CN113281367B - Method for detecting hydrogen peroxide or glucose - Google Patents

Abstract

The invention discloses a method for detecting hydrogen peroxide or glucose. The detection method comprises the following steps: catalyzing a target object by using the peroxidase activity of the modified ferroferric oxide nanoenzyme, and detecting the concentration of the target object by using a magnetic resonance technology; the target is hydrogen peroxide or glucose. The invention adopts the magnetic resonance technology to detect the concentration of hydrogen peroxide or glucose, and overcomes the defects that the complex steps of the colorimetric method in the prior art influence the detection result and the chromogenic substrate TMB is not easy to store. Meanwhile, by adopting the detection method, the detection limit of the C-SPIO nanoenzyme on the hydrogen peroxide is as low as 3.32 mu M; the detection limit of the SPIO nanoenzyme cluster on hydrogen peroxide is as low as 8.78 mu M, the detection limit of the SPIO nanoenzyme cluster and the detection limit of the SPIO nanoenzyme cluster are far lower than the hydrogen peroxide permission level of 15 mu M specified by the FDA in the United states, and the detection limit of the C-SPIO nanoenzyme cluster and the detection limit of the SPIO nanoenzyme cluster on glucose are also lower.

Description

A kind of detection method of hydrogen peroxide or glucose

technical field

The invention belongs to the technical field of detection, in particular to a detection method of hydrogen peroxide or glucose.

Background technique

Hydrogen peroxide (H ₂ O ₂ ), as an active product of biological metabolism, participates in various biological oxidase-catalyzed reactions. The monitoring of life activities can be realized by detecting the concentration of H ₂ O ₂ . _The catalytic metabolism of glucose oxidase can produce efficient _H2O2 molecules. For example, a molecule of glucose in the organism can be metabolized by glucose oxidase to produce a molecule of H ₂ O ₂ , and then detecting the content of H ₂ O ₂ can reflect the glucose metabolism in the human body and obtain the blood glucose concentration. Therefore, the development of a rapid, sensitive and selective _{H2O2 detection} method is of great significance for the prevention, diagnosis and monitoring of diabetes.

At present, the clinical detection methods for H ₂ O ₂ are mainly colorimetric method, electrochemical sensor and chemiluminescence sensor. In many methods, horseradish peroxidase (HRP) is the main material, which has high specificity and sensitivity to H ₂ O ₂ , and then decomposes H ₂ O ₂ to generate OH by combining with H ₂ O ₂ ; Such hydroxyl reactive radicals have high chemical activity and can further participate in subsequent reactions to generate optical absorption signals or electrochemical signals. However, the inherent limitations of natural enzymes such as HRP, such as poor thermal stability, high cost, and harsh storage conditions, hinder their wide application. Compared with natural enzymes, nanomaterials that have attracted much attention in recent years have been found to have natural enzymatic activity, so they are called nanozymes; due to their stable chemical properties, they exhibit stable catalytic performance and low preparation costs. Reusable and easy to store. At this stage, people have synthesized a variety of nanomaterials that can mimic the function of natural enzymes. For example, in 2007, it was first discovered that superparamagnetic iron oxide can mimic the activity of peroxidase. Its catalytic mechanism is similar to that of HRP, which can catalyze H ₂ O ₂ produces a color change with 3,3',5,5'-tetramethylbenzidine (TMB) substrate, and the H ₂ O ₂ concentration is detected by measuring the color change. In the follow-up, the researchers mixed the superparamagnetic iron oxide nanozyme with glucose oxidase (GOD) to catalyze the production of H ₂ O ₂ from glucose through GOD, and then the H ₂ O ₂ was further catalyzed by the superparamagnetic iron oxide nanozyme and The same color with TMB, so as to achieve the determination of glucose concentration.

The currently reported ferric oxide nanozymes (SPIO nanozymes) for the determination of H ₂ O ₂ concentration all use colorimetric methods, which utilize the peroxidase activity of SPIO nano enzymes to catalyze H ₂ O ₂ to oxidize the substrate 3,3',5,5'-Tetramethylbenzidine (TMB) color, according to the proportional relationship between the color product absorbance and hydrogen peroxide concentration to achieve the determination of H ₂ O ₂ . The detection process requires the catalytic decomposition of H ₂ O ₂ to generate ·OH, and ·OH to oxidize the substrate TMB to oxTMB; this method requires two-step reactions, which can easily affect the detection results. At the same time, the storage conditions of TMB are harsh, and it needs to be protected from light at low temperature, which further limits its practical application.

SUMMARY OF THE INVENTION

In view of the above-mentioned shortcomings of the prior art, the present invention provides a method for detecting hydrogen peroxide or glucose.

In order to achieve the above object, the technical scheme adopted in the present invention is: a method for detecting hydrogen peroxide or glucose, comprising the steps of: utilizing the peroxidase activity of the modified iron tetroxide nanozyme to catalyze the target substance, The resonance technique detects the concentration of the target; the target is hydrogen peroxide or glucose.

The invention utilizes the peroxidase activity of the modified iron tetroxide nanozyme to catalyze hydrogen peroxide or glucose to generate hydroxyl radicals (·OH), thereby affecting the proton nuclear spin relaxation process of surrounding H ₂ O molecules. The determination of hydrogen peroxide can be achieved according to the linear relationship between the change in its T2 relaxation time and the concentration of _hydrogen peroxide or glucose. The detection method using the magnetic resonance technology in the present invention overcomes the disadvantages of the complicated steps of the colorimetric method in the prior art that affect the detection result and the difficult preservation of the substrate TMB.

As a preferred embodiment of the present invention, the method for detecting hydrogen peroxide or glucose is specifically: the method for detecting hydrogen peroxide includes the following steps: using a modified iron tetroxide nanozyme to catalyze hydrogen peroxide, Magnetic resonance technology to detect the concentration of hydrogen peroxide;

The method for detecting glucose includes the following steps: combining the modified iron tetroxide nanozyme and glucose oxidase to catalyze glucose, and detecting the concentration of glucose by magnetic resonance technology.

As a preferred embodiment of the present invention, the modified ferric oxide nanozyme is a sodium citrate modified ferric oxide nanozyme or a polymer-modified ferric oxide nanozyme; the polymer is polyethylene One of imine, polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer, polyoxyethylene-polyoxypropylene ether block copolymer, polyethylene glycol-polylactic acid copolymer kind.

