Preparation method and application of NM88B-POR / Rf nanozyme

Abstract

The invention discloses a preparation method of a NM88B-POR / Rf nanozyme and its application. The method first in-situ dopes porphyrin in NM88B(Fe) (NM88B-POR), and then loads riboflavin (Rf) on NM88B-POR by electrodeposition, while realizing the sarcosine oxidase (SOx) nanozyme Rf and peroxidase nanozyme NM88B(Fe) enzyme cascade reaction to enhance the sarcosine (Sar) detection signal, and also enhances the capture of oxygen molecules in aqueous solution through the heme-like effect of the Fe-POR structure in NM88B-POR to construct a high oxygen microenvironment, thereby realizing highly sensitive electrochemical detection of Sar. The present invention establishes a new Sar enzyme cascade electrochemical detection strategy, and the heme-like Fe-POR structure in the constructed electrochemical nanozyme can also build a high-oxygen microenvironment by enhancing the capture of dissolved oxygen, solving the problem that the current Rf nanozyme can only biomimetic the SOx active center structure and ignores the fact that the capture of dissolved oxygen during the reaction process can significantly improve the reaction rate. The nanozyme ultimately achieves ultra-sensitive electrochemical detection of Sar through the synergistic effect of enzyme cascade and high-oxygen microenvironment.

Description

Technical Field

[0001] The present invention relates to the field of enzyme cascade nanozyme biomaterials, and in particular to a preparation method and application of a NM88B-POR / Rf nanozyme. Background Art

[0002] Prostate cancer (PCa) has become the most common malignant tumor of the male urogenital system and is usually detected by prostate-specific antigen (PSA) in serum. However, some other non-malignant diseases may also lead to elevated PSA concentrations, resulting in false positive results for early detection of PCa by PSA, thus limiting the accuracy of PSA testing. Sarcosine (Sar) is widely present in human muscle and other tissues, and its concentration increases significantly with the progression of prostate cancer, and the degree of change is not affected by other diseases. This characteristic makes Sar the most promising prostate cancer marker at present, and its highly sensitive detection is of great value for achieving highly sensitive determination of PCa. At present, the main methods for detecting Sar are chromatography, colorimetry and electrochemical methods, among which electrochemical detection is highly valued due to its simplicity and high sensitivity.

[0003] Sarcosine oxidase (SOx) can react with Sar by adsorbing oxygen molecules to produce H2O2, glycine and formaldehyde. Therefore, loading SOx on the electrode can achieve highly sensitive and anti-interference electrochemical detection of Sar. However, SOx biosensors can achieve highly sensitive electrochemical detection of Sar, but their practical applications are limited by the poor environmental tolerance and high cost of SOx natural enzymes. To solve this problem, some researchers have constructed a nanozyme based on riboflavin (Rf) to detect Sar using the structural similarity between the active center of riboflavin (Rf). The results show that nanozyme Rf can indeed detect Sar, but the current nanozyme simply simulates its active center structure and ignores the capture of dissolved oxygen, resulting in weak catalytic ability of the nanozyme. Therefore, the efficient construction of SOx nanozymes with a high oxygen microenvironment, improved dissolved oxygen adsorption capacity, and high-sensitivity Sar detection is a current research hotspot. Summary of the invention

[0004] In view of the problems that the current SOx nanozyme bionic structure has low activity, limited catalytic ability and weak signal response, the present invention provides a preparation method and application of NM88B-POR / Rf nanozyme. In the present invention, NM88B-POR, as a peroxidase nanozyme, converts H2O2 into OH, reacts with MB to produce a strong electrical signal, thereby converting a weak electrical signal reaction into a strong electrical signal reaction, thereby improving the sensitivity of Sar detection. At the same time, the Fe-POR heme structure in NM88B-POR can construct a high-oxygen microenvironment by enhancing the capture of dissolved oxygen, thereby improving the catalytic activity of SOx nanozyme, thereby improving the electrical signal and the sensitivity of Sar detection.

