CN115779971A - Cascade nanoenzyme and preparation method and application thereof - Google Patents
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Abstract
The invention provides a cascade nanoenzyme and a preparation method and application thereof, belonging to the technical field of biocatalysis. The cascade nanoenzyme comprises a two-dimensional metal organic framework nanosheet containing a ligand and gold nanoparticles combined with the two-dimensional metal organic framework nanosheet, wherein the ligand is at least one of heme and tetracarboxyl (phenyl) porphyrin. The cascade nanoenzyme is mainly used for detecting the content of glucose. The two-dimensional metal organic framework peroxide mimic enzyme nanosheet formed by metal salt and ligand is used as a carrier, and gold nanoparticles (AuNPs) are combined to the surface of the peroxide mimic enzyme nanosheet through electrostatic attraction to form artificial nanoenzyme, so that the peroxide mimic enzyme action of the two-dimensional metal organic framework nanosheet and the glucose oxidation mimic enzyme action of the AuNPs can be combined together to form a cascade reaction.
Description
Technical Field
The invention belongs to the technical field of biocatalysis, and particularly relates to a cascade nanoenzyme, and a preparation method and application thereof.
Background
In clinical practice, enzyme-linked immunosorbent assay (ELISA), which is a traditional method for quantitative measurement of components by the change of signals corresponding to the reaction system, is the most commonly used method for biological detection such as glucose detection. However, the development and development cycle of the corresponding kit is long, the cost is high, and the detection needs professional personnel to carry out and other factors, so that the wide application of the kit is limited.
Nanometer enzymes have attracted much attention in recent years due to their unique enzymatic catalytic properties. It is well known that monitoring glucose levels in brain tissue is important, glucose being an important chemical affecting the nervous system, involved in many physiological and pathological brain functions, such as learning and memory, and associated with cerebral ischemia.
However, the research on glucose detection technology by nanoenzymes is still in an early stage, and many potential problems are not solved yet, for example, most nanoenzymes have lower catalytic activity and poorer substrate selectivity compared with natural enzymes. Therefore, it is highly desirable to develop a high-sensitivity detection method with low manufacturing cost and convenient detection.
Disclosure of Invention
The invention provides a cascade nanoenzyme, aiming at solving the problems of high cost, easy inactivation and the like of a natural enzyme when the content of glucose is detected by a colorimetric method.
The invention provides a cascade nanoenzyme, which comprises a two-dimensional metal organic framework nanosheet containing a ligand and gold nanoparticles combined with the two-dimensional metal organic framework nanosheet, wherein the ligand is at least one of heme and tetracarboxyl (phenyl) porphyrin.
Further, the gold nanoparticles are bound to the surface of the two-dimensional metal organic framework nanosheets.
Further, the size of the cascade nanoenzyme is 1-5 μm;
preferably, the particle size of the gold nanoparticles is 1-10nm.
The invention also provides a preparation method of any one of the cascade nanoenzymes, which comprises the following steps:
adding tetrachloroalloy solution into solution of a ligand-containing two-dimensional metal organic framework nanosheet, mixing, adding tannic acid solution, washing, and centrifuging to obtain the cascade nanoenzyme.
Further, the ligand-containing two-dimensional metal organic framework nanosheet is prepared by the following steps:
dissolving metal salt and polyvinylpyrrolidone in a solvent, stirring, dropwise adding a ligand solution, carrying out ultrasonic treatment, and heating for reaction; and cooling, centrifuging and washing the reaction mixture to obtain the two-dimensional metal organic framework nanosheet containing the ligand.
Further, the dosage ratio of the metal salt to the polyvinylpyrrolidone is 0.01-0.02mmol: 15.0-20.0 mg;
the metal salt comprises Zn (NO) 3 ) 2 ·6H 2 O、Co(NO 3 ) 2 ·6H 2 O、Cd(NO 3 ) 2 ·4H 2 O、FeCl 3 ·6H 2 O、MnCl 4 ·4H 2 Any one of O;
the ligand in the ligand solution is tetracarboxyphenyl porphyrin or heme;
the molar ratio of the ligand to the metal salt is 1:1-5.