The sodium citrate modified ferric oxide nanozyme of the present invention is regarded as C-SPIO nanozyme; the modified ferric oxide nanozyme of the polymer is regarded as SPIO nanozyme cluster.

The C-SPIO nanozyme and the SPIO nanozyme cluster of the present invention can oxidize colorless TMB to blue oxTMB in the presence of hydrogen peroxide, and have an obvious absorption peak at 652 nm on the ultraviolet-visible spectrum , it is proved that both nanozymes have peroxidase-like activity, and the catalytic activity of C-SPIO nanozyme is stronger than that of SPIO nanozyme cluster.

As a preferred embodiment of the present invention, the described modified iron tetroxide nanozyme is used for the detection method of hydrogen peroxide, which is characterized in that it includes the following specific steps:

S1: Disperse the modified ferric oxide nanozyme in an acetate buffered saline solution to obtain a modified ferric oxide nanozyme suspension;

S2: adding hydrogen peroxide solutions of different concentrations to the modified ferric tetroxide nanozyme suspension obtained in S1 to react to obtain a mixed solution;

S3: use the magnetic resonance method to measure the T ₂ relaxation time of the mixed solution obtained in S2, and establish a standard curve of hydrogen peroxide concentration-T ₂ relaxation time;

S4: Prepare an unknown hydrogen peroxide concentration solution according to the method in S1-S2, measure the T2 relaxation time of the unknown _hydrogen peroxide concentration solution, and substitute the T2 relaxation time into the _hydrogen peroxide concentration _- T2 obtained by S3 A standard curve of relaxation times to calculate the concentration of hydrogen peroxide in an unknown solution.

As a preferred embodiment of the present invention, in the S1, when the modified ferric oxide nanozyme is a sodium citrate modified ferric oxide nanozyme, the pH value of the acetate buffered saline solution is 2.0-5.0; When the ferric oxide nanozyme is a polymer-modified ferric oxide nanozyme, the pH value of the acetate buffer salt solution is 2.5-4.5.

The C-SPIO nanozyme of the present invention has good catalytic activity for hydrogen peroxide in a wide pH range (pH=2.0-5.0), and the SPIO nanozyme cluster has a good catalytic activity for hydrogen peroxide in pH=2.5-4.5 better catalytic activity. It shows that C-SPIO nanozyme can react with H 2 O 2 at pH=2.0-5.0 and SPIO nano-enzyme cluster can react with H ₂ O ₂ and form ·OH in the range of pH=2.5-4.5. And C-SPIO nanozyme at pH=2.0-5.0, the detection limit of hydrogen peroxide is below 3.32μM; SPIO nanozyme cluster at pH=2.5-4.5, the detection limit of hydrogen peroxide is below 8.78μM , both of which are well below the permitted level of 15 μM for hydrogen peroxide prescribed by the US FDA.

More preferably, in the S1, when the modified ferric oxide nanozyme is a sodium citrate modified ferric oxide nanozyme, the pH value of the acetate buffered saline solution is 4.0; When the enzyme is a polymer-modified iron tetroxide nanozyme, the pH value of the acetate buffered saline solution is 3.5.

The C-SPIO nanozyme of the present invention has the best catalytic activity for hydrogen peroxide at pH=4.0, and the detection limit for hydrogen peroxide is the lowest; the SPIO nanozyme cluster is at pH=3.5. Hydrogen oxide has the best catalytic activity and the lowest detection limit for hydrogen peroxide.

As a preferred embodiment of the present invention, in the S1, the concentration of the modified ferric oxide nanozyme in the modified ferric oxide nanozyme suspension is 0.01-0.025mM.

As a preferred embodiment of the present invention, in the S2, the hydrogen peroxide solutions with different concentrations are hydrogen peroxide solutions with a concentration gradient.

More preferably, in the S2, the concentration gradient of hydrogen peroxide with different concentrations in the mixed solution is selected from 5-300 μM. The hydrogen peroxide concentration gradient was selected in the range of 5–300 μM, and the _hydrogen peroxide concentration was linearly related to the T relaxation time.

As a preferred embodiment of the present invention, in the S2, when the modified ferric oxide nanozyme is a sodium citrate modified ferric oxide nanozyme, the reaction temperature is 30-70 °C, and the reaction time is 10 min; When the functional ferric oxide nanozyme is a polymer-modified ferric oxide nanozyme, the reaction temperature is 40-50° C. and the reaction time is 10 min.

The C-SPIO nanozyme of the present invention has good catalytic activity for hydrogen peroxide in a wide catalytic temperature range (30-70° C.); when the SPIO nano-enzyme cluster is at a catalytic temperature of 40-50° C., it can catalyze hydrogen peroxide. Hydrogen oxide has good catalytic activity; it shows that the C-SPIO nanozyme can react with H 2 O 2 in the catalytic temperature range of 30-70℃, and the SPIO nano-enzyme cluster can react with H ₂ O ₂ in the catalytic temperature range of 40-50 ℃ , and formed OH, and the C-SPIO nanozyme has a detection limit of 3.32 μM or less for hydrogen peroxide at the catalytic temperature of 30-70 °C; The detection limit of hydrogen peroxide was below 8.78 μM, both of which were far below the permitted level of 15 μM for hydrogen peroxide stipulated by the US FDA.

More preferably, in the S2, the reaction temperature is 50°C and the reaction time is 10min.

The C-SPIO nano-enzyme and the SPIO nano-enzyme cluster of the present invention have the best catalytic activity for hydrogen peroxide and the lowest detection limit for hydrogen peroxide when the catalytic temperature is 50°C.

As a preferred embodiment of the present invention, the modified iron tetroxide nanozyme is used for the detection method of glucose, which is characterized in that it includes the following specific steps:

(1) Glucose oxidase is reacted with glucose solutions of different concentrations in phosphate buffer to obtain mixed solution A;

(2) adding the modified ferric oxide nanozyme and the mixed solution A obtained in step (1) to an acetate buffered saline solution to react to obtain a mixed solution B;

(3) use the magnetic resonance method to measure the T2 relaxation time of the mixed solution B obtained in step ( ₂ ), and establish a standard curve of glucose solution concentration _- T2 relaxation time;

(4) According to the method in steps (1) to ( ₂ ), prepare an unknown glucose concentration solution, measure the T2 relaxation time of the unknown glucose solution, and substitute the T2 relaxation time into the glucose concentration obtained in step ( ₃ ) − Standard curve of _T2 relaxation time to calculate the concentration of glucose in the unknown solution.