[0005] The technology of the present invention is achieved through the following technical solutions:

[0006] A method for preparing NM88B-POR / Rf nanozyme comprises the following steps:

[0007] (1) Preparation of NM88B-POR:

[0008] Dissolve ferric chloride hexahydrate and porphyrin in N,N-dimethylformamide, add 2-aminoterephthalic acid with stirring to obtain a mixed solution, continue stirring and add sodium hydroxide solution, mix well, transfer to a reactor, perform hydrothermal reaction at 80-100° C. for 10-12 hours, and obtain NM88B-POR after washing and drying.

[0009] (2) Preparation of NM88B-POR / Rf:

[0010] The NM88B-POR material obtained in step (1) is immersed in a riboflavin (Rf) solution having sarcosine oxidase (SOx)-like activity, and NM88B-POR / Rf is obtained after deposition by an electrodeposition method.

[0011] As a preferred technical solution, in step (1), the molar ratio of ferric chloride hexahydrate, porphyrin, 2-aminoterephthalic acid and sodium hydroxide is 1:0.5:0.5-1:0.8.

[0012] As a preferred technical solution, the amount of ferric chloride hexahydrate added in the step (1) is 40-60 mg of ferric chloride hexahydrate and 1-20 mg of porphyrin per 1 ml of N,N-dimethylformamide; the amount of 2-aminoterephthalic acid added in the step (1) is 30-40 mg of 2-aminoterephthalic acid per 1 ml of N,N-dimethylformamide.

[0013] As a preferred technical solution, the amount of sodium hydroxide solution added in step (1) is 0.01-0.2 mL of sodium hydroxide solution with a concentration of 1-2 mol / L per mL of the mixed solution.

[0014] As a preferred technical solution, in step (1), the time for stirring and mixing the ferric chloride hexahydrate and the porphyrin solution to be uniformly mixed is 5-10 minutes; in step (1), the time for stirring and mixing the ferric chloride hexahydrate and the porphyrin mixed solution with 2-aminoterephthalic acid to be uniformly mixed is 20-40 minutes.

[0015] As a preferred technical solution, the washing and drying in step (1) are washing with N,N-dimethylformamide and acetone for 2-4 times, then drying at 70-80°C for 10-12h, and then drying in vacuum at 70-90°C for 10-12h.

[0016] As a preferred technical solution, the riboflavin Rf concentration in step (2) is 2-40 mmol / L.

[0017] As a preferred technical solution, in step (2), the plastidial ratio of NM88B-POR to riboflavin Rf is 2.2:1.

[0018] As a preferred technical solution, the electrodeposition method in step (2) is a constant potential method, the voltage is 0.6-0.9V, and the deposition time is 60-120s.

[0019] The NM88B-POR / Rf prepared by the present invention is characterized in that the BET specific surface area of ​​the material is 30-40m 2 / g, as a flexible skeleton MOFs material, it has unique breathing properties, a stretchable structure in aqueous solution, and a specific surface area of ​​up to 3000m 2 / g, the crystal size is about 250-300nm, and its crystal structure is an irregular spindle-like structure.

[0020] The NM88B-POR / Rf of the present invention can be applied to the electrochemical analysis and detection of Sar.

[0021] The principle of the present invention is: first, porphyrin is in situ doped in NM88B (Fe) (NM88B-POR), and then Rf is loaded on NM88B-POR by electrodeposition. As a peroxide nanozyme, NM88B-POR can convert H2O2 into OH, and react with MB to produce a strong electrical signal, thereby converting a weak electrical signal reaction into a strong electrical signal reaction, thereby improving the sensitivity of Sar detection. At the same time, the Fe-POR heme structure in NM88B-POR can construct a high oxygen microenvironment by enhancing the capture of dissolved oxygen in water, thereby enhancing the catalytic activity of SOx nanozymes, thereby enhancing electrical signals and improving the sensitivity of Sar detection. The present invention ultimately achieves ultra-sensitive electrochemical detection of Sar through the synergistic effect of enzyme cascade and high oxygen microenvironment.

