Synthesis method and application of Tyr-Mn bionic nano-enzyme with multienzyme activity

Abstract

The invention discloses a synthesis method and application of a Tyr-Mn bionic nano-enzyme with multienzyme activity, and relates to the technical field of nano-enzymes, and the synthesis method comprises the following steps: mixing a Mn < 2 + > solution and a NaOH and L-tyrosine mixed solution, stirring, washing, and drying to obtain the Tyr-Mn bionic nano-enzyme. The Tyr-Mn bionic nano-enzyme can catalyze an oxidase substrate and a laccase substrate, the Tyr-Mn bionic nano-enzyme can be synthesized through a relatively simple method, the synthesized Tyr-Mn bionic nano-enzyme has excellent oxidase-like and laccase-like activity and can be used as a catalyst to catalyze the oxidase substrate and the laccase substrate, and when the Tyr-Mn bionic nano-enzyme is subjected to a catalytic reaction, the Tyr-Mn bionic nano-enzyme can catalyze the oxidase substrate and the laccase substrate. The affinity to a substrate is relatively high.

Description

Technical Field

[0001] The present invention relates to the technical field of nanozymes, and in particular to a synthesis method and application of a Tyr-Mn bionic nanozyme with multi-enzyme activity. Background Art

[0002] Nanozymes have attracted extensive attention in the past decades since the landmark discovery of Fe3O4 nanoparticles with peroxidase-like activity in 2007. Compared with natural biological enzymes, nanozymes have relatively low activities, which limits their further practical applications. Recently, considerable efforts have been made for various novel nanozymes with enhanced catalytic activities to meet the needs for practical applications. For example, Wu et al. reported a novel three-channel colorimetric sensor array based on Au2Pt bimetallic nanozymes for distinguishing antioxidants in food. Lu et al. reported a colorimetric sensing platform based on molybdenum-doped carbon dot nanozymes for monitoring ampicillin. However, on the one hand, most nanozymes exhibit a single mimetic enzyme activity, while multiple enzyme mimics bring about chain reactions, broadening the potential applications of enzymes. On the other hand, the enhanced activity of nanozymes can enhance the performance of their potential applications. Therefore, it is of great significance to explore the high activity and catalytic function of natural enzymes, which originate from their well-defined active sites, where the catalytic units and binding pockets synergistically produce ultrahigh catalytic activity. For example, cytochrome c oxidase (CcO) is the terminal enzyme in the respiratory electron transport chain, reducing molecular oxygen to water. The binuclear active site in CcO includes a high-spin heme associated with a CuB complex and a redox-active tyrosine. Tyrosine donates both electrons and protons in the OO bond scavenging step to form a tyrosine radical. Laccase is a member of the copper oxidase family and has been used as a green catalyst in the environmental and biochemical industries. Laccase has four copper sites, including type 1 (T1), type 2 (T2), and binuclear type 3 (T3) copper sites. The substrate is oxidized at the T1 Cu site, and electrons are transferred from the T1 Cu site to the T2 / T3 trinuclear Cu sites, and molecular oxygen is converted to water through a four-electron process. Natural enzymes with mild reaction conditions, high catalytic activity, and substrate specificity have been widely used in industry, medicine, and biology. However, the high cost of preparation and purification, low operational stability, sensitivity of catalytic activity to environmental conditions, and difficulties in recycling and reuse have limited their further application in the field of biosensing. Therefore, in order to overcome the shortcomings of natural enzymes, nanozymes are nanomaterials with the ability to mimic the activity of natural enzymes, which have attracted great attention due to their excellent stability, wide pH applicability, low cost, and modifiability. Recently, a variety of novel nanozymes with enhanced activity have been extensively studied to meet the demand for practical applications. For example, Cu-Pt nanozymes are used for blood glucose monitoring mediated by discriminant analysis of colorimetric signals.AuNP-GNP-CS nanozymes with peroxidase mimetic properties were used for highly sensitive and selective immunoassay of porcine gelatin; flower-like L-Cys-FeNiNPs nanozyme aptasensors were used for sensitive colorimetric detection of aflatoxin B1; CS@Pt NS with peroxidase mimetic properties were used to construct colorimetric and photothermal dual-readout biosensors for sensitive detection of cholesterol; Fe3O4@MnO2 NS with oxidase-like activity were synthesized by mimicking the activity of natural oxidases; Fe2NC nanozymes were prepared by simulating coordination environments, which have good stability at extreme pH values ​​and high temperatures; covalent organic framework nanosheets as enhancers of light-responsive oxidase-like nanozymes for multifunctional applications in colorimetric sensing, antibiotic degradation, and antimicrobial agents; portable sensor kits based on NiCr2O4 nanozymes for on-site quantification of nerve agent simulants. Recently, some copper-containing nanomaterials have been explored for their laccase-like activities, such as Cu2O, I-Cu, MI-Bpy-Cu, Cu-gC3N5, and ZrF-laccase biocatalysts. Inspired by the structure of natural oxidases, the catalytic function of natural enzymes originates from their well-defined active sites. The catalytic unit and binding pocket of the active site synergistically produce ultrahigh catalytic activity. Therefore, it is a very attractive approach to design nanozymes by mimicking the fine catalytic structure of natural enzymes. In order to make up for the defects of natural enzymes, it has become a current research hotspot to study artificial mimetic enzymes with mimetic enzyme activity, stable structure, and low cost by mimicking the active sites of natural enzymes. However, most biomimetic enzymes mainly mimic enzyme activity and coordination environment, and there are few studies in the amino acid microenvironment.