Further, the concentration of the tetrachloroalloy solution is 10-30mM;
the concentration of the solution of the two-dimensional metal organic framework nanosheet is 0.05-2mgmL -1 ；
The dosage ratio of the tetrachloroalloy solution to the solution of the ligand-containing two-dimensional metal organic framework nanosheet is 100-200 mu L: 1-15 mL;
the concentration of the tannic acid solution is 1-20mM.
The invention also provides the cascade nanoenzyme and application of the cascade nanoenzyme prepared by the preparation method in determination of glucose content.
Further, the specific assay method comprises:
1) Mixing the solution of the cascade nanoenzyme with a glucose solution to be detected, adding 3,3',5,5' -tetramethylbenzidine solution after the first reaction, and characterizing the obtained supernatant by using an ultraviolet-visible spectrum method after the second reaction to obtain an absorbance value;
2) Preparing glucose standard solutions with different concentrations, respectively measuring absorbance values according to the method in the step 1), and fitting a standard curve equation according to the concentration of each glucose standard solution and the corresponding absorbance value;
3) And (3) measuring the absorbance value of the glucose solution to be measured according to the step 1), and calculating the concentration of the glucose solution by combining the standard curve equation.
Further, the time of the first reaction is 4h; the time of the second reaction is 1-300s.
The invention has the following advantages:
the invention takes a two-dimensional metal organic framework peroxide mimic enzyme nano-sheet formed by metal salt and ligand as a carrier, and combines gold nanoparticles (AuNPs) on the surface of the peroxide mimic enzyme nano-sheet through electrostatic attraction to form artificial nano-enzyme, thus combining the peroxide mimic enzyme action of the two-dimensional metal organic framework nano-sheet and the glucose oxidation mimic enzyme action of the AuNPs together to form cascade reaction. The method can combine two activities of glucose oxidation mimic enzyme and peroxide mimic enzyme to construct a cascade reaction for detecting the content of glucose.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic diagram of synthesis of AuNPs/Zn-TCPP mixed nanosheets according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of AuNPs/Zn-TCPP mixed nanosheet cascade reaction according to an embodiment of the present invention;
FIG. 3 is a Transmission Electron Microscope (TEM) image of a 2D Zn-TCPP mixed nanosheet mimetic enzyme prepared by an embodiment of the present invention;
FIG. 4 is a Fourier infrared transform spectroscopy (FTIR) graph of a Zn-TCPP mixed nanosheet mimetic enzyme prepared by an embodiment of the present invention;
FIG. 5 is a Transmission Electron Microscope (TEM) image of AuNPs/Zn-TCPP mixed nanosheet mimic enzyme prepared in an embodiment of the present invention;
FIG. 6 shows different metal ions (Zn) according to the present invention 2+ 、Co 2+ Etc.) and different ligands (TCPP, hemin)Belongs to an organic framework peroxide nano enzyme activity contrast diagram;
FIG. 7 shows the reduction of different concentrations of HAuCl measured in accordance with the practice of the present invention 4 An ultraviolet-visible spectrum (UV-Vis) contrast diagram of the AuNPs/Zn-TCPP mixed nanosheet synthesized by the precursor simulating the enzyme activity;
FIG. 8 is a comparison graph of ultraviolet-visible spectra (UV-Vis) obtained by catalytic reaction of AuNPs/Zn-TCPP mixed nanosheets prepared according to examples of the present invention with glucose of different concentrations.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The present invention will be described in detail below with reference to the accompanying drawings.
An embodiment of the present invention provides a cascade nanoenzyme, which includes a two-dimensional metal-organic framework nanosheet including a ligand, and gold nanoparticles (AuNPs) combined with the two-dimensional metal-organic framework nanosheet, wherein the ligand is at least one of heme (hemin) and tetracarboxyl (phenyl) porphyrin (TCPP).