[0022] The NM88B-POR / Rf nanozyme prepared by the present invention has excellent structural stability, and exhibits higher activity and sensitivity in the catalysis and detection of Sar than other materials.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) The present invention constructs NM88B-POR / Rf based on the enzyme cascade mechanism. The original SOx nanozyme has a weak electrical signal during the reaction process, while NM88B-POR in the present invention, as a peroxidase nanozyme, can convert H2O2 into ·OH, which reacts with MB to produce a strong electrical signal, thereby converting the weak electrical signal reaction into a strong electrical signal reaction, thereby improving the sensitivity of Sar detection.

[0025] (2) Based on the SOx catalytic mechanism, the present invention constructs the Fe-POR heme structure that can significantly increase the reaction rate into the SOx nanozyme for the first time. The Fe-POR in NM88B-POR captures oxygen molecules in the aqueous solution to create a high oxygen microenvironment, thereby improving the catalytic activity of the SOx nanozyme and thus improving the sensitivity of Sar detection. While the present invention amplifies the electrical signal through the enzyme cascade mechanism to increase the detection limit of Sar by 2 orders of magnitude, it also increases the detection limit of Sar by another 2 orders of magnitude through the oxygen-carrying enhancement effect of the Fe-POR heme structure. Through the synergistic effect of the enzyme cascade and the high oxygen microenvironment, the NM88B-POR / Rf constructed by the present invention has stronger enzyme activity than natural enzymes and other nanozymes, increasing the detection limit of Sar by 4 orders of magnitude.

[0026] (3) The NM88B-POR / Rf prepared in the present invention is a flexible skeleton MOFs material. Its unique breathing pores are closed under dry conditions, making it impossible to accurately measure its specific surface area. However, in aqueous solution, due to the swelling of the NM88B skeleton, the specific surface area can reach 2000m 2 / g or more, this structure can increase the contact area between the reaction substrate and the NM88B-POR / Rf material, and enhance the nanozyme catalytic activity of the NM88B-POR / Rf material.

[0027] (4) Compared with natural enzymes, the NM88B-POR / Rf prepared in the present invention has excellent storage stability, circulation stability and anti-interference performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The catalytic activity diagram of NM88B-POR / Rf prepared in Example 1;

[0029] Figure 2 This is the XRD pattern of NM88B-POR / Rf prepared in Example 1.

[0030] Figure 3 (A) is the SEM image of NM88B / Rf prepared in Example 1, Figure 3 (B) is the SEM image of NM88B-POR / Rf prepared in Example 1.

[0031] Figure 4 This is a comparison chart of nitrogen adsorption-desorption curves of NM88B and NM88B-POR prepared in Example 1.

[0032] Figure 5 This is a diagram of the catalytic performance of NM88B-POR / Rf prepared in Example 1 under different pH conditions.

[0033] Figure 6 This is a diagram of the catalytic performance of NM88B-POR / Rf prepared in Example 1 at different material concentrations.

[0034] Figure 7 This is a diagram of the catalytic performance of NM88B-POR / Rf prepared in Example 1 at different Rf adsorption times.

[0035] Figure 8 This is a diagram of the catalytic performance of NM88B-POR / Rf prepared in Example 1 at different Rf deposition times.

[0036] Fig. 9 This is a graph showing the catalytic stability of NM88B-POR / Rf prepared in Example 1 at different storage times.

[0037] Fig.10 This is a comparison chart of the catalytic activities of NM88B and NM88B-POR prepared in Example 1 in different oxygen-containing environments.

[0038] Fig.11 This is a comparison chart of the catalytic activities of NM88B / Rf and NM88B-POR / Rf prepared in Example 1.

[0039] Fig.12 (A) is the detection curve of NM88B / Rf prepared in Example 1 for Sar, Fig.12 (B) is the fitting straight line and detection limit of NM88B / Rf prepared in Example 1 for Sar.