[0003] At present, most of the research on nanozymes is focused on the study of peroxidase-like enzymes, and there are relatively few studies based on oxidase-like enzymes. Oxidase-like enzymes can catalyze the oxidation of TMB in the absence of H2O2. This paper easily synthesizes Tyr-Mn with oxidase-like activity at room temperature by simulating the metal active center and tyrosine active site of natural cytochrome c oxidase. In addition, laccase nanozymes are currently limited to materials with copper as the active agent. Non-copper laccase nanozymes are rarely reported, and there are also few studies in amino acid microenvironments. This paper uses multiple copper active sites of natural laccase, redox Cu 2+ / Cu + Inspired by the electron transfer pathway and spatial amino acid microenvironment, Tyr-Mn was easily synthesized at room temperature, mimicking the metal active center and amino acid microenvironment of natural laccase.

[0004] New organic pollutants such as tetracycline hydrochloride and isoniazid cause great harm to the environment and pose a potential threat to human health, so it is necessary to monitor them. Tetracycline hydrochloride (TCH) is a broad-spectrum antibiotic and one of the most detected antibiotics in the environment. The accumulation of TCH, even in small concentrations, can cause several adverse health effects, such as hepatotoxicity and genotoxicity. The excessive use of TCH poses a threat to human health, so its use must be monitored and regulated. Tuberculosis (TB) is one of the major infectious diseases that seriously endangers human health. Isoniazid (INH) is highly selective and effective against Mycobacterium tuberculosis, making it a first-line drug for the treatment of tuberculosis. But when excessive amounts of INH are ingested, it can lead to drug resistance, neurotoxicity, and even liver damage. In recent years, many methods for detecting TCH and INH have been reported, such as high-performance liquid chromatography (HPLC), electrochemical detection, fluorescence spectroscopy, etc. Although these methods are effective in detecting these two substances, the complex and time-consuming process inevitably limits their application in practice. In contrast, colorimetry has attracted much attention for its simplicity, visualization, and rapid detection, and provides an alternative for the detection of these two substances. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a synthesis method and application of a Tyr-Mn bionic nanozyme with multi-enzyme activity. By a simple method, a bionic nanozyme Tyr-Mn with excellent oxidase-like and laccase-like activities can be synthesized, providing a new option for detecting TCH and INH.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A method for synthesizing Tyr-Mn bionic nanozyme with multi-enzyme activity, wherein Mn 2+ The solution is mixed with a mixed solution of NaOH and L-tyrosine, stirred, washed and dried to obtain the Tyr-Mn biomimetic nanozyme.

[0008] Furthermore, Mn 2+ The solution was prepared by dissolving 5 mmol of MnCl2·4H2O in 5 mL of deionized water and ultrasonically treating the solution for 5 min.

[0009] Furthermore, the preparation method of the mixed solution of NaOH and L-tyrosine is as follows: 5.2 mmol NaOH and 2.5 mmol L-tyrosine are dissolved in 25 mL deionized water.

[0010] Furthermore, Mn 2+The solution and the mixed solution of NaOH and L-tyrosine were mixed, stirred at 700 rpm for 90 min, then washed three times with deionized water and centrifuged, and dried in a vacuum oven at 60° C. overnight after centrifugation.

