide by electrostatic adsorption. Under N2 atmosphere, C=S, CN and -NH3 in TAA provide N and S sources for the prepared nanocomposite, and the S source is excessive. Excessive S source is conducive to the formation of vacant sites on the nanosheets. The morphology of NiCo2S4@N,S-rGO and NiCo2S4 was characterized by scanning electron microscopy and transmission electron microscopy. Due to the lack of r-GO to play a dispersing and anchoring role, NiCo2S4 appeared agglomerates and was composed of irregular particles ( Figure 2 a). N,S-rGO nanosheets contain abundant wrinkles ( Figure 2 b). For NiCo2S4@N,S-rGO, a large number of small particles are distributed on the surface of N,S-rGO nanosheets, thus avoiding the agglomeration of NiCo2S4 and providing more active sites. TEM and HRTEM images of NiCo2S4@N,S-rGO show that NiCo2S4 nanoparticles are uniformly dispersed on the N,S co-doped graphene sheets ( Figure 2 c and 2d).

[0055] HRTEM images show that N, S-rGO nanosheets are well integrated with NiCo2S4 particles, forming diffraction fringes of 0.28 nm, which are consistent with the (311) plane of NiCo2S4 ( Figure 2-2 e). The clear boundaries between NiCo2S4 and N, S-rGO indicate that N and S atoms are well dispersed on the surface of N, S-rGO. Corresponding selected area electron diffraction (SAED) Figure 2-2 f shows the polycrystallinity of NiCo2S4, which is related to the bright rings of (111), (220), (311), (400), (511) and (440) planes of NiCo2S4. In addition, EDS element mapping ( Figure 3 ) showed the presence of Ni, Co, S, C, and N, proving the formation of NiCo2S4@N and S-rGO.

[0056] Through XRD analysis, NiCo2S4 and NiCo2S4@N,S-rGO have very clear diffraction peaks ( Figure 4a). The XRD data at positions 16.34, 26.83, 36.83, 31.59, 38.32, 38.32, 47.41, 57.41, 50.46, and 55.33 agree well with the hexagonal phase (111), (220), (311), (400), (511), and (440) crystals of NiCo2S4 (JCPDS#20-0782). These XRD data provide strong evidence that the prepared nanocomposite does not change the structure of the original nanomaterial, which is consistent with the SAED test results.

[0057] By Raman spectroscopy analysis (500-2000cm -1 ), at 1350cm -1 and 1590cm -1 Two peaks were found at Figure 4 b), corresponding to the characteristic Raman modes of carbon in the d band (disordered carbon) and g band (ordered graphitic carbon), respectively. Compared with N,S-rGO, NiCo2S4@N,S-rGO composite has higher I D / I G The ratio indicates that the graphene in the composite material has more defect sites, which may strongly affect the electron transfer characteristics. Figure 5 ), the presence of various oxygen-containing functional groups (C=O, C=C, and CO) indicates that NiCo2S4 and N,S-rGO are successfully recombined. Functional groups such as C=O and CN provide various types of active sites for the peroxidase activity of the nanocomposite.

[0058] The compositions of NiCo2S4@N,S-rGO and NiCo2S4 were characterized by XPS spectroscopy ( Figure 6 ). Accordingly, the peaks at 872.8 and 855.0 eV belong to Ni 3+ , while the peaks at 868.7 and 851.7 eV are Ni 2+ ( Figure 6 b). Especially with S2 2- The corresponding 163eV peak indicates the presence of S vacancies in NiCo2S4@N,S-rGO. The peaks at 792.5 and 777.4eV belong to Co 3+ , the peaks at 796.3 and 779.4 eV belong to Co 2+ ( Figure 6 c). The multivalent states of Co and Ni in the complex are beneficial to the enhancement of conductivity, especially trivalent Co and Ni. The C1s core level spectrum shows that the binding energies of C=O, CN / CS, and C=S bonds are 287.3, 284.6, and 283.8 eV, respectively. Figure 6d). These peaks indicate that N and S heteroatoms are successfully doped into the carbon matrix, which is basically consistent with the doping results of N,S-rGO. At the same time, the high-resolution XPS spectra of S and N verify the effective incorporation of heteroatoms. In the N1s spectra of NiCo2S4@N,S-rGO and N,S-rGO, the product peaks at 400.9, 399.3 and 398.3 eV are graphitic N, pyrrolic N and pyridinic N, respectively. Figure 6 e). In the XPS spectrum of S2p, the peaks at 161.6 and 160.4 eV correspond to S 2- The 164.5eV peak of N,S-rGO and NiCo2S4@N,S-rGO is thiophene S ( Figure 6 f), indicating that S atoms are doped in the carbon-based heterocyclic structure.