The embodiment of the invention is developed around bionic construction and catalytic characteristic research of nano enzyme, gold nanoparticles are used as glucose oxidation mimic enzyme, two-dimensional Metal Organic Frameworks (MOFs) are used as peroxide mimic enzyme, the two materials are combined, the composite material is successfully prepared for the first time, and the two activities of the glucose oxidation mimic enzyme and the peroxide mimic enzyme are combined to construct a cascade reaction for detecting the content of glucose.
In an embodiment of the present invention, the gold nanoparticles are bonded to the surface of the two-dimensional metal organic framework nanosheet.
In one embodiment of the invention, the particle size of the cascade nanoparticles is 1-5 μm. Preferably, the cascade nanoparticles have a particle size of 1 to 10nm.
An embodiment of the present invention further provides a method for preparing any of the cascade nanoenzymes, including the following steps:
mixing tetrachloro alloy (HAuCl) 4 ) Adding the solution to a solution containing a ligandMixing the two-dimensional metal organic framework nanosheets, adding a Tannic Acid (TA) solution, washing, and centrifuging to obtain the cascade nanoenzyme.
In the embodiment of the invention, tannic acid is used as a reducing agent, so that tetrachloroalloy is subjected to in-situ reduction reaction on the surface of a two-dimensional metal organic framework peroxide mimic enzyme nanosheet to obtain the gold nanoparticle glucose oxidation mimic enzyme.
In a preferred embodiment of the present invention, the ligand-containing two-dimensional metal-organic framework nanosheet is prepared by the following steps:
dissolving metal salt and polyvinylpyrrolidone (PVP) in a solvent, stirring, dropwise adding a ligand solution, carrying out ultrasonic treatment, and heating for reaction; and cooling, centrifuging and washing the reaction mixture to obtain the two-dimensional metal organic framework nanosheet containing the ligand.
Specifically, the dosage ratio of the metal salt to the polyvinylpyrrolidone is 0.01-0.02mmol: 15.0-20.0 mg. In the embodiment of the invention, in the process of preparing the two-dimensional metal organic framework nanosheet, polyvinylpyrrolidone plays an important role in the formation of the two-dimensional nanosheet. Specifically, in the process of forming the nanosheet, PVP is attached to the surface of the 2D Zn-TCPP nanosheet, so that anisotropic growth of the nanosheet is limited, and an important role is played in forming the 2D Zn-TCPP nanosheet.
Specifically, the metal salt includes Zn (NO) 3 ) 2 ·6H 2 O、Co(NO 3 ) 2 ·6H 2 O、Cd(NO 3 ) 2 ·4H 2 O、FeCl 3 ·6H 2 O、MnCl 4 ·4H 2 O。
Specifically, the solvent is a mixture of DMF-ethanol. Wherein the volume ratio of DMF to ethanol is 3:1. the solvent also includes pyrazine. The dosage ratio of the pyrazine to the DMF-ethanol mixture is 0.01mmol:5-20mL. The dosage ratio of the metal salt to the solvent is 0.01-0.02mmol:5-20mL.
Specifically, the ligand in the ligand solution is tetracarboxyphenylporphyrin or heme. The molar ratio of the ligand to the metal salt is 1:1-5. Preferably, the ligand to metal salt molar ratio is 1:2-3.
Specifically, the solvent of the ligand solution is a DMF-ethanol mixture. The dosage ratio of the ligand to the DMF-ethanol mixture is 0.003 to 0.008mmol:1-10ml. Preferably, the ratio of the ligand to the DMF-ethanol mixture is 0.005mmol:4ml.
Specifically, the time of the ultrasonic treatment was 10 minutes. Specifically, the heating reaction is specifically as follows: heating to 70-90 deg.c and reacting for 20-30 hr. Preferably, the reaction is carried out for 24h by heating to 80 ℃. Specifically, the cooling is to room temperature. The washing is carried out by using ethanol.