As a preferred embodiment of the present invention, in the step (2), when the modified ferric oxide nanozyme is a sodium citrate modified ferric oxide nanozyme, the pH value of the acetate buffered saline solution is 2.0-5.0 , when the modified ferric oxide nano-enzyme is a polymer-modified ferric tetroxide nano-enzyme, the pH value of the acetate buffered saline solution is 2.5-4.5.

The combination of C-SPIO nanozyme and glucose oxidase of the present invention has good catalytic activity for glucose in a wide pH range (pH=2.0-5.0), and the combination of SPIO nanozyme cluster and glucose oxidase has a pH=2.5 -4.5 has good catalytic activity for glucose. The results show that C-SPIO nanozymes can react with H ₂ O ₂ at pH=2.0-5.0, and the SPIO nanozyme clusters can react with glucose oxidase in the range of pH=2.5-4.5, and form ·OH .

As a preferred embodiment of the present invention, in the step (2), when the modified ferric oxide nanozyme is a sodium citrate modified ferric oxide nanozyme, the pH value of the acetate buffered saline solution is 4.0; When the functional ferric oxide nanozyme is a polymer-modified ferric oxide nanozyme, the pH value of the acetate buffered saline solution is 3.5.

The C-SPIO nanozyme of the present invention is combined with glucose oxidase and has the best catalytic activity for glucose at pH=4.0, and the detection limit of glucose is the lowest; the SPIO nanozyme cluster is combined with glucose oxidase in When pH=3.5, the catalytic activity for glucose is the highest, and the detection limit for glucose is the lowest.

As a preferred embodiment of the present invention, in the step (2), when the modified ferric oxide nanozyme is a sodium citrate modified ferric oxide nanozyme, the reaction temperature is 30-70° C., and the reaction time is 10 min; when the modified ferric oxide nanozyme is a polymer-modified ferric oxide nanozyme, the reaction temperature is 40-50° C. and the reaction time is 10 min.

The C-SPIO nanozyme combined with glucose oxidase has good catalytic activity for glucose in a wide catalytic temperature range (30-70 °C); the combined SPIO nanozyme cluster and glucose oxidase have a catalytic temperature of At 40-50 °C, it has good catalytic activity for glucose; it shows that the combined C-SPIO nanozyme and glucose oxidase have a catalytic temperature range of 30-70 °C, and the combined SPIO nanozyme cluster and glucose oxidase have a catalytic temperature range of 30-70 °C. It can react with H ₂ O ₂ in the range of 40-50 °C and form ·OH.

As a preferred embodiment of the present invention, in the step (2), the reaction temperature is 50° C. and the reaction time is 10 min.

The C-SPIO nanozyme of the present invention and the cluster of SPIO nanozymes have a better effect on glucose when the catalytic temperature is 50°C. Good catalytic activity is the largest, and the detection limit for glucose is the lowest.

As a preferred embodiment of the present invention, the concentration of glucose oxidase in the mixed solution A is 0.4 mg/mL.

As a preferred embodiment of the present invention, the concentration of the modified iron tetroxide nanozyme in the mixed solution B is 0.01-0.025mM.

As a preferred embodiment of the present invention, in the step (1), the pH value of the phosphate buffer is 7.0, the reaction temperature is 37° C., and the time is 30 min.

As a preferred embodiment of the present invention, in the step (2), the glucose solutions with different concentrations are glucose solutions with a concentration gradient.

More preferably, in the step (2), the glucose concentration gradient of different concentrations in the mixed solution is selected from 5-300 μM. _The glucose concentration gradient was selected in the range of 5-300 μM, and the glucose concentration was linearly related to the T relaxation time.

As a preferred embodiment of the present invention, the preparation method of the sodium citrate modified iron tetroxide nanozyme comprises the following steps:

The first step: under the protection of argon, fully dissolve and mix iron acetylacetonate and triethylene glycol and heat to obtain solution A;

The second step: adding ethyl acetate to the solution A to form a precipitate, after dispersing the precipitate in anhydrous ethanol, it is mixed and reacted with an aqueous sodium citrate solution to obtain a sodium citrate modified iron tetroxide nanozyme.

As a preferred embodiment of the present invention, the ratio of the amount of the iron acetylacetonate substance to the volume of triethylene glycol is 1:30 mmol/L; the substance concentration ratio of sodium citrate to iron acetylacetonate is 1:10xx.

As a preferred embodiment of the present invention, in the first step, the heating temperature is 280°C and the heating time is 30min; in the second step, the reaction temperature is 100°C and the heating time is 30min.

As a preferred embodiment of the present invention, the preparation method of the modified iron tetroxide nanozyme of the polymer includes the following steps: mixing the polymer and the iron tetroxide nanozyme in chloroform to obtain a mixed solution; The mixed solution is added to water and shaken, and the chloroform in the mixed solution is removed to obtain the modified iron tetroxide nanozyme of the polymer; the weight ratio of the polymer to the iron tetroxide nanozyme is 1:1-2.

As a preferred embodiment of the present invention, the preparation method of the ferric oxide nanozyme comprises the following steps:

Under the protection of argon, iron acetylacetonate, 1,2-dodecanediol, oleic acid, oleylamine, benzyl ether and N-methylpyrrolidone were dissolved and mixed, and then heated and reacted; Centrifuge to obtain the ferric oxide nanozyme.

As a preferred embodiment of the present invention, the heating reaction is a microwave hydrothermal reaction, specifically: microwave hydrothermal reaction at 140° C. for 30 min, and microwave hydrothermal reaction at 180° C. for 120 min.

As a preferred embodiment of the present invention, the molar concentration ratio of the iron acetylacetonate, 1,2-dodecanediol, oleic acid, and oleylamine is iron acetylacetonate: 1,2-dodecanediol: oleic acid : oleylamine=2:10:6:6; the volume ratio of the benzyl ether and N-methylpyrrolidone is 20:3.

Compared with the prior art, the beneficial effects of the present invention are:

(1) The method for detection by magnetic resonance technology in the present invention overcomes the disadvantages that the complicated steps of the colorimetric method in the prior art affect the detection result and the chromogenic substrate TMB is not easy to store.