[0011] Application of any of the above-synthesized Tyr-Mn bionic nanozymes in catalyzing oxidase substrates.

[0012] Furthermore, the catalytic conditions are: pH 4.0, temperature 25°C, the added volume ratio of Tyr-Mn bionic nanozyme to oxidase substrate is 2:5, and the reaction time is 10 min; wherein the concentration of Tyr-Mn bionic nanozyme is 2 mg / mL, and the concentration of oxidase substrate is 7.5 mM.

[0013] The synthesized Tyr-Mn bionic nanozyme can also be used in catalyzing laccase substrates.

[0014] Furthermore, the catalytic conditions are: pH 6.0, temperature 25°C, the added volume ratio of Tyr-Mn bionic nanozyme and laccase substrate is 1:2, and the reaction time is 30 min; wherein the concentration of Tyr-Mn bionic nanozyme is 2 mg / mL, and the concentration of laccase substrate is 20 mM.

[0015] The synthesized Tyr-Mn biomimetic nanozyme can also be used in the detection of TCH and INH.

[0016] Furthermore, a colorimetric sensor based on Tyr-Mn bionic nanozyme was constructed and combined with the precise color recognition function of smartphones to establish a smartphone-assisted colorimetric sensing platform for on-site visual detection of TCH and INH.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention can synthesize Tyr-Mn bionic nanozyme by a relatively simple method. The synthesized enzyme has excellent oxidase-like and laccase-like activities and can be used as a catalyst to catalyze oxidase substrates and laccase substrates. In addition, the Tyr-Mn bionic nanozyme has a high affinity for the substrate when the catalytic reaction occurs.

[0019] (2) The Tyr-Mn bionic nanozyme prepared by the present invention can be used as a colorimetric sensor for detecting TCH and INH, and can also be combined with the precise color recognition function of a smartphone to quickly and accurately detect TCH and INH. When detecting TCH, the detection limit LOD is 1.48 μM; when detecting INH, the detection limit LOD is 151.4 nM. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the scanning electron microscope image of Tyr-Mn;

[0021] Figure 2 is the EDS diagram of Tyr-Mn;

[0022] Figure 3 is the infrared image of Tyr-Mn and Tyr;

[0023] Figure 4-7 This is the XPS analysis diagram of Tyr-Mn;

[0024] Figure 8 This is a test diagram of Tyr-Mn oxidase enzyme activity;

[0025] Fig. 9 , Fig.10 It is a kinetic analysis diagram with TMB as the oxidase substrate;

[0026] Fig.11 This is a test diagram of Tyr-Mn laccase enzyme activity;

[0027] Fig.12 , Fig.13 For the kinetic analysis of laccase with 2,4-DCP as substrate;

[0028] Fig.14 To analyze the feasibility of using Tyr-Mn oxidase activity to detect TCH;

[0029] Fig.15 To analyze the feasibility of using Tyr-Mn laccase activity to detect INH;

[0030] Fig.16 The UV-visible spectra of the effects of different TCH concentrations on the simulated activity of Tyr-Mn oxidase;

[0031] Fig.17 is the linear relationship between the absorbance of the oxidase system and the TCH concentration;

[0032] Fig.18 The UV-visible spectra of the effects of different INH concentrations on the simulated activity of Tyr-Mn laccase;

[0033] Fig.19 is the linear relationship between the absorbance of the laccase system and the INH concentration;

[0034] Fig. 20 This is a selective assay for detecting tetracycline hydrochloride based on Tyr-Mn oxidase activity;

[0035] Fig.21 It is a selective test for the detection of isoniazid in urine based on Tyr-Mn laccase activity;

[0036] Fig. 22 It is a test of the stability of Tyr-Mn oxidase activity;

[0037] Fig.23 It is the activity stability experiment of Tyr-Mn laccase;

[0038] Fig.24 a is the image of different concentrations of TCH and Tyr-Mn sensing solution after reaction under visible light, and the linear relationship between relative color information (R / B) and TCH concentration;

[0039] Fig.24 b is the simulated activity image of laccase after the reaction of different concentrations of INH and Tyr-Mn sensing solution under visible light, as well as the linear relationship between the relative color information (G / R) and the INH concentration. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and embodiments. The embodiments of the present invention include but are not limited to the following embodiments.