In one embodiment of the present invention, HAuCl 4 Is in a concentration of 10-30mM. Preferably, HAuCl 4 The concentration of (2) is 20mM. Within the above suitable range, the higher the concentration of the reduced gold ions, the larger the particle diameter of the obtained gold nanoparticles, and the lower the glucose oxidation catalytic activity. Conversely, the lower the concentration of reduced gold ions, the smaller the particle size of the formed gold nanoparticles, and the higher the glucose oxidation catalytic activity.
In one embodiment of the invention, the concentration of the solution of the two-dimensional metal organic framework nanosheet is 0.05-2mgmL -1 . Preferably 0.1mgmL -1 . Specifically, the solvent of the solution of the two-dimensional metal organic framework nanosheet is at least one of DMSO and ethanol.
In one embodiment of the present invention, HAuCl 4 The dosage ratio of the solution to the solution of the two-dimensional metal organic framework nanosheet is 100-200 mu L: 1-15 mL.
In one embodiment of the present invention, the concentration of the aqueous solution of Tannic Acid (TA) is 1 to 20mM. HAuCl 4 The volume ratio of the solution to the tannic acid solution was 4:1.
In one embodiment of the present invention, the washing is washing with water. The centrifugation is carried out for 10min by using 10000 r.p.m. The gold nanoparticle glucose oxidation mimic enzyme is combined on the surface of the two-dimensional metal organic framework nanosheet through electrostatic attraction.
The embodiment of the invention also provides application of any one of the cascade nanoenzymes in determination of glucose content. The limits of glucose detection of interest to those skilled in the art can be directly determined by standard curve equations.
Further, the specific assay method comprises:
1) Mixing the solution of the cascade nanoenzyme with a glucose solution to be detected, adding 3,3',5,5' -tetramethylbenzidine solution after the first reaction, and characterizing the obtained supernatant by using an ultraviolet-visible spectrum method after the second reaction to obtain an absorbance value;
2) Preparing glucose standard solutions with different concentrations, respectively measuring absorbance values according to the method in the step 1), and fitting a standard curve equation according to the concentration of each glucose standard solution and the corresponding absorbance value;
3) And (3) measuring the absorbance value of the glucose solution to be measured according to the step 1), and calculating the concentration of the glucose solution by combining the standard curve equation.
Preferably, the time of the first reaction is 4h; the time of the second reaction is 1-300s.
As shown in FIG. 2, in the first step, auNPs are used as a glucose oxidation mimic enzyme to oxidize glucose to produce hydrogen peroxide (H) as a product 2 O 2 ). Then, the reaction substrate is used in the second step, in which the 2D Zn-TCPP nanosheet functions as a peroxidase mimetic enzyme, and H is introduced 2 O 2 Oxidizing to OH, then adding a chromogenic substrate TMB (3,3 ',5,5' -tetramethyl benzidine), oxidizing to macroscopic blue oxidation type substances (oxTMB) by OH, and constructing a cascade reaction to quickly detect the glucose content. Cascaded catalytic properties exist between gold nanoparticles (AuNPs) and metal-organic framework nanoenzymes without the need for exogenous provision of substrates such as hydrogen peroxide (H) 2 O 2 ) And then catalytic detection can be carried out.
In one embodiment of the invention, the concentration of the solution of the cascade nanoenzyme is 1-500 μ g mL -1 . Preferably, the concentration of the solution of the cascade nanoenzyme is 200. Mu. GmL -1 。
3,3',5,5' -Tetramethylbenzidine (TMB) in dimethylsulfoxide at a concentration of 25mM. Wherein the solvent in the 3,3',5,5' -tetramethyl benzidine solution is dimethyl sulfoxide solution.
The volume ratio of the solution of the cascade nanoenzyme, the glucose solution, the 3,3',5,5' -tetramethylbenzidine solution can be 750 μ L: 100-200 μ L: 50-55 mu L.
The colorimetric detection constructed by the embodiment of the invention is stable, and still has higher catalytic activity after the nano enzyme is stored for a longer time.
The present invention will be described in detail with reference to examples.