[0040] Fig.13 (A) is the detection curve of NM88B-POR / Rf prepared in Example 1 for Sar, Fig.13 (B) is the fitting straight line and detection limit of NM88B-POR / Rf prepared in Example 1 for Sar.

[0041] Fig.14 This is a diagram showing the detection of Sar and other amino acids by NM88B-POR / Rf prepared in Example 1. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but the scope of protection claimed by the present invention is not limited to the scope of protection of the embodiments.

[0043] Example 1

[0044] A method for preparing NM88B-POR / Rf nanozyme, the steps are as follows:

[0045] (1) Preparation of NM88B-POR:

[0046] Dissolve 270 mg of ferric chloride hexahydrate and 180 mg of porphyrin in 5 mL of N,N-dimethylformamide, add 181 mg of 2-aminoterephthalic acid while stirring, continue stirring, add 0.4 mL of 2 mol / L sodium hydroxide solution, and mix well. Then transfer the mixture to a polytetrafluoroethylene reactor, heat it to 100 ° C at 1 ° C / min, keep the reaction at 100 ° C for 12 hours, and then cool it to room temperature. After the reaction is completed, the precipitate in the reactor is centrifuged, washed and dried to obtain NM88B-POR.

[0047] (2) Preparation of NM88B-POR / Rf:

[0048] The NM88B-POR obtained in step (1) was immersed in 100 mL of riboflavin (Rf) solution having 30 mmol / L sarcosine oxidase (SOx) activity, and NM88B-POR / Rf was obtained by electroplating at -0.6 V for 90 s using a constant potential method.

[0049] Example 2

[0050] A method for preparing NM88B-POR / Rf nanozyme, the steps are as follows:

[0051] (1) Preparation of NM88B-POR:

[0052] Dissolve 270 mg of ferric chloride hexahydrate and 180 mg of porphyrin in 5 mL of N,N-dimethylformamide, add 90.5 mg of 2-aminoterephthalic acid while stirring, continue stirring, add 0.4 mL of 2 mol / L sodium hydroxide solution, and mix well. Then transfer the mixture to a polytetrafluoroethylene reactor, heat it to 100 ° C at 1 ° C / min, keep it at 100 ° C for 12 hours, and then cool it to room temperature. After the reaction is completed, the precipitate in the reactor is centrifuged, washed and dried to obtain NM88B-POR.

[0053] (2) Preparation of NM88B-POR / Rf:

[0054] The NM88B-POR obtained in step (1) was immersed in 100 mL of riboflavin (Rf) solution having 30 mmol / L sarcosine oxidase (SOx) activity, and NM88B-POR / Rf was obtained by electroplating at -0.6 V for 90 s using a constant potential method.

[0055] Example 3

[0056] A method for preparing NM88B-POR / Rf nanozyme, the steps are as follows:

[0057] (1) Preparation of NM88B-POR:

[0058] Dissolve 270 mg of ferric chloride hexahydrate and 180 mg of porphyrin in 5 mL of N,N-dimethylformamide, add 181 mg of 2-aminoterephthalic acid while stirring, continue stirring, add 0.4 mL of 2 mol / L sodium hydroxide solution, and mix well. Then transfer the mixture to a polytetrafluoroethylene reactor, heat it to 80 ° C at 1 ° C / min, keep it at 80 ° C for 12 hours, and then cool it to room temperature. After the reaction is completed, the precipitate in the reactor is centrifuged, washed and dried to obtain NM88B-POR.

[0059] (2) Preparation of NM88B-POR / Rf:

[0060] The NM88B-POR obtained in step (1) was immersed in 100 mL of riboflavin (Rf) solution having 30 mmol / L sarcosine oxidase (SOx) activity, and NM88B-POR / Rf was obtained by electroplating at -0.6 V for 90 s using a constant potential method.

