CN114100586A

CN114100586A - Composite nano enzyme and preparation method and application thereof

Info

Publication number: CN114100586A
Application number: CN202111413859.1A
Authority: CN
Inventors: 刘婷婷; 王学东; 李佳妮; 叶翰章; 丁永立; 王琪; 王慧利; 王贵鑫; 陈坤
Original assignee: Suzhou University of Science and Technology
Current assignee: Suzhou University of Science and Technology
Priority date: 2021-11-25
Filing date: 2021-11-25
Publication date: 2022-03-01
Anticipated expiration: 2041-11-25
Also published as: CN114100586B

Abstract

The invention provides a composite nano enzyme and a preparation method and application thereof. The composite nano enzyme is a transition metal sulfide/nitrogen-sulfur Co-doped carbon material with catalase-like catalytic activity and oxidase-like catalytic activity, and the transition metal sulfide is Co_1‑xS or Ni₉S₈Part of the transition metal sulfideThe composite nanoenzyme with excellent peroxidase-like catalytic activity and peroxidase-like catalytic activity is used for cholesterol quantitative detection, has good selectivity and low interference effect on cholesterol, and can realize low-cost, rapid, simple and accurate detection.

Description

Composite nano enzyme and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a composite nano enzyme, a preparation method and application thereof.

Background

Cholesterol is an important raw material for forming cholic acid and synthesizing hormone, is a main component forming cell membranes, and is closely related to human health and diseases. Cholesterol in human serum includes free cholesterol and cholesterol esters. The cholesterol level in blood is an important parameter for early diagnosis of diseases such as cancer, Alzheimer's disease, type II diabetes, arteriosclerosis, coronary heart disease, cerebral thrombosis, hypertension, Parkinson's disease and the like. The current quantitative analysis techniques for total cholesterol mainly include enzymatic methods, colorimetric methods, molecular luminescence methods, electrochemical methods, chromatography methods and the like. The application of the electrochemical method and the chromatography requires special equipment and is difficult to popularize and use; the enzyme method has the advantages of high technology content, strong specificity, strict requirements on applicable conditions, higher cost and large error of detection results.

With the rapid development of the smart phone and the application of the program, the smart phone has strong image acquisition and analysis processing functions, and the operability and the portability of the image colorimetric detection system are greatly improved. Digital Imaging Colorimetry (DIC) is a simple and efficient Colorimetry, and is characterized in that Digital equipment (mobile phones, scanners, Digital cameras and the like) is used for collecting images of developing solutions, and then images of objects to be detected are converted into color values through appropriate color models, so that quantitative analysis is carried out on the objects to be detected. During the course of cholesterol detection, the color intensity of the solution after the catalytic reaction monotonically increases with the increase in cholesterol concentration. And (3) generating a calibration curve by drawing the relation between the gray value of the reaction solution and the corresponding cholesterol concentration, and further carrying out quantitative analysis on the cholesterol content in the actual sample. The mobile phone visual colorimetric method is convenient, low in cost, rapid, simple and accurate, and provides a promising application for field diagnosis.

However, the extraction cost of the biological enzyme for colorimetric detection of cholesterol is high, the use condition is harsh, and the biological enzyme is not suitable for long-time storage. The nano enzyme is an artificial mimic enzyme with catalytic function and unique physical and chemical properties of nano materials, and can mimic the activity of enzymes, such as oxidase, superoxide enzyme, catalase, superoxide dismutase, phosphatase and the like. The high catalytic activity and lower preparation cost of the nano enzyme are beyond the reach of the traditional mimic enzyme. The catalytic substrate reaction is similar to that of natural enzyme, and has a kinetic curve conforming to the Mie equation; the pH, temperature and substrate concentration affect its catalytic activity.

The reported metal, metal oxide, metal sulfide, metal organic frame and carbon-based nano material are basically applied in the fields of energy storage, photoelectricity, catalysis and the like. In recent years, the application of nano materials in the fields of biochemistry and environment is also gradually receiving attention. The nanometer material can be used as an oxidase-like enzyme to be applied to biochemical analysis, but is still in the laboratory research and development stage, wherein the cobalt-based material has the advantages of rich element valence and various structures, similar properties and good stability with the iron-based nanometer enzyme material discovered at the earliest time, and the like, but the application of the enzyme-like activity to the detection of cholesterol is rarely reported. The nano enzyme for cholesterol detection needs to have the characteristics of good stability, high catalytic efficiency, strong anti-interference capability and the like, so that more nano enzyme materials with excellent enzyme activity need to be developed, the variety of nano enzyme is enriched, more choices are provided for biochemical analysis of cholesterol, and the on-site efficient detection of cholesterol is realized by combining a mobile phone visual colorimetric method.

Disclosure of Invention

The invention aims to provide a method for preparing nano enzyme consisting of transition metal sulfide and nitrogen-sulfur co-doped carbon material.

It is another object of the present invention to provide a transition metal sulfide/nitrogen and sulfur co-doped carbon material having catalase-like catalytic activity and oxidase-like catalytic activity.

The invention also aims to provide application of the transition metal sulfide/nitrogen and sulfur co-doped carbon material in cholesterol detection.

The invention also aims to provide a method for detecting cholesterol by using a transition metal sulfide/nitrogen and sulfur co-doped carbon material colorimetric method.

The invention also aims to provide a method for visually detecting cholesterol by using the mobile phone.

It is another object of the present invention to provide a cholesterol measurement kit including a composite nanoenzyme having excellent peroxidase catalytic activity and oxidase catalytic activity.

In order to solve the technical problems, the invention adopts the following technical scheme:

the preparation method of the composite nano enzyme comprises the steps of preparing aerogel by using an organic matter containing N and S and a transition metal salt in the presence of a reducing agent and a cross-linking agent, calcining the aerogel at 400-800 ℃ under an inert atmosphere condition, washing with water, and drying to obtain the composite nano enzyme, wherein the transition metal salt is cobalt salt or nickel salt.