[0059] Figure 7 The results show distinct ESR signals for sulfur vacancies, with a g-factor of 2.004. Compared to NiCo2S4, NiCo2S4@N,S-rGO exhibits a higher ESR signal, indicating the presence of a large number of sulfur vacancies on its surface. The sulfur vacancies provide more active sites and contact sites, which facilitates electron transfer and enhances its catalytic activity.

[0060] The specific surface area and pore size distribution (PSDs) of NiCo2S4@N,S-rGO, NiCo2S4 and N,S-rGO were analyzed by N2 adsorption-desorption isotherm (BET). Figure 8 As shown in a, at higher relative pressures (0.45-1.0), all isotherms exhibit typical hysteresis loop IV curves, indicating their mesoporous structures. The SSA of NiCo2S4@N,S-rGO is 155.8m 2 / g, is NiCo2S4(54.2m 2 The pore size distribution of the three mesoporous materials is 7-9 nm. Figure 8 b. The enlarged specific surface area and mesoporous properties provide a large number of active sites and more charge transfer channels, which are beneficial for adsorption and catalytic reactions, thereby improving the enzyme-like activity of NiCo2S4@N,S-rGO.

[0061] In this example, chemicals and reagents were purchased from Aladdin (Shanghai, China): CoCl2·6H2O, Ni(CH3COO)2·4H2O, thioacetamide (TAA), p-benzoquinone (PBQ), isopropyl alcohol (IPA), disodium tetraacetate (EDTA-2Na), ethylene glycol (EG), acetic acid (HAC), dimethyl sulfoxide (DMSO), sodium acetate (NaAc), and TMB. Graphene oxide (GO) was obtained from Xianfeng Nanomaterials Technology Co., Ltd. (Nanjing, China). Ultrapure water (>18.2 MΩ) was generated using a Milli-Q gradient system (Bedford, MA, USA). All reagents were used without further purification.

[0062] A KQ-500DE digitally controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., Kunshan), an OTF-1200x tube furnace (Hefei Kejing, Hefei), a DHG-9070a electric blow drying oven (Xiwen Biotechnology, Shanghai), a B15-3 multifunctional constant speed magnetic stirrer (Shanghai Sile Instrument Co., Ltd., Shanghai, China), and a PB-10 pH meter (Sartorius Scientific Instruments, Nanjing, China) were used.

[0063] In this embodiment, the X-ray diffraction (XRD) pattern of the NiCo2S4@N,S-rGO nanomaterial was obtained by a Bruker D8 Advance X-ray diffractometer (λ=0.15418 nm) equipped with Cu Kα radiation. The morphology and structure of NiCo2S4@N,S-rGO were characterized by field emission environmental scanning electron microscopy (QUANTA FEG 250, FEI, USA) and transmission electron microscopy (TaloSF200S (Thermo Scientific, USA). The specific surface area of ​​NiCo2S4@N,S-rGO was calculated using the BET (Brunauer-Emmett-Teller, BET) method. Electron paramagnetic resonance (EPR) spectroscopy was performed on a Bruker EMX X-band spectrometer equipped with an Oxford variable temperature cryostat. High-resolution X-ray photoelectron spectroscopy (XPS) was recorded on an X-ray photoelectron spectrometer (ESCALAB 250Xi, Shimadzu Corporation, Japan) equipped with a 150 W (15 kV, 10 mA) standard monochromatic light source (AIKR). All XPS peaks were calibrated to C1s (284.8 eV). Fourier transform infrared (FT-IR) spectroscopy was performed using a Bruker Tensor II All UV-visible absorbances were measured on a UV-2600 spectrophotometer (Shimadzu, Japan).