[0041] 1. Synthesis and related experiments of Tyr-Mn biomimetic nanozymes

[0042] 1.1 Synthesis of Tyr-Mn biomimetic nanozyme

[0043] 5mmol (0.9896g) MnCl2·4H2O was dissolved in 5mL deionized water and ultrasonically treated for 5min to form solution A; 5.2mmol (0.208g) NaOH and 2.5mmol (0.4530g) L-tyrosine were dissolved in 25mL deionized water to form solution B; A was poured into B, the two solutions were quickly mixed, and stirred at 700rpm for 90min. The solution was washed with deionized water three times and centrifuged, and dried in a vacuum oven at 60℃ overnight to obtain Tyr-Mn biomimetic nanozyme.

[0044] 1.2. Evaluation of the performance of oxidase and laccase mimics

[0045] The oxidase mimetic activity of Tyr-Mn was evaluated by measuring the absorbance change at 652 nm. 10 μL 2 mg / mL Tyr-Mn and 25 μL 3,3,5,5-tetramethylbenzidine (TMB, 7.5 mM) were sequentially added to 1 mL HAc-NaAc buffer (0.1 M, pH = 4.0), the mixture was reacted at 25 ° C for 10 min, and the absorbance of the solution at 652 nm was recorded using a UV-2450 spectrophotometer. The effects of pH value, temperature, TMB dosage (7.5 mM) and reaction time of HAc-NaAc buffer on the oxidase mimetic activity of Tyr-Mn were studied. The laccase mimetic activity of Tyr-Mn was evaluated by measuring the absorbance change at 510 nm. 20 μL 2 mg / mL Tyr-Mn, 40 μL 2,4-dichlorophenol (2,4-DCP, 20 mM), and 30 μL 4-aminoantipyrine (4-AAP, 4 mM) were sequentially added to 1 mL HAc-NaAc buffer (0.1 M, pH = 6.0), and the mixture was reacted at 25°C for 30 min, and the absorbance at 510 nm was recorded using a UV-2450 spectrophotometer. The effects of pH value of HAc-NaAc buffer, temperature, amount of 2,4-DCP (20 mM) and 4-AAP (4 mM), and reaction time on the Tyr-Mn laccase mimetic activity were studied.

[0046] 1.3 Steady-state kinetic analysis of Tyr-Mn

[0047] The affinity of Tyr-Mn for TMB was studied by steady-state kinetics experiments. 10 μL of 2 mg / mL Tyr-Mn and different concentrations of TMB (0.0225-0.1125 mM) were added to 1 mL of HAc-NaAc (pH = 4.0, 0.1 M) buffer. The maximum reaction rate (V) was then calculated using the Michaelis-Menten equation based on the Lineweaver-Burk curve. max ) and the Michaelis-Menten constant (K m ). Michaelis-Menten equation: V = (V max ×[S] / (K m +[S])), in this formula, V maxis the maximum reaction rate, V is the apparent initial reaction rate, and [S] is the substrate concentration. The affinity of Tyr-Mn to 2,4-DCP was studied by steady-state kinetic experiments. 20 μL 2 mg / mL Tyr-Mn, different concentrations of 2,4-DCP (0.1-0.44 mM) and 30 μL 4-AAP (4 mM) were added to 1 mL HAc-NaAc (pH = 6.0, 0.1 M) buffer. The maximum reaction rate (V) was then calculated using the Michaelis-Menten equation based on the Lineweaver-Burk curve. max ) and the Michaelis-Menten constant (K m ). Michaelis-Menten equation: V = (V max ×[S] / (K m +[S])), in this formula, V max is the maximum reaction rate, V is the apparent initial reaction rate, and [S] is the substrate concentration.

[0048] 1.4. Colorimetric detection of TCH and INH

[0049] To detect TCH, 1 mL HAc-NaAc (0.1 M, pH = 4.0), different concentrations of TCH, and 10 μL Tyr-Mn (2 mg / mL) were added to a 2 mL centrifuge tube and incubated at 25 ° C for 45 min. Then, 15 μL TMB (7.5 mM) was added and reacted for 2 min. Finally, the absorbance of the mixed solution at 652 nm was recorded. To detect INH, 1 mL HAc-NaAc (0.1 M, pH = 6.0), different concentrations of INH, 20 μL Tyr-Mn (2 mg / mL), 30 μL 2,4-DCP (20 mM), and 15 μL 4-AAP (4 mM) were added to a 2 mL centrifuge tube and reacted at 25 ° C for 24 min. Then, the absorbance of the mixed solution at 510 nm was recorded.