[0061] Example 4

[0062] A method for preparing NM88B-POR / Rf nanozyme, the steps are as follows:

[0063] (1) Preparation of NM88B-POR:

[0064] Dissolve 270 mg of ferric chloride hexahydrate and 180 mg of porphyrin in 5 mL of N,N-dimethylformamide, add 181 mg of 2-aminoterephthalic acid while stirring, continue stirring, add 0.4 mL of 2 mol / L sodium hydroxide solution, and mix well. Then transfer the mixture to a polytetrafluoroethylene reactor, heat it to 100 ° C at 1 ° C / min, keep it at 100 ° C for 24 hours, and then cool it to room temperature. After the reaction is completed, the precipitate in the reactor is centrifuged, washed and dried to obtain NM88B-POR.

[0065] (2) Preparation of NM88B-POR / Rf:

[0066] The NM88B-POR obtained in step (1) was immersed in 100 mL of riboflavin (Rf) solution having 30 mmol / L sarcosine oxidase (SOx) activity, and NM88B-POR / Rf was obtained by electroplating at -0.6 V for 90 s using a constant potential method.

[0067] Example 5

[0068] A method for preparing NM88B-POR / Rf nanozyme, the steps are as follows:

[0069] (1) Preparation of NM88B-POR:

[0070] Dissolve 270 mg of ferric chloride hexahydrate and 180 mg of porphyrin in 5 mL of N,N-dimethylformamide, add 181 mg of 2-aminoterephthalic acid while stirring, continue stirring, add 0.4 mL of 2 mol / L sodium hydroxide solution, and mix well. Then transfer the mixture to a polytetrafluoroethylene reactor, heat it to 100 ° C at 1 ° C / min, keep it at 100 ° C for 12 hours, and then cool it to room temperature. After the reaction is completed, the precipitate in the reactor is centrifuged, washed and dried to obtain NM88B-POR.

[0071] (2) Preparation of NM88B-POR / Rf:

[0072] The NM88B-POR obtained in step (1) was immersed in 100 mL of riboflavin (Rf) solution having 30 mmol / L sarcosine oxidase (SOx) activity, and NM88B-POR / Rf was obtained by electroplating at -0.9 V for 90 s using a constant potential method.

[0073] Example 6

[0074] A method for preparing NM88B-POR / Rf nanozyme, the steps are as follows:

[0075] (1) Preparation of NM88B-POR:

[0076] Dissolve 270 mg of ferric chloride hexahydrate and 180 mg of porphyrin in 5 mL of N,N-dimethylformamide, add 181 mg of 2-aminoterephthalic acid while stirring, continue stirring, add 0.4 mL of 2 mol / L sodium hydroxide solution, and mix well. Then transfer the mixture to a polytetrafluoroethylene reactor, heat it to 100 ° C at 1 ° C / min, keep it at 100 ° C for 12 hours, and then cool it to room temperature. After the reaction is completed, the precipitate in the reactor is centrifuged, washed and dried to obtain NM88B-POR.

[0077] (2) Preparation of NM88B-POR / Rf:

[0078] The NM88B-POR obtained in step (1) was immersed in 100 mL of riboflavin (Rf) solution having 30 mmol / L sarcosine oxidase (SOx) activity, and NM88B-POR / Rf was obtained by electroplating at -0.6 V for 60 s using a constant potential method.

[0079] Material performance testing

[0080] The product prepared in Example 1 of the present invention was subjected to characterization analysis and performance test analysis.