Further preferably, the organic matter containing N and S is one or more of trithiocyanuric acid, thioacetamide, mercaptosuccinic acid or thiourea.

Further preferably, the cross-linking agent is one or more of sodium alginate, hexamethylenetetramine, or dicyanodiamine.

Preferably, the reducing agent has weak reducibility, and CoS can be obtained₂Reduction of etc. to Co_1-xS, the carbon material of nitrogen and sulfur co-doped carbon material can be provided.

Preferably, the reducing agent is one or more of citric acid, glucose and cysteine.

Preferably, the calcination is performed under the protection of nitrogen or argon.

Preferably, the transition metal salt is one or more of cobalt acetate, cobalt nitrate, nickel acetate or nickel nitrate.

Preferably, the mass ratio of the cross-linking agent, the organic matter containing N and S, the reducing agent and the transition metal salt is 1: (3-8): (0.1-0.8): (0.1-0.8).

Preferably, the drying temperature is 50-80 ℃.

Further preferably, the drying temperature is 55-75 ℃.

Further preferably, the preparation method of the aerogel comprises the following steps: mixing N and S-containing organic matters, a reducing agent, a cross-linking agent and water to form uniform primary mixed liquid, then mixing the mixed liquid and a transition metal salt aqueous solution to form uniform secondary mixed liquid, and dehydrating the secondary mixed liquid to form the aerogel.

Still further preferably, in the preliminary mixed solution, the concentration of the organic matter containing N and S is 40-60 g/L, the concentration of the reducing agent is 1-10 g/L, and the concentration of the crosslinking agent is 5-20 g/L.

Still more preferably, the concentration of the aqueous solution of the transition metal salt is 1-10 g/L;

still further preferably, the dehydration is dehydration by freeze drying.

Still further preferably, the calcination includes a primary calcination and a secondary calcination, the temperature of the successive calcination is lower than that of the secondary calcination, and the primary calcination specifically includes: heating to 400-600 ℃ at a heating rate of 1-3 ℃/min, and then carrying out heat preservation calcination at 400-600 ℃ for 20-50 min; the secondary calcination specifically comprises the following steps: after the primary calcining, continuously heating to 600-800 ℃ at the heating rate of 4-6 ℃/min, and then carrying out heat preservation calcining at 600-800 ℃ for 100-150 min.

The composite nano enzyme is a transition metal sulfide/nitrogen and sulfur Co-doped carbon material with catalase-like catalytic activity and oxidase-like catalytic activity, and the transition metal sulfide is Co_1-xS or Ni₉S₈And part of the transition metal sulfide is dispersedly attached to the surface of the nitrogen-sulfur co-doped carbon material, and the other part of the transition metal sulfide is doped in the nitrogen-sulfur co-doped carbon material.

Preferably, the transition metal sulfide is Co_1-xS, the specific surface area of the composite nano enzyme is 15-25 m²g^-1The mass ratio of the constituent elements N, S, C and Co of the composite nano enzyme is (15-40): (1-8): (40-80): (1-15).

Further preferably, the specific surface area of the composite nano enzyme is 15-20 m²g^-1。

Further preferably, the mass ratio of the constituent element N, S, C and Co of the composite nano enzyme is (20-40): (2-6): (45-70): (5-12).

Preferably, the transition metal sulfide is Co_1-xS, the element composition of the composite nano enzyme comprises N accounting for 20-40% of the total mass of the composite nano enzyme, S accounting for 1-5% of the total mass of the composite nano enzyme, C accounting for 40-70% of the total mass of the composite nano enzyme, and Co, Co and Co accounting for 5-12% of the total mass of the composite nano enzyme_1-xThe size of the S particles is less than 20 nm.

The invention also provides an application of the composite nano enzyme or the composite nano enzyme prepared by the preparation method in cholesterol detection.

The invention also provides a cholesterol detection method, which is based on a colorimetric method for testing cholesterol, and uses the composite nanoenzyme or the composite nanoenzyme prepared by the preparation method to catalyze the reaction between hydrogen peroxide and a chromogenic substrate, wherein 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.

Preferably, the temperature of the catalytic reaction of the composite nano-enzyme is 25-60 ℃, and further preferably 45-55 ℃.

Preferably, the concentration of the composite material nano enzyme in the reaction system is 0.5-2.5 mg/mL, and further preferably 0.8-1.2 mg/mL.

Preferably, the initial pH of the reaction system is 3 to 4, and more preferably 3.2 to 3.8.

Preferably, the detection method further comprises the steps of hydrolyzing cholesterol ester under the action of cholesterol esterase to generate cholesterol, and oxidizing cholesterol under the action of cholesterol oxidase to generate hydrogen peroxide. Free cholesterol can be quantitatively detected only by using cholesterol oxidase, and total cholesterol can be quantitatively detected by using cholesterol lipase and cholesterol oxidase.

Preferably, the test sample is one or more of serum, blood, urine, milk or edible oil.

Preferably, the detection method further comprises colorimetric data processing using digital imaging software.

The invention also provides a kit for detecting cholesterol, and the kit comprises the composite nano enzyme or the composite nano enzyme prepared by the preparation method.

Preferably, the kit further comprises hydrogen peroxide and 3,3',5,5' -tetramethylbenzidine.

Preferably, the kit further comprises cholesterol oxidase cholesterol and cholesterol lipase.