(2) The present invention detects the concentration of hydrogen peroxide by magnetic resonance technology, the detection limit of C-SPIO nanozyme for hydrogen peroxide is as low as 3.32 μM; the detection limit of SPIO nanozyme cluster for hydrogen peroxide is as low as 8.78 μM , the detection limits of both are well below the FDA-regulated hydrogen peroxide permit level of 15 μM.

(3) In the present invention, the concentration of glucose is detected by magnetic resonance technology, and the C-SPIO nanozyme is used in combination with glucose oxidase, and the detection limit of glucose is as low as 2.81 μM; the SPIO nanozyme cluster is used in combination with glucose oxidase , the detection limit for glucose was as low as 13.25 μM.

Description of drawings

Fig. 1 is the transmission electron microscope picture of the C-SPIO nanozyme prepared in Example 1;

Fig. 2 is the particle size distribution diagram of the C-SPIO nanozyme prepared in Example 1;

Fig. 3 is the transmission electron microscope image of the SPIO nanozyme cluster prepared in Example 9;

4 is a particle size distribution diagram of the SPIO nanozyme clusters prepared in Example 9;

Figure 5 is a graph showing the catalytic activity of C-SPIO nanozyme and SPIO nanozyme cluster to hydrogen peroxide under different pH conditions;

Figure 6 is a graph showing the catalytic activity of C-SPIO nanozyme and SPIO nanozyme cluster to hydrogen peroxide at different catalytic reaction temperatures;

Figure 7 shows the steady-state kinetics of C-SPIO nanozyme simulating peroxidase at different hydrogen peroxide concentrations or different TMB concentrations;

Figure 8 shows the steady-state kinetics of SPIO nanozyme clusters simulating peroxidase at different hydrogen peroxide concentrations or different TMB concentrations;

Fig. 9 is the linear fitting diagram of iron concentration and 1/T ₁ or 1/T ₂ of C-SPIO nanozyme of the present invention before adding hydrogen peroxide and after catalyzing reaction with hydrogen peroxide;

Figure 10 is a linear fitting diagram of iron concentration and 1/T ₁ or 1/T ₂ of SPIO nanozyme clusters before adding hydrogen peroxide and after catalytic reaction with hydrogen peroxide;

Figure 11 is a linear fitting diagram of the _hydrogen peroxide concentration and the T2 relaxation time of the catalytic reaction of C-SPIO nanozyme or SPIO nanozyme cluster in buffer solution in Example 1 of the present invention;

FIG. 12 is a linear fitting diagram of glucose concentration and T ₂ relaxation time of the reaction catalyzed by C-SPIO nanozyme or SPIO nanozyme cluster in buffer solution in Example 9 of the present invention.

Detailed ways

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific embodiments.

The SPIO nanozyme clusters described in Test Examples 1-4 are the SPIO nanozyme clusters prepared in Example 9.

Example 1

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of C-SPIO nanozyme comprises the following steps:

(1) under argon protection, 1mmol of ferric acetylacetonate and 30mL of triethylene glycol are fully dissolved and mixed and heated to obtain solution A;

(2) reflux solution A at 280 ° C for 30 min to obtain solution B;

(3) adding ethyl acetate to the solution B to form a precipitate, after dispersing the precipitate in absolute ethanol, mixing with an aqueous sodium citrate solution, and reacting at 100° C. for 30 min in a microwave reactor to obtain sodium citrate modified trioxide Iron nanozyme, counted as C-SPIO nanozyme.

The detection method of described hydrogen peroxide, comprises the steps:

(1) Disperse 25 μl of 0.5 mM C-SPIO nanozyme in 450 μl of acetate buffered saline solution with a pH value of 4.0 to obtain a C-SPIO nanozyme suspension;

(2) adding 25 μl of hydrogen peroxide solutions of different concentrations to the C-SPIO nanozyme suspension obtained in step (1) and reacting at 50° C. for 10 min to obtain a mixed solution; the concentrations of hydrogen peroxide in the mixed solution are respectively 5, 10, 25, 50, 75, 100, 150, 200, 300 μM;

(3) Use a magnetic resonance scanner to measure the T2 relaxation time of the mixed solution obtained by S2, and establish a standard curve of hydrogen peroxide concentration-T2 relaxation time.

The detection method of described glucose, comprises the steps:

(1) Add 10 μl, 10 mg/mL glucose oxidase and 40 μl glucose solutions of different concentrations to 200 μL, pH7.0 phosphate buffer solution and react at 37° C. for 30 min to obtain mixed solution A;

(2) Add 50 μL, 0.5 mM C-SPIO nanozyme and the mixed solution A obtained in step (1) to 740 μL, pH=4.0 acetate buffered saline solution, and react at 50°C for 10 min to obtain a mixed solution B;

(3) Use a magnetic resonance scanner to measure the T2 relaxation time of the mixed solution B obtained in step (2), and establish a standard curve of glucose solution concentration-T2 relaxation time.

Figure 1 is a transmission electron microscope image of the C-SPIO nanozyme prepared in Example 1, and Figure 2 is a particle size distribution diagram of the C-SPIO nanozyme prepared in Example 1. It can be seen from Figures 1 and 2 that C -SPIO nano-enzyme has good dispersibility, and the particle size distribution is uniform, and the particle size distribution is between 10-100nm.

Example 2

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of the C-SPIO nanozyme in this embodiment is the same as the preparation method of the C-SPIO nanozyme described in Example 1.

The only difference between the hydrogen peroxide detection method described in this embodiment and Embodiment 1 is that in step (1), the pH value of the acetate buffered saline solution is 2.0.

The only difference between the method for detecting glucose in this example and Example 1 is that in step (2), the pH value of the acetate buffered saline solution is 2.0.

Example 3

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of the C-SPIO nanozyme described in this example is the same as the preparation method of the C-SPIO nanozyme described in Example 1.

The only difference between the hydrogen peroxide detection method described in this embodiment and Embodiment 1 is that in step (1), the pH value of the acetate buffered saline solution is 3.0.

The only difference between the method for detecting glucose in this example and Example 1 is that in step (2), the pH value of the acetate buffered saline solution is 3.0.