[0081] (I) Enzyme activity test of materials:

[0082] Prepare 1 mol / L sodium acetate-acetic acid buffer solution (pH=7.0), 30% hydrogen peroxide (H2O2) solution, 10 mmol / L 3,3',5,5'-tetramethylbenzidine (TMB) solution and 1 mg / mL NM88B-POR / Rf aqueous dispersion respectively. Use the proportions in Table 1 and Table 2 below to configure the reaction system. After the reaction is completed for 5 minutes, scan the absorbance of each system by ultraviolet time, and then determine the catalytic activity of the material by drawing and fitting the Michaelis curve. The test results are as follows: Figure 1 As shown. It was calculated that the Michaelis constant (Km) of NM88B-POR / Rf with H2O2 as substrate was 0.38mM, which was much lower than the Km (3.70mM) of horseradish peroxidase (HRP) reported in similar experiments, indicating that NM88B-POR has a higher affinity for H2O2, while the Km of NM88B-POR with TMB as substrate was 1.67mM, which was much higher than the Km (0.43mM) of TMB reported in similar experiments, indicating that its affinity with TMB was poor. At the same time, the Km of H2O2 as substrate is much smaller than the Km of TMB as substrate, indicating that TMB is not a substrate catalyzed by NM88B-POR. This shows that NM88B itself can act as a peroxidase-like enzyme with strong peroxidase activity.

[0083] Table 1 Reaction system with H2O2 concentration as independent variable

[0084]

[0085] Table 2 Reaction system with TMB concentration as independent variable

[0086]

[0087] (II) XRD characterization of materials

[0088] The crystal structure of NM88B-POR / Rf obtained by the treatment of the present invention was characterized by using Japanese D / Max 2500V X-ray powder diffraction. Figure 2 As shown in the figure, the diffraction peaks of the original NM88B at 2θ=9.29° and 10.55° are relatively typical, which is consistent with the literature reports, indicating that NM88B(Fe) with good crystallinity was synthesized. The (002) plane diffraction peak of NM88B-POR is stronger than that of the original NM88B, which is due to the fact that the addition of porphyrin affects the growth of the NM88B crystal surface.

[0089] (III) Surface morphology of materials

[0090] The surface morphology of NM88B-POR / Rf treated by the present invention was characterized by using a Japanese Hitachi S-3400N low-power scanning electron microscope. Figure 3 As shown, NM88B / Rf( Figure 3 A) The morphology shows a spindle-shaped structure with uniform size, about 500 nm in size. The morphology of NM88B / Rf changed after doping with porphyrin, that is, the surface of NM88B-POR / Rf was rougher than before, and the particle size was shorter and thicker than before ( Figure 3 B), about 300nm.

[0091] (IV) Characterization of the specific surface area and pore structure parameters of the material.

[0092] The specific surface area and pore structure of NM88B and NM88B-POR treated by the present invention were characterized by using the American Micromeritics ASAP 2460 specific surface and porosity analyzer. The results are as follows: Figure 4 As shown in Table 3.

[0093] Table 3 Specific surface area and pore structure parameters of materials

[0094]

[0095] Depend on Figure 4 As shown in Table 3, the specific surface area (SSA) of NM88B is 29.3 m 2 / g, this is because NM88B is a typical flexible skeleton MOF with unique breathing properties and a stretchable structure in aqueous solution. According to the current public data, its SSA is as high as 3000m 2 / g. The SSA of NM88B-POR is 36.1m 2 / g, with obvious mesopore and micropore structures. The increase of mesopores may be due to the defects of NM88B crystal caused by porphyrin doping during the growth process.

[0096] (V) Optimization of detection conditions

[0097] The peak current of experimental conditions such as pH value, NM88B-POR / Rf material concentration, Rf adsorption time and electrodeposition time were optimized through DPV curve to achieve the best enzyme catalytic activity of NM88B-POR / Rf nanozyme.

[0098] (1) The enzyme catalytic activity of NM88B-POR / Rf was tested at different pH values ​​of 5.0-8.0. Figure 5 In comparison, NM88B-POR / Rf has the highest catalytic activity at pH = 7.0.

[0099] (2) Material concentration performance test: NM88B-POR / Rf was tested for its enzyme catalytic activity at different material concentrations (1.00-2.00 mg / mL). The results are as follows: Figure 6 In comparison, NM88B-POR / Rf has the highest catalytic activity when the material concentration is 1.50 mg / mL.