Compared with the prior art, the invention has the following advantages:

the composite nanoenzyme prepared by the preparation method is a transition metal sulfide/nitrogen-sulfur co-doped carbon material, the nitrogen-sulfur co-doped carbon material has rich pore structures, the surface of the composite material has rich defect sites, part of transition metal sulfide nanoparticles are dispersedly attached to the surface of the nitrogen-sulfur co-doped carbon material, and the other part of transition metal sulfide nanoparticles and the nitrogen-sulfur co-doped carbon material greatly improve the catalytic activity of catalase-like enzyme and the catalytic activity of oxidase-like enzyme, are used for quantitatively detecting cholesterol, have good selectivity and low interference effect on the cholesterol, and can realize low-cost, quick, simple and accurate detection. The invention enriches the variety of the composite nano-enzyme and provides more choices for cholesterol detection.

Drawings

FIG. 1 shows NSC/Co_1-xS, a schematic diagram of a synthesis process of the nano composite material;

FIG. 2(a) SEM image of NSC; (b) NSC/Co_1-xSEM image of S; (c) NSC/Co_1-xA TEM image of S; (d) NSC/Co_1-xAn HRTEM image of S; (e, f) NSC/Co_1-xThe distribution diagram of the elements of S,

FIG. 3(a) NSC and NSC/Co_1-xAn XRD spectrum of S; (b) NSC and NSC/Co_1-x(ii) a raman spectrogram of S; (c) NSC and NSC/Co_1-xNitrogen adsorption-desorption isotherms and pore size distribution curves (inset images) of S; (d) NSC and NSC/Co_1-xAn EPR spectrum of S;

FIG. 4 shows NSC/Co_1-xHigh resolution XPS spectra of S composites (a) N1S, (b) S2p, (C) C1S and (d) Co 2 p;

FIG. 5(a) is a photograph showing the UV-VIS absorption spectrum and color change of different reaction systems after incubation for 20 minutes. Reaction conditions on NSC/Co_1-xInfluence of class S peroxidase Activity: (b) (ii) temperature; (c) the pH value; and (d) NSC/Co_1-x(ii) the concentration of S;

FIG. 6 NSC/Co_1-xS under different atmosphere conditions (N)₂Air and O₂) Ultraviolet absorption spectrum of oxidase-like activity (reaction system consisting of TMB, NaAc-HAc and NSC/Co)_1-xS composition).

FIG. 7(a) TMB and (b) H₂O₂NSC/Co of_1-xThe Michaelis-Menten diagram of S; (c) TMB and (d) H₂O₂The Lineweaver-Burk plot of (a);

FIG. 8(a) three active scavengers at NSC/Co_1-xS catalyzes the role in the oxidation process of TMB; (b, c and d) NSC/Co_1-xAn EPR spectrum of S; (e) NSC/Co_1-xA mechanism for detecting peroxidase-like and oxidase-like activities of S;

FIG. 9(a) UV-visible spectra of different cholesterol concentrations (0.2-10mmol) of the sensing system detected at 652 nm. (b) Inset corresponds to a linear fit curve, error bars represent the standard deviation of triplicate analysis (n-3);

FIG. 10NSC/Co_1-xS-catalyzed colorimetry to quantify cholesterol selectivity/interference (note: error bars represent standard deviations of three assays (n-3));

fig. 11 is a schematic diagram of the procedure for cholesterol quantification using the "sitting identity" application on the smartphone.

FIG. 12(a) uses NSC/Co-based_1-xCarrying out cholesterol determination on the smart phone with the S-catalyzed colorimetric reaction; (b) regression equation of absorbance and cholesterol concentration (0.2-3.0 mmol);

FIG. 13NSC/Ni₉S₈SEM image of (d).

Detailed Description

The present invention will be further described with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not mentioned are conventional conditions in the industry. The technical features of the embodiments of the present invention may 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, and the implementation conditions not indicated are generally the conditions in routine experiments.

In the following examples, Sodium Alginate (SA), Trithiocyanuric Acid (TA), Citric Acid (CA), cobalt acetate tetrahydrate, hydrogen peroxide (H)₂

O

₂30%), acetic acid (CH)₃COOH, not less than 98.0 percent) and sodium acetate (CH)₃COONa ≥ 99.0%), ethanol ≥ 99.7%, dimethyl sulfoxide (DMSO ≥ 99.0%), p-benzoquinone (PBQ), ethylenediamine tetraacetic acid (EDTA), sucrose (Suc), D-histidine (His), galactose (Gal) and D-serine (Ser), and other series of analytical or chromatographic grade reagents are purchased from Adams (Shanghai, China). Isopropyl alcohol (IPA, 99.5% or more) is freely extracted from great (Shanghai)And (4) supplying. The cholesterol (cholesterol) is a mixture of cholesterol (Cho,>95.0%) and 3,3', 5' -Tetramethylbenzidine (TMB) were from Aladdin (Shanghai). Cholesterol Lipase (Chol, ≧ 300units/g) from Rhawn (Shanghai) and cholesterol oxidase (Chox) from Macklin (Shanghai). All reagents were used as received without further purification. Ultrapure water (>18.2M Ω) was generated by a Milli-Q gradient system (bedford, ma, usa).

Example 1

Comparison of composite nanoenzymes and N, S Co-doped C Material

Preparation of composite nanoenzyme

The synthesis process of the NSC/Co1-xS nano composite material is shown in figure 1, and comprises the following steps:

1) 5.0g of TA, 1.0g of SA and 0.5g of CA were dissolved in 100mL of deionized water and magnetically stirred at room temperature for 12 hours to obtain a homogeneous solution (A). Subsequently, 0.5g of Co (CH)₃COO)₂·4H₂Solution B was prepared by dissolving O in 100mL of deionized water. Slowly adding the solution B into the solution A, magnetically stirring the obtained mixed solution for 12 hours to form a uniform phase, and then dehydrating by freeze drying to form light yellow aerogel.