Example 4

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of the C-SPIO nanozyme in this example is the same as the preparation method of the C-SPIO nanozyme described in Example 1.

The only difference between the hydrogen peroxide detection method described in this embodiment and Embodiment 1 is that in step (1), the pH value of the acetate buffered saline solution is 5.0.

The only difference between the method for detecting glucose in this example and Example 1 is that in step (2), the pH value of the acetate buffered saline solution is 5.0.

Example 5

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of the C-SPIO nanozyme in this example is the same as the preparation method of the C-SPIO nanozyme described in Example 1.

The only difference between the hydrogen peroxide detection method described in this example and Example 1 is: in step (2), the reaction temperature of the C-SPIO nanozyme suspension obtained in step (2) and the hydrogen peroxide solution is 30 °C.

The only difference between the glucose detection method described in this example and Example 1 is: in step (2), the C-SPIO nanozyme and the mixed solution A obtained in step (1) are added to the acetate buffered saline solution, The reaction temperature was 30°C.

Example 6

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of the C-SPIO nanozyme in this example is the same as the preparation method of the C-SPIO nanozyme described in Example 1.

The only difference between the hydrogen peroxide detection method described in this example and Example 1 is: in step (2), the reaction temperature of the C-SPIO nanozyme suspension obtained in step (2) and the hydrogen peroxide solution is 40 ℃ °C.

The only difference between the glucose detection method described in this example and Example 1 is: in step (2), the C-SPIO nanozyme and the mixed solution A obtained in step (1) are added to the acetate buffered saline solution, The reaction temperature was 40°C.

Example 7

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of the C-SPIO nanozyme in this example is the same as the preparation method of the C-SPIO nanozyme described in Example 1.

The only difference between the hydrogen peroxide detection method described in this example and Example 1 is: in step (2), the reaction temperature of the C-SPIO nanozyme suspension obtained in step (1) and the hydrogen peroxide solution is 60 °C °C.

The only difference between the glucose detection method described in this example and Example 1 is: in step (2), the C-SPIO nanozyme and the mixed solution A obtained in step (1) are added to the acetate buffered saline solution, The reaction temperature was 60°C.

Example 8

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of the C-SPIO nanozyme in this example is the same as the preparation method of the C-SPIO nanozyme described in Example 1.

The only difference between the hydrogen peroxide detection method described in this example and Example 1 is: in step (2), the reaction temperature of the C-SPIO nanozyme suspension obtained in step (1) and the hydrogen peroxide solution is 70 °C °C.

The only difference between the glucose detection method described in this example and Example 1 is: in step (2), the C-SPIO nanozyme and the mixed solution A obtained in step (1) are added to the acetate buffered saline solution, The reaction temperature was 70°C.

Example 9

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of SPIO nanozyme cluster includes the following steps:

(1) under argon protection, add 2mmol iron acetylacetonate, 10mmol 1,2-dodecanediol, 6mmol oleic acid, 6mmol oleylamine, 20mL benzyl ether, 3mL N-methylpyrrolidone in microwave reaction tank, fully Dissolve and mix to obtain a mixture;

(2) program the mixture in step (1) in a microwave hydrothermal synthesizer, first react at 140°C for 30min, and then react at 180°C for 120min;

(3) after the reaction finishes, cool to room temperature, add 80 mL of absolute ethanol, centrifuge at 8000 rpm for 10 min, collect the precipitate, and obtain SPIO nanoparticles;

(4) Dissolve 2.0 mg of polyethyleneimine and 4.0 mg of SPIO nanoparticles in 400 μL of chloroform to obtain solution a;

(5) Add solution a dropwise to 6 mL of ultrapure water under ultrasonic conditions for 30s of ultrasonic time to prepare solution b;

(6) shake solution b at 100rpm for 24h, and remove chloroform by rotary evaporation to obtain PEI-SPIO nanozyme clusters;

The only difference between the hydrogen peroxide detection method described in this example and Example 1 is that the C-SPIO nanozyme is replaced by a PEI-SPIO nanozyme cluster.

The only difference between the glucose detection method described in this example and Example 1 is that the C-SPIO nanozyme is replaced with a PEI-SPIO nanozyme cluster.

Figure 3 is a transmission electron microscope image of the SPIO nanozyme cluster prepared in Example 9, and Figure 4 is a particle size distribution diagram of the SPIO nanozyme cluster prepared in Example 9. It can be seen from Figures 3 and 4 , SPIO nano-enzyme clusters have good dispersibility, and the particle size distribution is uniform, and the particle size distribution is between 50-80.

Example 10

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of SPIO nanozyme cluster includes the following steps:

(1) the preparation method of SPIO nanoparticles is identical with the preparation method of SPIO nanoparticles in Example 9;

(2) Dissolve 2.0 mg of polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer (P123) and 4.0 mg of SPIO nanoparticles in 400 μL of chloroform to obtain solution a;

(3) Under ultrasonic conditions, solution a was added dropwise to 6 mL of ultrapure water, and the ultrasonic time was 2 min to obtain solution b;

(4) The solution b was shaken at 100 rpm for 24 h, and the chloroform was removed by rotary evaporation to obtain the P123-SPIO nanozyme cluster.

The only difference between the hydrogen peroxide detection method described in this example and Example 1 is that the C-SPIO nanozyme is replaced by a P123-SPIO nanozyme cluster.

The only difference between the glucose detection method described in this example and Example 1 is that the C-SPIO nanozyme is replaced by a P123-SPIO nanozyme cluster.

Example 11

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of SPIO nanozyme cluster includes the following steps:

(1) the preparation method of SPIO nanoparticles is identical with the preparation method of SPIO nanoparticles in Example 9;

(2) Dissolve 2.0 mg of polyoxyethylene-polyoxypropylene ether block copolymer (F127) and 4.0 mg of SPIO nanoparticles in 400 μL of chloroform to obtain solution a;

(3) Under ultrasonic conditions, solution a was added dropwise to 6 mL of ultrapure water, and the ultrasonic time was 2 min to obtain solution b;

(4) The solution b was shaken at 100 rpm for 24 h, and the chloroform was removed by rotary evaporation to obtain F127-SPIO nanozyme clusters.

The only difference between the hydrogen peroxide detection method described in this example and Example 1 is that the C-SPIO nanozyme is replaced by the F127-SPIO nanozyme cluster.