[0050] 1.5. Construction of Smartphone-Assisted Sensing Platform

[0051] Achieve on-site detection of TCH and INH. Design a smartphone-assisted TCH and INH detection method. Use Tyr-Mn and TMB colorimetric system to detect TCH. Use Tyr-Mn, 2,4-DCP, and 4-AAP colorimetric system to detect INH. Obtain RGB values ​​of different colorimetric systems by taking smartphone photos, then study the relationship between RGB values ​​and TCH and INH concentrations, and construct a linear relationship for TCH and INH analysis.

[0052] 1.6. Actual sample analysis

[0053] Fish pond water and Jialing River water were obtained from Nanchong. Rabbit blood was obtained from a local farmer's market. Each sample was spiked with 0, 5, 9, and 13 μM TCH and measured three times. In addition, the standard addition method of the actual sample was analyzed by a smartphone-assisted method. Each sample was spiked with 0, 30, 60, and 90 μM TCH and measured three times. Urine samples were collected from healthy volunteers, and the urine was pretreated and centrifuged at 9000 rpm. The samples were then filtered through a 0.22 μm pore size filter membrane to remove impurities and diluted 10 times. For subsequent experiments. Each sample was spiked with 0, 1, 8, and 15 μM INH and measured three times. In addition, the standard addition method of the actual sample was analyzed by a smartphone-assisted method. Each sample was spiked with 0, 3, 18, and 33 μM INH and measured three times.

[0054] 2. Results and Discussion

[0055] 2.1 Characterization of Tyr-Mn

[0056] Tyr-Mn was simply synthesized by stirring at room temperature and observed using a scanning electron microscope (SEM). Figure 1 As shown in the figure, it can be seen that the synthesized Tyr-Mn nanomaterial is a nanorod with nanospheres evenly distributed on the surface. At the same time, the EDS spectrum of Tyr-Mn is as follows Figure 2 As shown, C, O, N, and Mn all exist in Tyr-Mn, indicating the successful synthesis of Tyr-Mn.

[0057] Fourier transform infrared spectroscopy was used to verify the possibility of interaction between species based on the appearance, disappearance, or shift of signals associated with functional groups. The FT-IR results for Tyr and Tyr-Mn are shown in Figure 2. Figure 3 As shown. Tyr exhibits the following characteristic vibration peaks: OH (3205cm -1 )、NH(1589cm -1 )、C=O(1600cm -1 )、CO(1244cm -1 )、Ph(700-750cm -1 ). The characteristic absorption peak of Tyr also exists in the infrared spectrum of Tyr-Mn, indicating the presence of Tyr in the composite material.

[0058] Figures 4 to 7 XPS analysis of Tyr-Mn and high-resolution XPS of Mn 2p ( Figure 4 ) Display Mn 2+ , Mn 3+ and Mn 4+The Mn 2p peak can be divided into Mn 2p3 / 2 (634-646 eV), and the Mn 2p3 / 2 peak is divided into three peaks at 641.2 eV, 642.0 eV, and 643.11 eV, which are similar to Mn 2+ , Mn 3+ , Mn 4+ Correspondingly, the binding energy of Mn is 653.53 eV, which is 2p1 / 2, and Mn 4+ Related. High-resolution XPS of O 1s ( Figure 5 ) shows peaks at 530.44eV, 531.7eV, and 532.59eV, which belong to the binding energies of C=O, OC=O, and C=O. High-resolution XPS of N 1s ( Figure 6 ) shows that the peak at 401.95 eV belongs to the binding energy of NH. The high-resolution XPS of C 1s ( Figure 7 ) shows peaks at 284.7 eV, 286.1 eV, and 288.8 eV, belonging to the binding energies of CC, CN, and C=O. All the above characterizations prove the successful synthesis of Tyr-Mn.