Example 1The preparation method of the cascade nanoenzyme comprises the following steps:
an example of the synthesis process is shown in FIG. 1;
(1) Synthesis of 2D Zn-TCPP nanosheet
Zn (NO) is synthesized by surfactant-assisted synthesis 3 ) 2 ·6H 2 O (3.0mg, 0.01mmol), pyrazine (0.8mg, 0.01mmol) and PVP (20.0 mg) were dissolved in 12mL of a DMF-ethanol mixture (V: V = 3:1), and then 4mL of a DMF-ethanol mixture (V: V = 3:1) containing TCPP (4.4mg, 0.005mmol) was added dropwise with vigorous stirring. After 10 minutes of sonication, the resulting reaction solution was heated to 80 ℃ and held for 24 hours. The reaction mixture was cooled and centrifuged (8000r.p.m. for 10 min) to obtain a dark brown precipitate, which was washed with ethanol multiple times, and the obtained 2D Zn-TCPP nanosheets were redispersed in DMSO.
The nanosheets obtained were characterized and their Transmission Electron Microscopy (TEM) image is shown in fig. 3 and fourier infrared transform spectroscopy (FTIR) image is shown in fig. 4.
From the figure 3, the obtained 2D Zn-TCPP mixed nanosheet mimic enzyme has an obvious two-dimensional structure and a small thickness, so that a larger specific surface area can be provided, more catalytic active sites can be exposed, and the catalytic activity of the nanoenzyme can be effectively improved.
From FIG. 4, the obtained 2D Zn-TCPP mixed nanosheet mimics PVP and Zn (NO) used by the enzyme 3 ) 2 Has strong interaction, is used as a surfactant to be attached to the surface of the 2D Zn-TCPP mixed nanosheet, and plays an important role in forming a 2D structure of the mixed nanosheet.
(2) 2D Zn-TCPP nanosheet and gold nanoparticle combination
100 mu of LHAuCl 4 (10 mM) 10mLZn-TCPP nanoplatelets (0) were added.1mgmL -1 ) After that, the mixture was stirred for 1min, and then 25. Mu.L of ice-cold freshly prepared reducing agent tannic acid solution (16.7 mM) was added. The mixture was then immediately washed twice with water and then centrifuged at 10000r.p.m for 10min to collect the mixed nanoplatelets. And re-dispersing the obtained AuNPs/Zn-TCPP hybrid nanosheets in water. A Transmission Electron Microscope (TEM) image thereof is shown in FIG. 5.
From fig. 5, the obtained AuNPs/Cu-TCPP (M) mixed nanosheet double mimic enzyme has been successfully synthesized, and the AuNPs are loaded onto the 2D nanosheets through in situ synthesis.
Example 2Preparation method of binuclear enzyme
The difference from example 1 is that Zn (NO) is added in step (1) 3 ) 2 ·6H 2 O (3.0mg, 0.01mmol) was replaced with Co (NO) 3 ) 2 ·6H 2 O (4.4mg, 0.015mmol) to prepare the 2D Co-TCPP nano-sheet.
Example 3Preparation method of binuclear enzyme
The same as example 1, except that in the step (1), "4 mL of the solution containingTCPP(4.4 mg, 0.005mmol) of DMF-ethanol mixture (V: V = 3:1) "was replaced with" 4mL of DMF-ethanol mixture (V: V = 3:1) containing Hemin (34mg, 0.005mmol) was added dropwise with vigorous stirring "to prepare Zn-Hemin nanoenzyme.
Example 4Preparation method of binuclear enzyme
The same as example 2, except that in the step (1), "4 mL of the solution containingTCPP(4.4 mg, 0.005mmol) of DMF-ethanol mixture (V: V = 3:1) "was replaced with" 4mL of DMF-ethanol mixture (V: V = 3:1) containing Hemin (34mg, 0.005mmol) was added dropwise under vigorous stirring "to prepare 2D Co-Hemin nanosheets.
Example 5Preparation method of binuclear enzyme
The difference from example 1 is that "Zn (NO) is added in step (1) 3 ) 2 ·6H 2 Replacement of O (3.0 mg, 0.01mmol) "with" FeCl 3 ·6H 2 O (3.0 mg, 0.01mmol) ", and preparing the Fe-TCPP nanosheet.