[0100] (3) Rf adsorption time performance test: The enzyme catalytic activity of NM88B-POR / Rf was tested under different Rf adsorption time conditions (5-15 min). The results are as follows: Figure 7 In comparison, NM88B-POR / Rf has the highest catalytic activity when the Rf adsorption time is 10 min.

[0101] (4) Rf electrodeposition time performance test: The enzyme catalytic activity of NM88B-POR / Rf was tested under different Rf electrodeposition time conditions (30-120s). The results are as follows: Figure 8 In comparison, NM88B-POR / Rf has the highest catalytic activity when the Rf electrodeposition time is 90 s.

[0102] (VI) Storage stability characterization

[0103] like Fig. 9 As shown, NM88B-POR / Rf still has 91.3% of the initial enzyme activity after being stored at room temperature for 7 days. However, according to the literature, natural SOx and peroxidase have basically lost their activity after being stored for 7 days, indicating that NM88B-POR / Rf has excellent storage stability.

[0104] (VII) Characterization of oxygen carrying capacity of materials

[0105] In order to verify the oxygen-carrying capacity of Fe-POR structure to mimic heme in NM88B-POR / Rf nanozyme and provide a high oxygen microenvironment for Rf-catalyzed Sar, the oxygen-carrying capacity of NM88B and NM88B-POR was tested by IT experiment. Fig.10 As shown. Under different initial oxygen concentration conditions, the peak current values ​​of NM88B and NM88B-POR tend to balance with the increase of detection time. The peak current difference before (solid line) and after (dashed line) the addition of Sar is defined as the current reaching equilibrium (ΔI), which is used to represent the ability of the nanozyme to capture dissolved oxygen in the solution. With the increase of initial oxygen concentration, the utilization rate of dissolved oxygen by NM88B only increased by 0.92, 1.10, and 1.75 times (corresponding to Fig.10 A, C, E), but NM88B-POR consumed 5.7, 29.2, and 50.7 times more dissolved oxygen at high oxygen concentration than at low oxygen concentration (corresponding to Fig.10 B, D, F), which shows that the heme-like Fe-POR structure of NM88B-POR can significantly improve the oxygen capture ability of the nanozyme as a mimic of heme, and provide an efficient high-oxygen microenvironment for Rf. The Sar electrical signals detected by NM88B-POR / Rf and NM88B / Rf under the same conditions show that the response signal of NM88B-POR / Rf is 2.3 times higher than that of NM88B / Rf. This shows that the Fe-porphyrin structure can significantly improve the oxygen capture ability of the nanozyme, provide an efficient dissolved oxygen microenvironment for Rf, and thus improve the catalytic activity of the nanozyme on Sar ( Fig.11 ).

[0106] (VIII) Characterization of material testing performance

[0107] The NM88B / Rf and NM88B-POR / Rf obtained by the present invention were used to detect Sar of different concentrations.

[0108] Sar solutions with a concentration gradient of 10 pmol / L-1 mmol / L were prepared, 50 μL of each solution was mixed with sodium acetate-acetic acid buffer (pH = 7, 850 μL), and then 100 μL of 1 mg / mL aqueous dispersions of NM88B / Rf and NM88B-POR / Rf were added, respectively, and reacted at 25°C for 10 min, followed by detection. The results were as follows: Fig.12 (A), (B) (NM88B / Rf) and Fig.13 (A), (B) (NM88B-POR / Rf).

[0109] The Sar detection limit of NM88B / Rf was calculated to be 0.33nmol / L, and that of NM88B-POR / Rf was 3.31pmol / L, indicating that the incorporation of porphyrin significantly increased the Sar detection limit of the material. Fig.10 and 11 The reason is that the incorporation of porphyrin gives the material a Fe-POR structure, which enables the NM88B-POR / Rf material to have the oxygen-carrying capacity of heme, while the oxidation process of Sar requires the participation of oxygen. Therefore, the incorporation of porphyrin can promote the oxidation of Sar, increase the sensitivity of the NM88B-POR / Rf material to Sar, and improve the detection limit. At the same time, the detection performance of the NM88B-POR / Rf material for Sar is significantly better than that of natural SOx and other SOx nanozyme materials reported so far.