2) The prepared aerogel was transferred to a ceramic boat, placed in a tube furnace, and N was used₂The airflow expels the air (-20 minutes). Then, in N₂Heating to 500 deg.C at a heating rate of 2 deg.C/min under protection, maintaining the temperature for 30min, heating to 700 deg.C at a heating rate of 5 deg.C/min, and maintaining the temperature for 120 min. After cooling down the tube furnace to room temperature, the calcined aerogel was collected, washed/rinsed three times with Milli-Q ultrapure water, the resulting black product was dried in a vacuum oven at 60 ℃ for 6 hours, and the final product was labeled NSC/Co_1-xS nanocomposite.

Preparation N, S Co-doping of the C Material (NSC)

1) 5.0g TA, 1.0g SA and 0.5g CA were added to 100mL deionized water and magnetically stirred at room temperature for 12h to form a homogeneous phase, and this mixture was dehydrated by freeze-drying to form an aerogel. Subsequently, the prepared aerogel was transferred to a ceramic boat, placed in a tube furnace, and N was used₂The airflow expels the air (-20 minutes).

2) Quartz porcelain boat in N₂Heating to 500 deg.C at a heating rate of 2 deg.C/min under protection, maintaining the temperature for 30min, heating to 700 deg.C at a heating rate of 5 deg.C/min, and maintaining the temperature for 120 min. After cooling the tube furnace to room temperature, the calcined aerogel was collected and washed/rinsed three times with Milli-Q ultrapure water. Finally, the resulting black product was dried in a vacuum oven at 60 ℃ for 6 hours and the final product was labeled NSC.

NSC and NSC/Co_1-xCharacterization of S nanocomposites

NSC and NSC/Co were obtained using a field emission scanning electron microscope and X-ray energy spectrometer (FESEM, EDS, FEI Quanta) and Transmission Electron microscope (TEM, FEI Tecnana F20)_1-xMorphological characteristics and elemental distribution of S. The crystal structure of the sample was determined using an X-ray diffractometer (XRD, D8-Advance, Bruker). X-ray photoelectron spectroscopy (XPS) was performed using a Saimer science K-Alpha instrument. Specific surface area (BET) and pore size distribution curves were obtained from a TriStar II Plus specific surface pore size Analyzer, Mic. Raman analysis was performed using an Invia Reflex, LabRam HR Evolution Instrument. Enzyme kinetics and UV-vis were performed on a UV-5500PC spectrophotometer (Metash). Bruker EMXPLUS was used to measure S vacancies and free radicals.

SEM images of NSCs showed that the material surface was smooth and porous (fig. 2 a). In use of Co_1-xAfter the attachment of the S nanoparticles to the NSCs, some small particles were observed to adhere to the surface of the porous carbon layer (fig. 2b). According to NSC/Co_1-xTEM image of S, much Co_1- _xThe S nanoparticles were uniformly dispersed on the carbon layer and had a diameter of about 10-20nm (fig. 2 c). In HRTEM images, we observed significant lattice fringes with a spacing of 0.29nm, corresponding to Co_1-xThe (102) crystal plane of S, with a spacing of 0.34nm, corresponds to the (002) crystal plane of C (FIG. 2 d). The EDS elemental diagram shows that Co, N and S atoms are all present in the carbon layer, enabling recombination of the material and doping of the atoms (fig. 2e, f).

NSC/Co testing by XRD_1-xS nanocomposite crystal type and structure. A broad diffraction peak at 20-30 deg., associated with amorphous carbon. NSC/Co_1-xThe diffraction peaks of S are concentrated at 30.5,35.2,46.8 and 54.3 degrees, belonging to Co_1-xThe (100), (101), (102) and (110) facets of S match well with standard PDF card (PDF No.42-0826) (FIG. 4).

Raman spectroscopic analysis showed NSC and NSC/Co_1-xS is 1334cm^-1(D band) and 1570cm^-1(band G) has two characteristic peaks (fig. 3 b). Generally, the D band is characteristic of disordered carbon or graphite structural defects, while the G band is associated with a level of graphitization. NSC and NSC/Co_1-xD-G peak intensity ratio of S (I)_D/I_G) 1.41 and 1.34, respectively, indicating that there are a large number of defects in the porous structure. NSC/Co_1-xI of S_D/I_GThe slightly lower value indicates that the addition of cobalt sulfide during pyrolysis reduces the degree of disorder. N is a radical of₂The results of the adsorption measurements (FIG. 3c) show that NSC/Co_1-xThe specific surface areas of S and NSC were 18.47 and 31.52m, respectively²g^-1The main pore diameters are all 7-9nm (inset in figure 3c), and belong to mesoporous structures. These observations indicate that Co_1-xS nanoparticles have been embedded into the pores of the carbon layer. The electron spin resonance (EPR) signal at g 2.003 points to NSC/Co_1-xThe S vacancies of the S structure (fig. 3d) provide strong evidence for the presence of S vacancies on the surface of the prepared nanocomposite.