Example 6Preparation method of binuclear enzyme
The difference from example 1 is that "Zn (NO) is added in step (1) 3 ) 2 ·6H 2 Replacement of O (3.0mg, 0.01mmol) "with" MnCl 4 ·4H 2 O (4.4-5.0 mg, 0.015mmol) ", and preparing the Mn-TCPP nanosheet.
Examples 7 to 8Preparation method of binuclear enzyme
The difference from example 1 is that, in step (2), HAuCl 4 The concentrations of the solutions were 15mM and 20mM, respectively.
Test example 1
1. Taking the 2D Zn-TCPP nano-sheet as a peroxide mimic enzyme test sample, and carrying out the determination of the activity of the synthesized peroxide mimic enzyme
mu.L of the 2D metal organic framework peroxide nanosheet suspension (612. Mu.g/mL) obtained in examples 1-6 was added to 0.10M acetic acid buffer (pH 5.0) containing hydrogen peroxide (2-10. Mu.L, 200 mM) and TMB (4-10. Mu.L, 25 mM), respectively. The final volume of the mixture was adjusted to 200 μ L with 0.10M acetate buffer (pH 5.0) and then incubated at room temperature for 2min. Then, the resulting reaction solution was subjected to absorption spectrum (at 652 nm) measurement on an ultraviolet-visible spectrophotometer. The results are shown in FIG. 6.
From fig. 6, it can be seen that the absorbance of the Zn-TCPP two-dimensional metal organic framework nanosheet synthesized by using zinc ions as metal nodes and TCPP as a ligand tends to be relatively stable with time, i.e., the Zn-TCPP two-dimensional metal organic framework nanosheet has better activity compared with other nanomaterials. Therefore, we selected Zn-TCPP nanoplates with better catalytic activity for further activity determination.
2. Determination of glucose oxidation mimic enzyme-peroxide mimic enzyme cascade reaction activity after synthesis of AuNPs/Zn-TCPP mixed nanosheet
750 μ L of AuNPs/Cu-TCPP (M) mixed nanoplates (200 μ g mL) obtained in examples 7 to 8, respectively -1 ) Mixed with 200 μ L glucose (1M) and the mixed solution incubated for 4h. Then, 50. Mu.L of TMB (25 mM) was added to the above solution, followed by incubationDifferent reaction times were used. And finally, removing the AuNPs/Cu-TCPP hybrid nanosheets from the solution by using a centrifuge, and then characterizing the obtained supernatant by using an ultraviolet-visible spectrum. The results are shown in FIG. 7.
FIG. 7 shows the reduced HAuCl 4 Effect of precursor concentration on nanoenzymes, reduced HAuCl 4 The higher the concentration of the precursor is, the larger the particle size of the gold nanoparticles (AuNPs) obtained by reduction is, and the lower the catalytic activity of the double mimic enzyme is.
Test example 2Application of cascade nano enzyme in determination of glucose content
The specific determination method comprises the following steps:
1) Mixing the solution of the cascade nanoenzyme obtained in the embodiment 1 with a glucose solution to be detected, adding 3,3',5,5' -tetramethylbenzidine solution after the first reaction, and characterizing the obtained supernatant by using an ultraviolet-visible spectrum method after the second reaction to obtain an absorbance value;
2) Preparing glucose standard solutions (5 mM,10mM, 2mM, 50mM and 100mM) with different concentrations, respectively measuring absorbance values according to the method in the step 1), and fitting a standard curve equation according to the concentration of each glucose standard solution and the corresponding absorbance value; as shown in particular in fig. 8.
3) And (3) measuring the absorbance value of the glucose solution to be measured according to the step 1), and calculating the concentration of the glucose solution by combining the standard curve equation.