[0110] (IX) Characterization of the anti-interference performance of materials

[0111] The specificity of NM88B-POR / Rf obtained by the present invention was evaluated for four common interfering substances, including serine (Ser), lysine (Lys), cysteine ​​(Cys) and glucose (Glu). The results are as follows: Fig.14 As shown - even though the concentration of these interferents (100.0 μM) is much higher than Sar (1.0 μM), the current response is still similar to the blank control, indicating that there is almost no interference from Ser, Lys, Cys and Glu. In addition, when 1.0 mM Sar is mixed with these four interfering substances, there is no obvious change in the signal compared to only doping Sar. These results show that the NM88B-POR / Rf processed by the present invention has good detection specificity for Sar.

[0112] The examples given in the present invention are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A method for preparing NM88B-POR / Rf nanozyme, characterized in that: The steps include: (1) Preparation of NM88B-POR: Dissolve ferric chloride hexahydrate and porphyrin in N,N-dimethylformamide, add 2-aminoterephthalic acid with stirring to obtain a mixed solution, continue stirring and add sodium hydroxide solution, mix well, transfer to a reactor, perform hydrothermal reaction at 80-100°C for 10-24 h, and obtain NM88B-POR after washing and drying; (2) Preparation of NM88B-POR / Rf: The NM88B-POR material obtained in step (1) is immersed in a riboflavin Rf solution having sarcosine oxidase SOx activity, and NM88B-POR / Rf is obtained after deposition by an electrodeposition method.

2. The method for preparing the NM88B-POR / Rf nanozyme according to claim 1, characterized in that: In the step (1), the molar ratio of ferric chloride hexahydrate, porphyrin, 2-aminoterephthalic acid and sodium hydroxide is 1:0.5:0.5-1:0.

8.

3. The method for preparing the NM88B-POR / Rf nanozyme according to claim 1, characterized in that: In the step (1), 40-60 mg of ferric chloride hexahydrate and 1-20 mg of porphyrin are added to every 1 ml of N,N-dimethylformamide; the amount of 2-aminoterephthalic acid added is 30-40 mg of 2-aminoterephthalic acid per 1 ml of N,N-dimethylformamide; and the amount of sodium hydroxide solution added is 0.01-0.2 mL of sodium hydroxide solution with a concentration of 1-2 mol / L per milliliter of the mixed solution.

4. The method for preparing the NM88B-POR / Rf nanozyme according to claim 1, characterized in that: In the step (1), the time for uniformly mixing the ferric chloride hexahydrate and the porphyrin solution is 5-10 min; and the time for uniformly mixing the 2-aminoterephthalic acid is 20-40 min.

5. The method for preparing the NM88B-POR / Rf nanozyme according to claim 1, characterized in that: The washing and drying in step (1) are washing with N,N-dimethylformamide and acetone for 2-4 times, then drying at 70-80° C. for 10-12 h, and then drying under vacuum at 70-90° C. for 10-12 h.

6. The method for preparing the NM88B-POR / Rf nanozyme according to claim 1, characterized in that: The concentration of the riboflavin Rf solution in step (2) is 2-40 mmol / L.

7. The method for preparing the NM88B-POR / Rf nanozyme according to claim 1, characterized in that: In the step (2), the plastidial ratio of NM88B-POR to riboflavin Rf is 2.2:

1.

8. The method for preparing the NM88B-POR / Rf nanozyme according to claim 1, characterized in that: The electrodeposition method in step (2) is a constant potential method with a voltage of 0.6-0.9 V and a deposition time of 60-120 s.

9. The NM88B-POR / Rf nanozyme prepared by the preparation method described in any one of claims 1 to 8 is used in the electrochemical analysis and detection of sarcosine.

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