NSC/Co by XPS_1-xThe elemental composition and morphology of the S nanocomposite was evaluated. NSC/Co_1-xThe mass ratios of N, S, C and Co in S were 20.6%, 4.15%, 68.5% and 6.81%, respectively. Furthermore, the single total peak and the three partial peaks in the high resolution N1s spectrum provide additional evidence for the presence of N in the nanocomposite (fig. 4 a). XPS peaks with binding energies of 398.2, 399.6 and 400.0eV belong to pyridine type, pyrrole type and graphite type N, respectively (FIG. 4 a). The purpose of N doping is to increase NSC/Co_1-xThe electron transfer speed of the S nano composite material is increased, so that the catalytic performance is improved. Meanwhile, S2p XPS spectra have peaks at 163.6eV and 164.8eV, respectively for the C-S2 p3/2 and 2p1/2 orbits, and a peak at 169.0eV, which is the satellite signal (FIG. 4 b). These findings demonstrate the presence of sulfur atoms and the presence of carbon-sulfur bonds in the composite. C1S XPS spectra gave 4 peaks at 284.7, 286.1, 287.1 and 288.2eV, corresponding to C-C, C ═ N, -C-S-C-and C ═ O, respectively (fig. 4C). Formation of the C ═ N and-C-S-C-groupsThe bright N and S atoms were successfully doped into the carbon matrix. Finally, the Co 2p spectrum shows two broad peaks centered around 780.2eV and 795.8eV, belonging to Co 2p3/2 and Co 2p1/2, respectively (FIG. 4 d). The Co 2p3/2 peak was further deconvoluted at 779.8eV and 781.2eV into two peaks, assigned to Co (III) and Co (II), respectively. Similarly, the Co 2p1/2 peak deconvoluted into two peaks, corresponding to Co (III) at 794.8eV and Co (II) at 796.5eV, respectively. These results confirmed the coexistence of Co (III) and Co (II), with Co_1-xThe Co valence of S is consistent, therefore, the interconversion between Co (III) and Co (II) can obviously improve the prepared NSC/Co_1-xCatalytic activity of S nanocomposite.

NSC/Ni₉S₈Preparation of nanocomposites

The preparation method is basically the same as NSC/Co_1-xS nanocomposite, with the difference that Co (CH) in step 2₃COO)₂·4H₂Conversion of O to Ni (CH)₃COO)₂·4H₂O。

Scanning Electron Microscopy (SEM) on synthetic NSC/Ni₉S₈Performing a morphology analysis, NSC/Ni as shown in FIG. 13₉S₈The material is composed of a carbon layer and metal particles, wherein the carbon layer is rich in pores and is successfully doped with the cubic metal particles.

Example 2

NSC/Co_1-xEnzymatic activity of S nanocomposite

In NSC/Co_1-xAfter the reaction of S with hydrogen peroxide, the catalytic oxidation of TMB was measured with an ultraviolet spectrophotometer. Briefly, 60. mu.L of 1mg/mL NSC/Co_1-xThe S suspension was added to 1700 μ L of NaAc-HAc buffer (20mM, pH 3.6). Then, 200. mu.L of 6mM TMB and 40. mu.L of 50mM hydrogen peroxide were added to the above solution in this order. After incubation at 50 ℃ for 20min, the absorbance of the synthetic solution at 652nm was recorded.

6 different experimental treatments were designed to detect TMB as chromogenic substrate (NSC/Co)_1-xPeroxidase-like activity of S (fig. 5 a). NSC/Co_1-xS+H₂O₂+ TMB treatment showed the strongest absorbance at 652nm (up to-2.50), followed by NSC + H₂O₂+ TMB processing (2.0). In sharp contrast thereto, in H₂O₂Little absorbance at 652nm was produced during the treatment, but the color remained white. In the other five treatments, the color of the solution changed to blue (NSC/Co)_1-xS+H₂O₂+TMB，NSC+H₂O₂+TMB，NSC/Co_1-xS + TMB, NSC + TMB and H₂O₂+TMB)。TMB+H₂O₂The system also exhibited a relatively weak absorption at 652nm (OD. about.0.4), probably due to a small amount of decomposed H₂O₂. Importantly, these data are NSC/Co_1-xS has a strong peroxidase-like activity providing strong evidence.

To evaluate NSC/Co_1-xWhether S nanocomposite has oxidase-like activity at O₂Or N₂Under gas atmosphere in NSC/Co_1-xPresence and absence of S H₂O₂The color change of the solution was observed. At O₂Under the atmosphere, there was a bright blue color (FIG. 6). Based on the above data, we speculate that Co is present_1-xS nanoparticles anchored on the surface of NSC, in NSC/Co_1-xThe S nanocomposite material has a concerted catalytic action. Notably, NSC/Co_1-xThe S nano enzyme has peroxidase-like and oxidase-like activities.

The activity of the nanoenzyme is closely related to the experimental conditions, so three key variables are selected for optimization: temperature, pH and NSC/Co_1-xAnd S adding amount.

NSC/Co as the temperature increases from 10 ℃ to 50 ℃_1-xThe absorbance at 652nm of the S system gradually increased from 0.12 to 1.58. However, as the temperature was further increased from 50 ℃ to 70 ℃, the absorbance at 652nm dropped sharply to 0.18 (fig. 5 b). We attributed the decrease in OD at higher temperatures to the instability of the nanomaterials at higher temperatures. For solution pH, ODs increased from 1.25 to 1.59 as pH increased from 3.2 to 3.6, and then decreased dramatically as solution pH increased from 3.6 to 6.0 (fig. 5 c). Because of H₂O₂The aqueous solution of (A) is weakly acidic, and an increase in pH promotes H₂O₂Resulting in an oxidizing powerAnd (4) descending. When NSC/Co_1-xWhen the change in the concentration of S was increased from 0 to 6.0mg/mL, the absorbance monotonically increased from 1.0 to 3.1. At higher NSC/Co_1-xThe S concentration (6.0-10.0mg/mL) and the absorbance at 652nm remained almost unchanged (FIG. 5 d). This indicates that the catalytic action of nanoenzymes reaches a plateau and that higher doses beyond this do not cause a greater response due to complete substrate consumption. In addition, in the ultraviolet detection system, too high OD value (C:)>2.0) causes the absorbance to saturate resulting in a sharp drop in the color recognition responsivity. Therefore, we chose 1.0mg/mL as NSC/Co_1-xOptimum concentration of S nanocomposite. In summary, the optimized assay conditions were set at 50 ℃, initial pH 3.6 and NSC/Co_1-xThe S concentration was 1.0 mg/mL.