Wherein, the volume ratio of the solution of the cascade nanoenzyme, the glucose solution, the 3,3',5,5' -tetramethyl benzidine solution can be 750 μ L: 100-200 μ L: 50-55 mu L. The concentration of the solution of the cascade nano enzyme is 200 mu gmL -1 . The concentration of the dimethylsulfoxide solution of 3,3',5,5' -Tetramethylbenzidine (TMB) was 25mM.
FIG. 8 is a linear graph of UV-visible absorbance for glucose at different concentrations, which is a linear plot of the value of absorbance versus the UV-visible absorbance of glucose concentration, and the linear equation is A =0.00032c +0.072 (c represents the glucose concentration in mM, A represents the absorbance at a wavelength of 652 nm), the correlation coefficient is 0.9981, and the detection limit is: 0.4mM.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A cascade nanoenzyme, comprising a two-dimensional metal-organic framework nanosheet comprising a ligand, and a gold nanoparticle bound to the two-dimensional metal-organic framework nanosheet, wherein the ligand is at least one of heme and tetracarboxyl (phenyl) porphyrin.
2. The cascade nanoenzyme of claim 1,
the gold nanoparticles are combined on the surface of the two-dimensional metal organic framework nanosheet.
3. The cascade nanoenzyme of claim 1,
the size of the cascade nanoenzyme is 1-5 mu m;
preferably, the particle size of the grade gold nanoparticles is 1-10nm.
4. The method for preparing a cascade nanoenzyme according to any one of claims 1 to 3, comprising the steps of:
adding tetrachloroalloy solution into solution of a ligand-containing two-dimensional metal organic framework nanosheet, mixing, adding tannic acid solution, washing, and centrifuging to obtain the cascade nanoenzyme.
5. The production method according to claim 4,
a ligand-containing two-dimensional metal organic framework nanosheet is prepared by the following steps:
dissolving metal salt and polyvinylpyrrolidone in a solvent, stirring, dropwise adding a ligand solution, carrying out ultrasonic treatment, and heating for reaction; and cooling, centrifuging and washing the reaction mixture to obtain the ligand-containing two-dimensional metal organic framework nanosheet.
6. The production method according to claim 5,
the dosage ratio of the metal salt to the polyvinylpyrrolidone is 0.01-0.02mmol: 15.0-20.0 mg;
the metal salt comprises Zn (NO) 3 ) 2 ·6H 2 O、Co(NO 3 ) 2 ·6H 2 O、Cd(NO 3 ) 2 ·4H 2 O、FeCl 3 ·6H 2 O、MnCl 4 ·4H 2 Any one of O;
the ligand in the ligand solution is tetracarboxyphenyl porphyrin or heme;
the molar ratio of the ligand to the metal salt is 1:1-5.
7. The method according to claim 4,
the concentration of the tetrachloroalloy solution is 10-30mM;
the concentration of the solution of the two-dimensional metal organic framework nanosheet is 0.05-2mgmL -1 ；
The dosage ratio of the tetrachloroalloy solution to the solution of the ligand-containing two-dimensional metal organic framework nanosheet is 100-200 mu L: 1-15 mL;
the concentration of the tannic acid solution is 1-20mM.
8. The method of
Use of the cascade nanoenzyme of any one of claims 4 to 7 or the cascade nanoenzyme prepared by the preparation method of any one of claims 4 to 7 in determination of glucose content.
9. The use according to claim 8, wherein the specific assay method comprises:
1) Mixing the solution of the cascade nanoenzyme with a glucose solution to be detected, adding 3,3',5,5' -tetramethylbenzidine solution after the first reaction, and characterizing the obtained supernatant by using an ultraviolet-visible spectrum method after the second reaction to obtain an absorbance value;
2) Preparing glucose standard solutions with different concentrations, respectively measuring absorbance values according to the method in the step 1), and fitting a standard curve equation according to the concentration of each glucose standard solution and the corresponding absorbance value;
3) And (3) measuring the absorbance value of the glucose solution to be measured according to the step 1), and calculating the concentration of the glucose solution by combining the standard curve equation.
10. Use according to claim 9,
the time of the first reaction is 4h; the time of the second reaction is 1-300s.
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