[0064] Example 2

[0065] Peroxidase-like and oxidase-like activities of NiCo2S4@N,S-rGO nanocomposites

[0066] The UV-visible absorption of 8 reaction systems was detected at 652 nm. Figure 9 As shown in Figure 1a, when only TMB (system 1#) was present, no adsorption peak at 652 nm was observed. Similarly, when only hydrogen peroxide (system 2#) was present, the solution was colorless. In the presence of both hydrogen peroxide and TMB, system 3# exhibited a light blue color, indicating that hydrogen peroxide could oxidize TMB to TMBox. When N,S-rGO (system 4#), NiCo2S4 (system 5#), or NiCo2S4@N,S-rGO (system 6#) were added to the TMB+H2O2 system, the solutions all exhibited varying degrees of blue. Although system 4# exhibited a light blue color, it was darker than system 3#, likely because the N and S doping resulted in more active sites in the graphene, promoting the oxidation of TMB. In sharp contrast, the addition of NiCo2S4 (system 5#) produced a dark blue color, indicating that NiCo2S4 could promote the oxidation of TMB. Due to the co-existence of TMB, hydrogen peroxide, and NiCo2S4@N,S-rGO, system 6 produced the deepest blue color, reflecting the strongest characteristic peak at 652nm. These results indicate that the NiCo2S4@N,S-rGO nanocomposite has a superior ability to oxidize TMB to TMBox, which is due to its large SSA and abundant vacancy sites. Notably, systems 7# (TMB+NiCo2S4@N,S-rGO) and 8# (TMB+NiCo2S4) exhibited varying degrees of blue color, indicating that NiCo2S4 and NiCo2S4@N,S-rGO are also likely to have oxidase-like activity.

[0067] System optimization (TMB+H2O2+NiCo2S4@N,S-rGO)

[0068] In order to achieve the best catalytic efficiency, the important parameters in system 6# (TMB+H2O2+NiCo2S4@N,S-rGO) were optimized, including incubation temperature, solution pH value and nanocomposite concentration. Figure 9 As shown in Figure 2, the absorbance at 652 nm increases with increasing temperature from 20°C to 40°C, but further increasing the temperature (40°C-60°C) results in a decrease in the absorbance intensity. Too low a temperature is not conducive to the catalytic oxidation of TMB by NiCo2S4@N,S-rGO, but too high a temperature will lead to the inactivation of TMB. Therefore, the deepest blue in system 6# appears at 40°C ( Figure 9(Illustration in b). Under alkaline conditions, hydrogen peroxide can decompose into O2 and H2O2, which greatly reduces its catalytic activity. Under strong acidic conditions, the electron cloud density of the N atom in TMB decreases, thereby weakening the intermolecular interaction between NiCo2S4@N,S-rGO and the substrate. Therefore, too high or too low solution pH is not conducive to the oxidation reaction of TMB. Figure 9 As can be clearly seen in Figure c, the absorbance at 652 nm drops sharply from pH 4.0 and is almost absent from pH 6.0 to 8.0. In subsequent experiments, the appropriate solution pH was 4.0. Within the concentration range of 0 to 10 mg / mL, the addition of the nanocomposite resulted in a gradual increase in the absorbance at 652 nm ( Figure 9 d). Specifically, when the concentration of the NiCo2S4@N,S-rGO composite material varied between 0 and 1.0 mg / mL (0, 0.1, 0.5, and 1.0 mg / mL), the absorbance (OD) at 652 nm increased significantly, with a slight increase between 1.0 and 10.0 mg / mL. In the UV-Vis detection system, excessive OD values ​​(>2.0) led to a sharp decrease in the color recognition response rate due to saturation absorbance. Based on these findings, a 1.0 mg / mL concentration of the NiCo2S4@N,S-rGO nanocomposite was the appropriate peak level for subsequent experiments.

[0069] Steady-state kinetics of NiCo2S4@N,S-rGO

[0070] The steady-state kinetics of the colorimetric sensor based on the prepared nanocomposite were studied using the typical Michaelis-Menten equation. TMB and hydrogen peroxide were selected as substrates, and each experiment was repeated three times by changing the concentration of one substrate ( Figure 10 ). As we all know, K m The lower the value, the higher the affinity between the nanozyme and the substrate. As shown in Table 1, the K m The values ​​ranged from 0.3326 to 0.3372. Therefore, the affinity of the nanocomposite was increased by approximately 40% and 10.6 times compared to NiCo2S4 and N,S-rGO, respectively. Furthermore, NiCo2S4@N,S-rGO exhibited higher affinity than standard HRP and previously reported Cu-Ag / rGO and WS2 / rGO (Table 1). These data indicate that NiCo2S4@N,S-rGO significantly enhances peroxidase-like activity.