Under optimized conditions, NSC/Co_1-xS Steady state kinetic analysis was performed on TMB at concentrations of 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0 mM. Kinetic analysis was performed using hydrogen peroxide as substrate, using a series of initial hydrogen peroxide levels (0.1, 0.3, 0.5, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0, 15.0mM) with constant TMB concentrations, all at 6 mM. The Optical Density (OD) at 652nm was measured with a UV-5500PC spectrophotometer. The catalytic parameters for steady state kinetic analysis were obtained by fitting the time-scanned OD data in the mie equation. Michaelis-Menten equation (1) describes the relationship between conversion rate for a given substrate and enzyme substrate concentration (NSC/Co in this case)_1-xS)：

Wherein K_mIs the Michaelis constant, V_maxFor the maximum reaction rate, [ S ]]Is the TMB concentration.

We investigated compounds with different concentrations of TMB (0.2-2.0mM) and H₂O₂(0.1-1.5mM) NSC/Co_1-xSteady state kinetic performance of S nanoenzyme. NSC/Co_1-xThe Michaelis-Menten curve model for S is shown in FIGS. 7a and b. Michaelis constant (K)_m) And maximum reaction velocity (V)_max) Is based on LineweaCalculated by ver-Burk (FIGS. 7c and d). NSC/Co_1-xKm value TMB of S system is 2.98, H₂O₂Is 2.92. Thus, it can be concluded that NSC/Co_1-xThe S system has good catalytic performance, wherein H₂O₂Exhibit slightly higher catalytic capacity (Table 1)

Table 1. Comparison of catalytic kinetic parameters of different nanoenzymes

Note: k_mThe Michaelis constant; HRP, horseradish peroxidase; NSP-CQDs, N, S, p-codoped carbon Quantum dots, K for other catalysts in Table 1_mThe parameters are from the literature.

Example 3

NSC/Co_1-xCatalytic mechanism of S System

Reactive Oxygen Species (ROS) capture assay

Previous studies have shown that isopropyl alcohol (IPA), Ethylene Diamine Tetraacetic Acid (EDTA) and p-benzoquinone (PBQ) have the ability to scavenge hydroxyl radicals (OH) and electron holes (h), respectively⁺) And superoxide radical (. O)^2-) The ability of the cell to perform. To study the ROS species involved in the catalytic reaction, we separately performed 2mL of H₂O₂The system of TMB was added with 200. mu.L of capture agent and 100. mu.L of NSC/Co_1-xS nanocomposite, and the absorbance was measured at 652nm after incubating the mixed solution at 50 ℃ for 20 minutes.

To clarify NSC/Co_1-xCatalytic mechanism of S participation, three radical species (h)⁺OH and O₂ ^-) Captured by their respective scavengers (EDTA, IPA and PBQ) (fig. 8 a). In this method, the response signal is reduced if the radical species responsible for the catalytic reaction is captured by the scavenger. Most notably, h is added⁺The scavenger EDTA is an absorbance value display which causes 652nmSignificant decrease indicates h⁺Plays an important role in catalytic reaction. After the addition of PBQ, the OD at 652nm slightly decreased, indicating that part of O was present₂ ^-Participate in the catalytic system. Finally, the presence of IPA had no significant effect on the 652nm OD, suggesting that OH does not play a significant role in the catalytic process.

Further, NSC/Co was inferred by EPR analysis_1-xFree radical systems may be present in S (FIGS. 8 b-d). h is⁺OH and O₂ ^-All the characteristic signals are detected, and NSC/Co is proved_1-xS nano enzyme capable of catalyzing H₂O₂Generation of h⁺OH and O₂ ^-。h⁺Is strongest, followed by O₂ ^-And OH, consistent with results obtained with ROS scavenger experiments. Due to NSC/Co_1-xThe multivalent state of Co in the S nanocomposite promotes electron transfer, thereby improving redox ability and promoting a catalytic process.

The absence of electrons in the bond between Co (II) and Co (III) generates a hole, corresponding to h⁺The presence of a signal (fig. 8 b). Under acidic conditions, O₂Conversion to O₂ ^-TMB was oxidized to oxTMB, which is consistent with the EPR results (8 c). As shown in FIG. 8d, a characteristic signal of OH was detected, this being NSC/Co_1-xS-nanoenzyme catalysis H₂O₂The generation of OH provides convincing evidence. Therefore, we hypothesize that OH and O are produced₂ ^-By NSC/Co_1-xS has an enzyme-like activity in it as shown in FIG. 7 e. Taken together, these findings infer NSC/Co_1-xS exhibits not only excellent peroxidase-like activity but also potential oxidase-like activity.

Example 4

Colorimetric cholesterol assay

To obtain a cholesterol calibration curve, 100. mu.L of cholesterol (0.2-10 mM), 50. mu.L of cholesterol esterase (1mg/mL), and 50. mu.L of cholesterol oxidase (2mg/mL) were reacted with 50. mu.L of PBS buffer (20mM, pH 7) at 37 ℃ for 30 minutes. The reacted solution was added to a solution containing 200. mu.L of TMB (6mM) and 60. mu.L of NSC/Co_1-xS (1mg/ml) and 1490 μ L NaAc-HAc buffer solution (20mM, pH 3.6). After incubation at 50 ℃ for 20 minutes, the absorbance at 652nm of the resulting solution was recorded.

In FIGS. 9a and 9b, the Linear Range (LR) and limit of detection (LOD) of cholesterol were 0.2-6.0mM and 0.05mM, respectively. These performance indicators were compared to several cholesterol determination methods reported in the literature, such as electrochemical, chemiluminescent, and colorimetric methods. Although the electrochemical method based on Au/CdS (0.012mM) has a low LOD, the method has the disadvantages of high energy consumption and low current efficiency. Chemiluminescence methods, such as NG/CIS/ZnS (LR 0.31-5.0mM, LOD 0.22mM) have a wide linear range, but are less specific for the target analyte. NSC/Co as shown in Table 2_1-xThe S nanoenzyme biosensor achieved lower or comparable performance.