The only difference between the glucose detection method described in this example and Example 1 is that the C-SPIO nanozyme is replaced by the F127-SPIO nanozyme cluster.

Example 12

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of SPIO nanozyme cluster includes the following steps:

(1) the preparation method of SPIO nanoparticles is identical with the preparation method of SPIO nanoparticles in Example 9;

(2) Dissolve 2.0 mg of polyethylene glycol-polylactic acid copolymer (PEG-PLA) and 4.0 mg of SPIO nanoparticles in 400 μL of chloroform to prepare solution a;

(3) Under ultrasonic conditions, solution a was added dropwise to 6 mL of ultrapure water, and the ultrasonic time was 2 min to obtain solution b;

(4) The solution b was shaken at 100 rpm for 24 h, and chloroform was removed by rotary evaporation to prepare PEG-PLA nanozyme clusters.

The only difference between the hydrogen peroxide detection method described in this example and Example 1 is that the C-SPIO nanozyme is replaced by a PEG-PLA nanozyme cluster.

The only difference between the glucose detection method described in this example and Example 1 is that the C-SPIO nanozyme is replaced with a PEG-PLA nanozyme cluster.

Example 13

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of the PEI-SPIO nanozyme cluster in this embodiment is the same as the preparation method of the PEI-SPIO nanozyme cluster described in Example 9.

The only difference between the hydrogen peroxide detection method described in this embodiment and Embodiment 9 is that in step (1), the pH value of the acetate buffered saline solution is 2.5.

The only difference between the method for detecting glucose in this example and Example 9 is that in step (2), the pH value of the acetate buffered saline solution is 2.5.

Example 14

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of the PEI-SPIO nanozyme cluster described in this example is the same as the preparation method of the PEI-SPIO nanozyme cluster described in Example 9.

The only difference between the hydrogen peroxide detection method described in this example and Example 9 is that in step (1), the pH value of the acetate buffered saline solution is 3.5.

The only difference between the method for detecting glucose in this example and Example 9 is that in step (2), the pH value of the acetate buffered saline solution is 3.5.

Example 15

The embodiment of the detection method of hydrogen peroxide or glucose of the present invention is as follows:

The preparation method of the PEI-SPIO nanozyme cluster in this example is the same as the preparation method of the PEI-SPIO nanozyme cluster described in Example 9.

The only difference between the hydrogen peroxide detection method described in this embodiment and Embodiment 9 is that in step (1), the pH value of the acetate buffered saline solution is 4.5.

The only difference between the method for detecting glucose in this example and Example 9 is that in step (2), the pH value of the acetate buffered saline solution is 4.5.

Test Example 1

This test example is to test the optimal catalytic pH value of the C-SPIO nanozyme and the SPIO nanozyme cluster for the detection of hydrogen peroxide. The UV-Vis spectrophotometer is used to detect the pH value in the range of 2.5 to 8.0. In the internal detection system, the catalytic effect of C-SPIO nanozyme and SPIO nanozyme cluster on hydrogen peroxide.

Test method: Mix 25μL, 100mM hydrogen peroxide solution, 10μL, 1.0mM TMB solution and 25μL C-SPIO nanozyme solution or SPIO nanozyme cluster with iron concentration of 0.5mM, by adding 0.1M of different pH values. Acetic acid-sodium acetate buffer was reacted at 40°C for 10 min; the total volume of the reaction was controlled at 500 μL by adding buffer; the pH values of citric acid-sodium citrate buffer were monitored by UV-Vis spectrophotometer respectively at 2, 3, Catalytic effect on hydrogen peroxide at 4, 5, 6, 7, 8 and 9.

Figure 5(A) shows the catalytic activity of C-SPIO nanozymes for hydrogen peroxide under different pH conditions, and Figure 5(B) shows the catalysis of SPIO nanozyme clusters for hydrogen peroxide under different pH conditions Activity graph; it can be seen from the graph that the C-SPIO nanozyme has good catalytic activity towards hydrogen peroxide in a wide pH range (pH=2-5), and has the maximum activity at pH=4.0. The SPIO nanozyme clusters have good catalytic activity for hydrogen peroxide at pH=2.5-4, and have the maximum activity at pH=3.5. The results show that the C-SPIO nanozyme can react with H 2 O 2 at pH=2-5, and the SPIO nanozyme cluster can react with H ₂ O ₂ and form ·OH at pH=2.5-4.

Test Example 2

This test example is to test the optimal catalytic temperature of the C-SPIO nanozyme and the SPIO nanozyme cluster for the detection of hydrogen peroxide, using a UV-Vis spectrophotometer to detect pH values in the range of 2.5 to 8.0. In the detection system, the catalytic effect of C-SPIO nanozyme and SPIO nanozyme cluster on hydrogen peroxide.

Test method: Mix 25μL, 100mM hydrogen peroxide solution, 10μL, 1.0mM TMB solution and 25μL C-SPIO nanozyme solution or SPIO nanozyme cluster solution with iron concentration of 0.5mM, by adding 0.1M, pH value The buffer reaction of 4 was catalyzed for 10 min at different temperatures; the total volume of the reaction was controlled at 500 μL by adding buffer; the temperature was monitored by a UV-Vis spectrophotometer at 20 °C, 30 °C, 40 °C, 50 °C, Catalytic effect on hydrogen peroxide at 60°C and 70°C.

Figure 6(A) shows the catalytic activity of C-SPIO nanozymes for hydrogen peroxide at different catalytic reaction temperatures, and Figure 6(B) shows the catalytic activity of SPIO nanozyme clusters for hydrogen peroxide under different catalytic reactions. Catalytic activity graph; it can be seen from the graph that the C-SPIO nanozyme has good catalytic activity for hydrogen peroxide in a wide catalytic temperature range (30-70 °C), and has the maximum activity at 50 °C. The SPIO nanozyme clusters have good catalytic activity for hydrogen peroxide when the catalytic temperature is 40-50 °C, and the maximum activity is at 50 °C. The results show that C-SPIO nanozymes can react with H ₂ O ₂ and form OH in the catalytic temperature range of 30-70 °C, and the SPIO nano-enzyme clusters can react with H 2 O 2 in the catalytic temperature range of 40-50 °C.