[0071] Table 1 Comparison of Michaelis-Menten constants for the catalytic oxidation of H2O2 by NiCo2S4@N,S-rGO and NiCo2S4 N,S-rGO

[0072]

[0073]

[0074] Example 3

[0075] Detection of Glucose Based on NiCo2S4@N,S-rGO

[0076] Mechanism of NiCo2S4@N,S-rGO

[0077] So far, various mechanisms have been proposed to explain the catalytic oxidation of TMB by nanozymes, which mainly involve various reactive oxygen species (ROS), such as ·OH, ·O2 - and h + Here, we used three ROS scavengers (IPA, PBQ and EDTA) to remove ·OH, O2 - and h + Compared with the control group (no scavenger), the color of the system only slightly decreased after adding IPA, while more obvious fading occurred after adding PBQ and EDTA at the same time ( Figure 11 a). In particular, the system becomes almost colorless with the addition of EDTA. These observations suggest that the mechanism of TMB oxidation catalyzed by the nanocomposite may be due to the co-involvement of three ROS species in the system. Therefore, ESR was employed to further investigate the catalytic reaction mechanism. 5,5-Dimethyl-1-pyrrolidine N-oxide (DMPO) can transiently capture ROS, forming DMPO / radical spin adducts, which can produce signals of varying shapes. Figure 11 b shows the standard quartet peaks with a signal ratio of 1:2:2:1, which is a graph of DMPO / ·OH. Obviously, the high noise and weak signal intensity indicate that only a small amount of ·OH is produced in this system. Figure 11 c shows that DMPO / ·O2 - The curve diagram shows that the O2 - Compared with the DMPO / OH graph, the lower noise and higher signal intensity indicate that O2 - The content of photogenerated holes (h + ) is shaped like Figure 11 As shown in d, the strong signal provides strong evidence that h + These ESR observations are highly consistent with those of ROS scavengers. Therefore, the catalytic activity of NiCo2S4@N,S-rGO is generated by the three free radicals and is expressed as h + For the main.

[0078] It is speculated that the mechanism of NiCo2S4@N,S-rGO for glucose detection involves three aspects:

[0079] (1) For this catalytic reaction, TMB molecules can be adsorbed on the surface of the nanocomposite through π-π interactions and electrostatic attraction. The co-doping of N,S-doped reduced graphene oxide nanosheets provides abundant functional groups, which promotes the interaction with TMB and thus improves the catalytic efficiency. At the same time, the large SSA of NiCo2S4@N,S-rGO increases the active sites of its precursor NiCo2S4.

[0080] (2) Under visible light irradiation, the electrons of NiCo2S4 are excited in the conduction band (CB), generating holes in the valence band (VB), and the r-GO ene doped with N and S promotes the transfer of CB to VB. With the participation of electrons (e-), hydrogen peroxide is converted into O2 and further forms O2 - In contrast, TMB is - or h + Loss of e in the reaction - Forming TMBOx.

[0081] (3) The co-doping of N and S, the abundant S vacancies and the complex chemical valence of NiCo2S4@N,S-rGO promote electron transfer and accelerate the reaction process. Figure 12 As shown, engineering the catalyst interface structure facilitates electronic interactions, redistributing charge within heterogeneous catalysts and creating an internal electric field. This further induces the formation of ion pores, which act as bridges and transfer ions to reactants, thereby enhancing catalytic activity. Furthermore, this effect is influenced by the size effect of the nanomaterial on the interface support: the large amount of charge transfer per atom in small nanomaterials significantly enhances this effect.

[0082] Since the activity of NiCo2S4@N,S-rGO peroxidase is related to the concentration of hydrogen peroxide, hydrogen peroxide can be quantitatively determined by measuring the ODs at 652 nm. Figure 10 a and 10b show the absorbance at 652 nm. The hydrogen peroxide concentration has a good linear relationship between 0.04 and 50 mM, and the correlation coefficient (R 2 ) was 0.9989, and the lower limit of detection (LOD) was 12 μM. At three levels (50, 500, and 1000 μM), the relative recoveries were in the range of 98.9-102.4%, with relative standard deviations (RSDs) of 2.6-4.5%, indicating high experimental precision (Table 2).