TABLE 2 comparison of the NSC/Co1-xS System with other Cholesterol quantification catalytic systems

Material	LRs(mM)	LODs(mM)	Method
				Au/CdS QDs/TNTs	0.024-1.2	0.012	Electrochemistry method
NG/CIS/ZnS	0.312-5	0.222	Chemiluminescence
				Copper oxide: GNS	0.1-0.8	0.078	Chromatography method
n-cd	2.5-7.5	0.68	Chromatography method
				AgNPs+ChO_x	0.1-1.5	0.04	Chromatography method
Cu₂(OH)₃Cl-CeO₂	0.1-2	0.05	Chromatography method
				NSC/Co_1-xS	0.2-6	0.05	Chromatography method

Note: LOD is 3 sigma/k; QDs, quantum dots; TNTs, TiO₂A nanotube; NG, nitrogen doping graphene; CIS, CuInS, CNS graphene nanospheres; CDs, carbon dots.

LOD (0.05mM) higher than copper oxide: GNS (0.078mM), N-CD (0.68mM), AgNPs + ChO_x(0.04mM) and Cu₂(OH)₃Cl-CeO₂(0.05 mM). Therefore, the comparative performance indexes show that the constructed nano enzyme biosensor is beneficial to biological componentsPractical application in the field of sub-analysis.

NSC/Co_1-xSelectivity/interference limitation of S-based colorimetric cholesterol quantitation

To explore possible interfering effects during cholesterol detection, the concentration of various macromolecules and ions was increased to 10 mM: galactose (Gal), sucrose (Suc), glucose (Glu), d-histidine (His), d-serine (Ser), NaCl, KCl and MgSO₄. In this interference assessment, the cholesterol concentration in the system was kept constant at 1.0 mM. The experimental procedure for the various macromolecules and ions is the same as the cholesterol detection procedure.

In NSC/Co_1-xS+TMB+H₂O₂Monosaccharide, amino acid and metal ions are added into the reaction system in an amount of 10mM to evaluate the selectivity and potential interference effect of the new colorimetric method. The potentiating level of interfering compounds/ions was set at 10 times the cholesterol concentration (1.0 mM). The range of 652nm ODs from interfering solutes was 0.15 to 0.30, while the value of cholesterol without interfering solutes was-1.1 (FIG. 10). This comparison shows that the interfering effect of macromolecules and ions is generally low, even at higher concentrations of interfering solutes. Therefore, it is presumed that the newly developed NSC/Co-based_1-xThe colorimetric detection platform of the S nano enzyme has good selectivity/low interference effect on cholesterol analysis.

Example 5

Determination of total cholesterol in human serum

Human serum samples (6) were provided at a hospital and stored at-80 ℃ prior to use. Prior to the experiment, frozen serum samples were thawed at 4 ℃ and then centrifuged at 10000rpm for 20 minutes to remove large clumps. The supernatant was diluted 10-fold with PBS buffer (20mM, pH 7.0) to obtain a pretreated serum sample. mu.L of the pretreated serum sample, 50. mu.L of cholesterol esterase (1mg/mL) and 50. mu.L of cholesterol oxidase (2mg/mL) were added to 50. mu.L of PBS buffer (20mM, pH 7), respectively, and incubated at 37 ℃ for 30 minutes. The subsequent experimental procedure was the same as the detection of cholesterol described above.

Visual total cholesterol detection based on smart phone

A model based on NSC/Co is developed_1-xAnd an Android smart phone application program (APP) of the S colorimetric sensor is used for detecting total cholesterol in blood. We designed a "thining identification" application (accession number 2020SR0058103) in 2018 (download address http:// iwater. wmu. edu. cn/info/1042/1191. htm). When the "thigh identification" icon is selected, a new page appears with four option buttons, "IMAGE", "identification", "MODEL", and "DETAILS" (FIG. 11 a). When the "IMAGE" button is selected, two icons "Capture" and "Gallery" appear (FIG. 11 b). The detection image can be selected from available images containing blue reaction solutions representing different cholesterol concentrations (fig. 11 c). At the bottom of the home screen, when you select the "options" button, a new page appears. The user can select "linear regression" or "polynomial regression" on the page as a calibration option, and can also adjust the parameters of image recognition (fig. 11 d). Selecting the "MODEL" button returns to the main screen, at which point the user may select the "IDENTIFY" button and the application will automatically measure the intensity of the colors in the image (FIG. 11 e). By adjusting "param 1" and "param 2", the software can identify all target colors, but cannot identify any non-target colors. When you enter the concentration for each sample and select "MODEL," a standard curve of cholesterol concentration versus target color (gray scale) is generated (FIG. 11 f). After the calculation is complete, the "identify" button at the bottom of the screen is selected and a new page is opened (FIG. 9 g). Subsequently, the "IMAGE" button can be selected, selecting the test sample picture. Finally, by selecting "IDENTIFY", the screen will display the corresponding gray value and its corresponding cholesterol concentration (fig. 11 h).

After the key parameters are optimized and the analysis performance of the novel colorimetric method is researched, a smartphone-based colorimetric application program (app) is developed to quantitatively determine the cholesterol concentration in human serum. As shown in fig. 12a, the mobile phone screen, the color intensity after the catalytic reaction monotonically increases with the increase in cholesterol concentration (fig. 12b) a calibration curve was generated by plotting the gray value of the reaction solution versus the corresponding cholesterol concentration. The gray values were linearly related to the cholesterol concentration in the range of 0.2-6.0mM (FIG. 12b). The regression equation obtained isY＝66.3-0.667X+0.002X²(R²0.969), wherein X, Y are the reciprocal of the color grayscale value and the cholesterol concentration, respectively. The LOD of cholesterol provided by the application was 65 μ M (LOD 3 s/k). As shown in Table 3, the cholesterol concentration detected by APP is comparable to the detection result of the standard cholesterol detection kit. The relative recovery rate of cholesterol (RRs) was 93.6-104.1%, and RSDs was 1.7-4.7% (Table 3). The performance indexes show that the APP colorimetric method is convenient, low in cost, rapid, simple and accurate, and provides several promising applications for field diagnosis.