[0059] 2.2 Evaluation of the activity of oxidase and laccase mimics

[0060] In order to systematically study the oxidase mimetic activity of Tyr-Mn, TMB was oxidized as a typical oxidase substrate in different systems. Figure 8 As shown, Tyr-Mn and TMB are colorless, while the typical absorption peak at 652nm in the Tyr-Mn+TMB system corresponds to blue oxTMB, indicating that Tyr-Mn has intrinsic oxidase mimetic activity. In order to make Tyr-Mn have better oxidase mimetic activity, the catalytic conditions such as pH value, temperature, TMB dosage (7.5mM) and reaction time of HAc-NaAc buffer were optimized. When the pH value is in the range of 3.5-6.5, Tyr-Mn has a certain catalytic activity, but only when pH = 4.0, Tyr-Mn has the best oxidase mimetic activity. The optimum temperature for the catalytic reaction is 25°C, the amount of TMB (7.5mM) is 25μL, and the reaction time is 10min. In order to explore the catalytic mechanism of oxidase mimetic enzymes in Tyr-Mn, the steady-state kinetics of the material in HAc-NaAc buffer solution (0.1M, pH = 4.0) were studied. The concentration of TMB changes within an appropriate range, such as Fig. 9 , Fig.10 As shown in Table 1, the catalytic reaction process of Tyr-Mn oxidase conforms to the representative Michaelis-Menten curve. In addition, the maximum reaction rate (V max ) and the Michaelis-Menten constant (K m) is calculated by the Michaelis-Menten equation based on the Lineweaver-Burk curve. K m Used to assess the ability of an enzyme to bind to its substrate with affinity. m It indicates that the enzyme affinity with the substrate is very strong. When the catalyst is Tyr-Mn, the K m The value is 0.058 mM, which is lower than some reported nanozymes (HRP, V2C nanosheets, Mn-PBA, Mn-CDs / AgNPs). This result shows that Tyr-Mn has a stronger affinity for TMB than other nanozymes. max The value is 18.95×10 -8 MS -1 , which is higher than some reported nanozymes (HRP, V2C nanosheets, Mn-PBA, CoOOH NSs, Fe3O4@MnO2). In summary, Tyr-Mn shows a lower K than the same nanozymes. m value and higher V max , proving that Tyr-Mn is a good oxidase mimetic enzyme.

[0061] Table 1 Comparison of kinetic parameters of Tyr-Mn and other nanozymes when TMB is used as substrate

[0062] catalyst Substrate Michaelis constant (mM) <![CDATA[Maximum reaction rate (10 -8 M s -1 )]]> HRP TMB 0.434 10 <![CDATA[V2C nanosheets]]> TMB 0.163 4.075 Mn-PBA TMB 0.13 16.9 CoOOH NSs TMB 0.034 7.76 Mn-CDs / AgNPs TMB 0.244 22.6 <![CDATA[Fe3O4@MnO2]]> TMB 0.022 10.52 Tyr-Mn TMB 0.058 18.95

[0063] In order to systematically study the laccase mimetic activity of Tyr-Mn, 2,4-dichlorophenol (2,4-DCP) was used as a typical laccase substrate in different systems. In this process, 2,4-DCP was oxidized by Tyr-Mn to generate semiquinone free radicals, which then reacted with 4-aminoantipyrine (4-AAP) to generate 4-N-(1,4-benzoquinoneimine)antipyrine, which has a strong characteristic absorbance at 510nm. Fig.11As shown in the figure, Tyr-Mn+2,4-DCP, Tyr-Mn+4-AAP, and 2,4-DCP+4-AAP are colorless, while the typical absorption peak of 4-N-(1,4-benzoquinoneimine)antipyrine at 510nm in the Tyr-Mn+2,4-DCP+4-AAP system corresponds to 4-N-(1,4-benzoquinoneimine)antipyrine, indicating that Tyr-Mn has intrinsic laccase mimetic activity. In order to make Tyr-Mn have better laccase mimetic activity, the catalytic conditions such as reaction time, pH value of HAc-NaAc buffer solution, temperature, amount of 2,4-DCP (20mM) and amount of 4-AAP (4mM) were optimized. In order to explore the catalytic mechanism of laccase mimetic enzyme in Tyr-Mn, the steady-state kinetics of the material in HAc-NaAc buffer solution (0.1M, pH=6.0) were studied. The concentration of 2,4-DCP was varied within an appropriate range, such as Fig.12 , Fig.13 As shown in Table 2, the catalytic reaction process of Tyr-Mn laccase conforms to the representative Michaelis-Menten curve. In addition, the maximum reaction rate (V max ) and the Michaelis-Menten constant (K m ). When Tyr-Mn is used as the catalyst, the K of 2,4-DCP m The value was 0.3116 mM, which was lower than that of natural laccase and some reported nanozymes. The results showed that the affinity of Tyr-Mn for 2,4-DCP was stronger than that of natural laccase and other nanozymes. max The value is 7.426×10 -8 MS -1 , which is higher than that of natural laccase and some reported nanozymes. In summary, Tyr-Mn showed a lower K than that of natural laccase and the same nanozymes. m value and higher V max , proving that Tyr-Mn is a good laccase mimetic enzyme.