[0083] Table 2 H2O2 recovery rate based on NiCo2S4@N,S-rGO for colorimetric determination

[0084]

[0085] Glucose can be oxidized by GOx to produce hydrogen peroxide, which is closely related to the peroxidase activity of natural enzymes or nanozymes. Therefore, traditional HRP can be replaced by NiCo2S4@N,S-rGO for glucose colorimetric determination. As the glucose concentration increases, the increase in hydrogen peroxide content is accompanied by an increase in 652nm ODs and an increase in blue color. Under the optimized conditions (solution pH = 4.0, 1.0mg / mL nanocomposite, incubation at 40℃ for 20min), the linear equation y = 0.00141x + 0.1176 and the correlation coefficient R 2 A relationship diagram between ODs at 625 nm and glucose concentration was constructed for 0.9788 ( Figure 13 a). The linear range (LR) of glucose detection was 1.0–200 μM, with limits of quantification (LOQ) and detection (LOD) of 1.0-fold and 0.3 μM (S / N = 3) and 10 μM, respectively. These analytical performance indicators were compared with those of several glucose determination methods using sensors based on other nanomaterials (Table 3).

[0086] Table 3 Reliability of the colorimetric determination of glucose in human serum samples based on NiCo2S4@N,S-rGO

[0087]

[0088] Obviously, the LOD (0.30 μM) of the NiCo2S4@N,S-rGO nanozyme biosensor is higher than that of COOOH nanosheets (1.20 μM), r-CD (2.00 μM), CoSe2 / rGO (0.55 μM), and NiCo2O4 (1.62 μM). In addition, the NiCo2S4@N,S-rGO-based detection method provides a relatively wider LR (1.0-200 μM) compared with the other methods mentioned above. Therefore, these comparative performance indicators indicate that the constructed nanozyme biosensor is beneficial for practical applications in the field of biomolecule analysis.

[0089] Table 4 Comparative analysis of the performance of colorimetric assay based on NiCo2S4@N,S-rGO and other nanozyme glucose colorimetric assays

[0090] Nanomaterials LRs (μM) LODs (μM) CoOOH nanoflakes 5.3-500 1.20 r-CDs 10-400 2.00 <![CDATA[CoSe2 / rGO]]> 5-800 0.55 <![CDATA[NiCo2O4]]> 2-100 1.62 <![CDATA[NiCo2S4@N,S-rGO]]> 1-200 0.30

[0091] Selectivity / anti-interference effect and reproducibility of colorimetric determination of glucose based on nanocomposites

[0092] The coexisting components in human serum samples include fructose, galactose, sucrose, serine, histidine, ascorbic acid, K + , Ca 2+ 、Na + These components were added to the diluted serum samples at a concentration of 5.0 μg / mL. Figure 14 As shown in Figure 3, the interfering macromolecules / ions have little effect on the 625nm ODs. It is worth noting that the absorbance at 625nm decreased to a certain extent after the addition of ascorbic acid compared with the blank sample, reflecting the fading of the blue color. This phenomenon may be due to the reducing property of ascorbic acid, which consumes H2O2, one of the main products of the GOx catalytic reaction. Figure 14 As shown in b. The relative activity was still as high as 81.9% after 6 times, and the relative activity was 58.6% after 10 times. It can be seen that the prepared nanozyme can be reused as a colorimetric sensor at least 6 times. The feasibility of the constructed sensor was evaluated by detecting the glucose content in human serum samples. The glucose concentrations in the two sera were 15.62 and 5.29 μM, respectively, and the blood glucose meter was 16.12 and 5.42 μM, respectively. Therefore, the relative recoveries obtained spanned the range of 2.14-3.12%, with RSDs of 97.4-101.8% (Table 3). These data findings indicate that this colorimetric method based on NiCo2S4@N,S-rGO nanozyme can provide satisfactory experimental accuracy and precision for glucose determination in human serum samples.