TABLE 3

The present invention has been described in detail in order to enable those skilled in the art to understand the invention and to practice it, and it is not intended to limit the scope of the invention, and all equivalent changes and modifications made according to the spirit of the present invention should be covered by the present invention.

Claims

1. The preparation method of the composite nano enzyme is characterized by comprising the steps of preparing aerogel by using an organic matter containing N and S and a transition metal salt in the presence of a reducing agent and a cross-linking agent, calcining the aerogel at 400-800 ℃ in an inert atmosphere, washing with water, and drying to obtain the composite nano enzyme, wherein the transition metal salt is cobalt salt or nickel salt.

2. The preparation method of claim 1, wherein the organic matter containing N and S is one or more of trithiocyanuric acid, thioacetamide, mercaptosuccinic acid or thiourea;

and/or the cross-linking agent is one or more of sodium alginate, hexamethylenetetramine or dicyanodiamine;

and/or the reducing agent is one or more of citric acid, glucose and cysteine;

and/or, the calcination is carried out under the protection of nitrogen or argon;

and/or the transition metal salt is one or more of cobalt acetate, cobalt nitrate, nickel acetate or nickel nitrate.

And/or the mass ratio of the cross-linking agent, the organic matter containing N and S, the reducing agent and the transition metal salt is 1: (3-8): (0.1-0.8): (0.1 to 0.8);

and/or the drying temperature is 50-80 ℃.

3. The method for preparing the aerogel according to claim 1, wherein the method for preparing the aerogel comprises the following steps: mixing N and S-containing organic matters, a reducing agent, a cross-linking agent and water to form uniform primary mixed liquid, then mixing the mixed liquid and a transition metal salt aqueous solution to form uniform secondary mixed liquid, and dehydrating the secondary mixed liquid to form the aerogel.

4. The preparation method according to claim 3, wherein in the preliminary mixed solution, the concentration of the organic matter containing N and S is 40-60 g/L, the concentration of the reducing agent is 1-10 g/L, and the concentration of the crosslinking agent is 5-20 g/L;

and/or the concentration of the aqueous solution of the transition metal salt is 1-10 g/L;

and/or, the dehydration adopts freeze drying dehydration.

5. The preparation method according to claim 1, wherein the calcination comprises a primary calcination and a secondary calcination, the temperature of the primary calcination is lower than that of the secondary calcination, and the primary calcination specifically comprises: heating to 400-600 ℃ at a heating rate of 1-3 ℃/min, and then carrying out heat preservation calcination at 400-600 ℃ for 20-50 min; the secondary calcination specifically comprises the following steps: after the primary calcining, continuously heating to 600-800 ℃ at the heating rate of 4-6 ℃/min, and then carrying out heat preservation calcining at 600-800 ℃ for 100-150 min.

6. The composite nanoenzyme is characterized in that the composite nanoenzyme is a transition metal sulfide/nitrogen and sulfur Co-doped carbon material with catalase-like catalytic activity and oxidase-like catalytic activity, and the transition metal sulfide is Co_1-xS or Ni₉S₈And part of the transition metal sulfide is dispersedly attached to the surface of the nitrogen-sulfur co-doped carbon material, and the other part of the transition metal sulfide is doped in the nitrogen-sulfur co-doped carbon material.

7. The composite nanoenzyme of claim 6, wherein the transition metal sulfide is Co_1- _xS, the element composition of the composite nano enzyme comprises N accounting for 20-40% of the total mass of the composite nano enzyme, S accounting for 1-5% of the total mass of the composite nano enzyme, C accounting for 40-70% of the total mass of the composite nano enzyme, and Co, Co and Co accounting for 5-12% of the total mass of the composite nano enzyme_1-xThe size of S particles is less than 20nm, and the specific surface area of the composite nano enzyme is 15-25 m²g^-1。

8. Use of the composite nanoenzyme prepared by the preparation method of any one of claims 1 to 5 or the composite nanoenzyme of claim 6 or 7 in cholesterol detection.

9. A method for detecting cholesterol, characterized in that cholesterol is measured based on a colorimetry, and a composite nanoenzyme prepared by the preparation method according to any one of claims 1 to 5 or the composite nanoenzyme according to claim 6 or 7 catalyzes a reaction between hydrogen peroxide and a chromogenic substrate, wherein 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.

10. The detection method according to claim 9, wherein the temperature of the catalytic reaction of the composite nano-enzyme is 25-60 ℃;

and/or the concentration of the composite nano enzyme in the reaction system is 0.5-2.5 mg/mL;

and/or the initial pH of the reaction system is 3-4;

and/or, the detection method also comprises the steps of hydrolyzing cholesterol ester under the action of cholesterol esterase to generate cholesterol, and oxidizing the cholesterol under the action of cholesterol oxidase to generate hydrogen peroxide;

and/or the test sample is one or more of serum, blood, urine, milk or edible oil

And/or, the detection method further comprises the step of carrying out colorimetric data processing by using digital imaging colorimetric software.

11. A kit for detecting cholesterol, comprising the composite nanoenzyme prepared by the preparation method according to any one of claims 1 to 5 or the composite nanoenzyme according to claim 6 or 7.

12. The kit of claim 11, wherein the kit further comprises hydrogen peroxide, 3',5,5' -tetramethylbenzidine, cholesterol oxidase, and cholesterol esterase.