Test Example 3

This test example is to test the affinity of C-SPIO nanozyme and SPIO nanozyme cluster to chromogenic substrate and hydrogen peroxide.

testing method:

(1) Add acetic acid-sodium acetate buffer solution with pH 4.0, 10 μL, 1 mM TMB, 25 μL hydrogen peroxide solution of different concentrations, 25 μL, 0.5 mMC-SPIO nanozyme solution or SPIO nanozyme cluster into the cuvette The total volume was controlled at 500 μL by acetic acid-sodium acetate buffer solution, wherein the concentration of hydrogen peroxide solution in the C-SPIO nanozyme reaction system was 0.5, 3, 5, 10, 15, 20 and 30 mM, respectively; The concentrations of the hydrogen peroxide solution in the enzyme cluster reaction system were 1, 2, 3, 5, 10, 15, and 30 mM, respectively; using the time scan mode, the absorbance at a wavelength of 652 nm was read every 15 seconds.

(2) Add 10 μL of different concentrations of TMB, 25 μL, 10 mM hydrogen peroxide, 25 μL, 0.5 mMC-SPIO nanozyme solution or SPIO nanozyme cluster solution into the cuvette, by adding a concentration of 0.1 M, pH The acetic acid-sodium acetate buffer solution of 4.0 will control the total volume at 500 μL; among them, the concentrations of TMB in the C-SPIO nanozyme reaction system are 0.25, 0.5, 1.2, 2.4, 3.2, and 5 mM, respectively; the SPIO nanozyme cluster reaction The concentrations of TMB in the system were 0.4, 0.8, 1.2, 1.6, 2.4, 3.2, and 4 mM, respectively; the time scanning mode was used, and the absorbance value at a wavelength of 652 nm was read every 15 seconds.

Changes in absorbance were monitored using a UV-2600 UV-Vis spectrophotometer. The Michaelis-Menten constant was calculated using the Lineweaver-Burk curve: 1/v= _Km / _Vmax (1/[S]+1/ _Km ). where v is the initial concentration, _Vmax is the maximum reaction rate, and [S] is the substrate concentration. K _m is the Michelis-Menten constant, which is an indicator of the affinity of the enzyme to its substrate. The smaller the K _m value, the more affinity the enzyme has for the substrate.

Figure 7(A) shows the steady-state kinetics of C-SPIO nanozyme simulating peroxidase at different hydrogen peroxide concentrations; Figure 7(B) shows C-SPIO nanozyme simulating peroxidase at different TMB concentrations It can be seen from the figure that the Vmax of C-SPIO nanozyme to hydrogen peroxide is 0.716, and the _Vmax of C-SPIO _nanozyme to TMB is 0.029; Michaelis-Menten was calculated using Lineweaver-Burk curve constant, respectively, the Km of C-SPIO nanozyme to hydrogen peroxide is 3.70mM, which is the same as the Km=3.70mM of natural horseradish peroxidase (HRP) to hydrogen peroxide, and the Km of TMB is 0.67mM, Km = 0.43 mM higher than native horseradish peroxidase (HRP) for TMB.

Figure 8(A) shows the steady-state kinetics of SPIO nanozyme clusters simulating peroxidase at different hydrogen peroxide concentrations; Figure 8(B) shows SPIO nanozyme clusters simulating peroxidase at different TMB concentrations The steady-state kinetics of the enzyme; it can be seen from the figure that the SPIO nanozyme cluster has _Vmax = 1.9565 for hydrogen peroxide, and the SPIO nanozyme cluster has a _Vmax = 1.1285 for TMB; using Lineweaver-Burk curve The Michaelis-Menten constant was calculated, and the Km of the SPIO nanozyme cluster for hydrogen peroxide was 2.97 mM, which was lower than the Km of natural horseradish peroxidase (HRP) = 3.70 mM, and the Km of TMB was 2.86 mM. , higher than the natural horseradish peroxidase (HRP) Km=0.43mM.

Test Example 4

This test example is to test the relationship between the iron concentration and the magnetic resonance signal before and after the catalytic reaction of C-SPIO nanozyme and SPIO nanozyme cluster with hydrogen peroxide, respectively.

testing method:

(1) Different concentrations of C-SPIO nanozyme and SPIO nanozyme cluster were added to acetic acid-sodium acetate buffer solution with pH 4, respectively, and the T ₁ and T ₂ relaxation times were measured using a magnetic resonance scanner; The concentration of iron in the C-SPIO nanozyme or SPIO nanozyme cluster is 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM.

(2) Different concentrations of C-SPIO nanozyme and SPIO nanozyme cluster were added to acetic acid-sodium acetate buffer solution with pH 4, and 100 μM hydrogen peroxide was added to make the total reaction volume 500 μL. The reaction was performed at 50 °C for ₁₀ min, and the T1 and T2 relaxation times were measured using a magnetic resonance scanner _; the iron concentrations in the C-SPIO nanozyme or SPIO nanozyme cluster were 0.4mM, 0.5mM, 0.6mM.

9 is a linear fitting diagram of iron concentration and 1/T ₁ or 1/T ₂ of the C-SPIO nanozyme of the present invention before adding hydrogen peroxide and after catalyzing the reaction with hydrogen peroxide. It can be seen from the figure that the slope of 1/T ₂ and Fe concentration is the relaxation rate r ₂ . After adding hydrogen peroxide to the detection system, the relaxation rate r ₂ decreases, indicating that the C-SPIO nanozyme reacts with hydrogen peroxide The resulting hydroxyl radicals will affect the relaxation time of the protons of hydrogen nuclei in the surrounding water molecules, resulting in a decrease in the relaxation rate.

Figure 10 is a linear fitting diagram of iron concentration and 1/T ₁ or 1/T ₂ of the SPIO nanozyme cluster of the present invention before adding hydrogen peroxide and after the catalytic reaction with hydrogen peroxide. It can be seen from the figure that the slope of 1/T ₂ and Fe concentration is the relaxation rate r ₂ . After adding hydrogen peroxide to the detection system, the relaxation rate r ₂ decreases, indicating that the SPIO nanoenzyme clusters are closely related to hydrogen peroxide. The hydroxyl radicals generated after the reaction will affect the relaxation time of hydrogen nuclei protons in the surrounding water molecules, resulting in a decrease in the relaxation rate.

Test Example 5

This test example is to test the detection limit of C-SPIO nanozyme and SPIO nanozyme cluster to detect hydrogen peroxide and glucose.