[0093] The present invention adopts a simple calcination method to prepare a nanocomposite material (NiCo2S4@N,S-rGO), and uses a series of characterization techniques such as XRD, BET, HRTEM, SEM, FT-IR and ESR to characterize its structure in detail. NiCo2S4@N,S-rGO has a peroxidase activity significantly higher than that of peroxidase precursors (NiCo2S4 and N,S-rGO), which is due to the co-doping of N and S, the rich S vacancies and multivalent states of the nanocomposite material. The nanomaterial has a typical mesoporous structure (pore size of 7-9nm), and its specific surface area is 2 times that of NiCo2S4, thereby generating more active sites and charge transfer channels. Through ROS scavenging experiments, it was confirmed that the enhancement of peroxidase mimic activity is the result of ROS (·OH, ·O2 - and h + ) combined effect, where h +The nanocomposite-based sensor has a dominant position in TMB-based colorimetric reactions. Under optimized conditions, the LODs for hydrogen peroxide and glucose were 12 μM and 0.3 μM, respectively, comparable to those of conventional blood glucose meters. Overall, this invention provides more options and strategies for the rapid, accurate, and low-cost detection of hydrogen peroxide and glucose in complex human fluid samples.

[0094] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of NiCo2S4@N,S-rGO nanomaterials in visual glucose detection. The NiCo2S4@N,S-rGO nanomaterials have peroxidase-like activity. The preparation method of the NiCo2S4@N,S-rGO nanomaterials is as follows: Ni(CH3COO)2·4H2O, CoCl2·6H2O and TAA are dissolved in ethylene glycol to obtain solution A, GO is added to ethylene glycol to obtain a suspension, the suspension is dropwise added to the solution A, and the resulting mixed solution is calcined to obtain the NiCo2S4@N,S-rGO nanomaterials.

2. The use according to claim 1, characterized in that The NiCo2S4@N,S-rGO nanomaterial is a composite nanomaterial in which NiCo2S4 nanoparticles are uniformly dispersed on N,S co-doped graphene sheets. The specific surface area of ​​the NiCo2S4@N,S-rGO nanomaterial is 120~200m 2 / g, the NiCo2S4@N,S-rGO nanomaterial has a porous structure with a pore size of 5~10nm.

3. The use according to claim 1, characterized in that The calcination temperature is 280-320°C; and / or the calcination is carried out under a N2 atmosphere; and / or the calcination is heated to the desired temperature at a heating rate of 3-8°C / min; and / or the feed mass ratio of Ni(CH3COO)2·4H2O, CoCl2·6H2O, TAA and graphene oxide is (2-5):(3-8):(15-20):

1.

4. A method for visual detection of glucose, characterized in that: Glucose is tested based on a colorimetric method. NiCo2S4@N,S-rGO nanomaterials are used to catalyze the oxidation of glucose to produce hydrogen peroxide, and simultaneously catalyze the reaction of hydrogen peroxide with a chromogenic substrate. The chromogenic substrate is 3,3',5,5'-tetramethylbenzidine. The preparation method of the NiCo2S4@N,S-rGO nanomaterial is as follows: Ni(CH3COO)2·4H2O, CoCl2·6H2O, and TAA are dissolved in ethylene glycol to obtain solution A, GO is added to ethylene glycol to obtain a suspension, the suspension is dropwise added to the solution A, and the resulting mixed solution is calcined to obtain the NiCo2S4@N,S-rGO nanomaterial.

5. The glucose visualization detection method according to claim 4, characterized in that: The temperature of the catalytic reaction of the NiCo2S4@N,S-rGO nanomaterial is 35-50°C.

6. The method for visualizing glucose detection according to claim 4, wherein: The concentration of the NiCo2S4@N,S-rGO nanomaterial in the reaction system is 0.5~2.0 mg / mL.

7. The method for visualizing glucose detection according to claim 4, wherein: The initial pH value of the reaction system is 3~4.

8. The glucose visualization detection method according to claim 4, characterized in that: After the reaction is completed, the absorbance of the reaction system is tested, and the absorbance range is 625-675 nm; and / or the test sample is one or more of blood, serum or urine.

9. A glucose visualization detection kit, characterized in that: The glucose visualization detection kit includes a NiCo2S4@N,S-rGO nanomaterial and a chromogenic substrate. The preparation method of the NiCo2S4@N,S-rGO nanomaterial is as follows: Ni(CH3COO)2·4H2O, CoCl2·6H2O and TAA are dissolved in ethylene glycol to obtain solution A, GO is added to ethylene glycol to obtain a suspension, the suspension is dropwise added to the solution A, and the obtained mixed solution is calcined to obtain the NiCo2S4@N,S-rGO nanomaterial. The chromogenic substrate is 3,3',5,5'-tetramethylbenzidine.

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