According to the detection limit formula LOD=3S/M, the detection limit of C-SPIO nanozyme or SPIO nanozyme cluster to the target was calculated.

Wherein S is the standard deviation of the blank sample, M is the slope of the linear curve of the T2 relaxation time and the concentration of the target substance _; the target substance is hydrogen peroxide or glucose; the C-SPIO nano-enzyme detection system in the present invention detects hydrogen peroxide The S=0.2811 of the C-SPIO nano-enzyme detection system for glucose detection; the S=0.5821 for the detection of hydrogen peroxide by the SPIO nano-enzyme cluster detection system; the S=0.5940 for the C-SPIO nano-enzyme detection system to detect glucose.

Table 1 Embodiment 1-8 Detection limit of hydrogen peroxide detected by C-SPIO nano-enzyme detection system

M Detection limit/μM Example 1 0.254 3.32 Example 2 0.193 4.37 Example 3 0.219 3.85 Example 4 0.225 3.75 Example 5 0.195 4.32 Example 6 0.220 3.83 Example 7 0.235 3.59 Example 8 0.182 4.63

Table 2 Embodiment 1-8 The detection limit of glucose detected by C-SPIO nano-enzyme detection system

M Detection limit/μM Example 1 0.157 2.81 Example 2 0.113 3.90 Example 3 0.136 3.24 Example 4 0.140 3.15 Example 5 0.118 3.74 Example 6 0.120 3.67 Example 7 0.122 3.61 Example 8 0.110 4.01

Table 3 Embodiment 9-15 Detection limit of hydrogen peroxide detected by SPIO nanozyme cluster detection system

M Detection limit/μM Example 9 0.1987 8.79 Example 10 0.1982 8.81 Example 11 0.1990 8.78 Example 12 0.1988 8.78 Example 13 0.1959 8.91 Example 14 0.1962 8.90 Example 15 0.1958 8.92

Table 4 Embodiment 9-15 Detection limit of glucose detected by SPIO nanozyme cluster detection system

M Detection limit/μM Example 9 0.1345 13.25 Example 10 0.1340 13.30 Example 11 0.1342 13.28 Example 12 0.1344 13.26 Example 13 0.1310 13.60 Example 14 0.1335 13.35 Example 15 0.1321 13.49

FIG. 11 is a linear fitting diagram of the hydrogen peroxide concentration and the T ₂ relaxation time of the reaction catalyzed by C-SPIO nanozyme or SPIO nanozyme cluster in buffer in Example 1 of the present invention.

FIG. 12 is a linear fitting diagram of glucose concentration and T ₂ relaxation time of the reaction catalyzed by C-SPIO nanozyme or SPIO nanozyme cluster in buffer solution in Example 9 of the present invention.

It can be seen from Tables 1-4 and Figures 11 and 12 that the C-SPIO nanozyme has a pH range of pH=2-5, and the detection limit of C-SPIO nanozyme for hydrogen peroxide is as low as 3.32 μM. It is far lower than the permitted level of hydrogen peroxide regulated by the US FDA of 15 μM; and the detection limit of C-SPIO nanozyme for glucose is also lower at 2.81 μM at pH=2-5. The pH range of SPIO nanozyme clusters is pH=2.5-4.5, and the detection limit of SPIO nanozyme clusters for hydrogen peroxide is as low as 8.78μM, which is far lower than the permitted level of hydrogen peroxide 15μM stipulated by the US FDA; Moreover, the detection limit of SPIO nanozyme cluster for glucose was also lower at 13.25μM when pH=2.5-4.5. From the above results, it can be seen that the detection limit of C-SPIO nanozyme for hydrogen peroxide or glucose is significantly lower than that of the SPIO nanozyme cluster, because the particle size of C-SPIO nanozyme is smaller and its catalytic activity is higher. , so its detection limit for hydrogen peroxide and glucose is lower.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that, The technical solutions of the present invention may be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims (3)

1. a detection method of glucose, is characterized in that, comprises the steps: utilize the peroxidase activity catalysis target of modified ferric oxide nano-enzyme, detect the concentration of target by magnetic resonance technology; Described target The compound is glucose; the modified ferric oxide nanozyme is a polymer-modified ferric oxide nanozyme; the polymer is polyethyleneimine, polyethylene oxide-polypropylene oxide-polycyclic One of ethylene oxide triblock copolymer, polyoxyethylene-polyoxypropylene ether block copolymer, polyethylene glycol-polylactic acid copolymer; The modified iron tetroxide nanozyme is used for the detection method of glucose, comprising the following specific steps: (1) Glucose oxidase is reacted with glucose solutions of different concentrations in phosphate buffer to obtain mixed solution A; (2) adding the modified ferric oxide nanozyme and the mixed solution A obtained in step (1) to an acetate buffered saline solution to react to obtain a mixed solution B; (3) use the magnetic resonance method to measure the T2 relaxation time of the mixed solution B obtained in step ( ₂ ), and establish a standard curve of glucose solution concentration _- T2 relaxation time; (4) According to the method in steps (1) to ( ₂ ), prepare an unknown glucose concentration solution, measure the T2 relaxation time of the unknown glucose solution, and substitute the T2 relaxation time into the glucose concentration obtained in step ( ₃ ) − The standard curve of T ₂ relaxation time is used to calculate the concentration of glucose in the unknown solution; in the step (2), the pH value of the acetate buffered saline solution is 2.5-4.5. 2. the detection method that modified iron tetroxide nano-enzyme is used for glucose as claimed in claim 1, is characterized in that, in described step (1), the pH value of phosphate buffer is 7.0, and reaction temperature is 37°C for 30min; in the step (2), the reaction temperature is 40-50°C, and the reaction time is 10min; the concentration of the modified iron tetroxide nanozyme in the mixed solution B is 0.01-0.025mM . 3. the detection method of hydrogen peroxide or glucose as claimed in claim 1, is characterized in that, the preparation method of the ferric tetroxide nanozyme of described polymer modification, comprises the steps: by polymer and ferric tetroxide The nanozyme is mixed in chloroform to obtain a mixed solution, the mixed solution is added to water and shaken, and the chloroform in the mixed solution is removed to obtain the modified ferric oxide nanozyme of the polymer; the polymer is mixed with ferric tetroxide. The weight ratio of nanozymes is 1:1-2.

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