[0064] Table 2 Comparison of kinetic parameters of Tyr-Mn, natural laccase and other nanozymes

[0065] catalyst Substrate Michaelis constant (mM) <![CDATA[Maximum reaction rate (10 -8 M s -1 )]]> Laccase 2,4-DCP 0.40 5.85 ATP-Cu 2,4-DCP 0.207 3.67 GSH-Cu 2,4-DCP 6.37 3.83 Fe1@CN-20 2,4-DCP 0.078 4.15 <![CDATA[Cu2O v @Ce-TCPP(US)]]> 2,4-DCP 0.2 1.3 Tyr-Mn 2,4-DCP 0.3116 7.426 Laccase 2,4-DCP 0.40 5.85

[0066] 2.3. Feasibility analysis of detection of TCH and INH based on Tyr-Mn dual enzyme activity

[0067] The effects of TCH and INH on the mimetic activity of Tyr-Mn oxidase and laccase were systematically studied. First, the effect of TCH on the mimetic activity of Tyr-Mn oxidase was explored. Fig.14 As shown in Figure 2, the addition of TCH significantly reduced the absorbance of oxTMB at 650 nm, indicating that TCH can inhibit the oxidase mimetic activity of Tyr-Mn. Fig.15 As shown, the addition of INH significantly reduced the absorbance of 4-N-(1,4-benzoquinoneimine)antipyrine, with a strong characteristic absorbance at 510 nm. The results showed that INH could inhibit the laccase mimetic activity of Tyr-Mn.

[0068] 2.4. TCH and INH detection based on Tyr-Mn platform

[0069] (1) Based on the above feasibility study, a colorimetric sensor based on Tyr-Mn oxidase mimic activity was proposed for the detection of TCH. The TCH detection conditions were optimized, with the optimal incubation time of 45 min, the optimal reaction time of 2 min, the optimal pH value of the buffer of 4.0, and the optimal TMB (7.5 mM) dose of 15 μL. Under the optimal detection conditions, the color of the sensing solution changed with the increase in the amount of TCH added. When the TCH concentration increased from 1-20 μM, the absorbance of the system at 652 nm gradually decreased with the increase in TCH concentration ( Fig.16 ). It shows a good linear relationship in this range ( Fig.17 ). The detection limit (LOD) calculated by 3σ / k is 0.303μM. And compared with other applied TCH detection methods, the method provided in this embodiment has a good detection effect. Fig. 20 As shown in the figure, except for TCH which caused a significant decrease in absorbance at 652 nm, other antibiotics did not cause significant changes in absorbance. Therefore, the Tyr-Mn based colorimetric sensor showed good selectivity for TCH, and the stability of the sensor was also an indicator for evaluating the analytical method. Fig. 22 As shown, the relative oxidase-simulating activity of the Tyr-Mn-based colorimetric sensor remained above 80% within 30 days, indicating that the colorimetric sensor prepared in this example has good stability.

[0070] (2) A colorimetric sensor based on Tyr-Mn laccase activity was constructed for sensitive detection of INH. The experimental conditions for INH detection were optimized, with the optimal reaction time being 24 min, the optimal pH value of the buffer solution being 6.0, the optimal 2,4-DCP (20 mM) dose being 30 μL, and the optimal 4-AAP (4 mM) dose being 15 μL. Under the optimal detection conditions, the color of the sensing solution changed with the increase in the amount of INH added. When the INH concentration increased from 0.2-20 μM, the absorbance of the system at 510 nm gradually decreased with the increase in INH concentration ( Fig.18), which shows a good linear relationship in this range ( Fig.19 ). The detection limit (LOD) was calculated by 3σ / k to be 63.13 nM. And compared with other applied INH detection methods, the method provided in this embodiment has a good detection effect. Fig.21 As shown in Figure 2, except for INH which caused a significant decrease in absorbance at 510 nm, other substances commonly found in urine did not cause significant changes in absorbance. Therefore, the Tyr-Mn-based colorimetric sensor showed good selectivity for INH. The stability of the sensor is also an indicator for evaluating the analytical method. Fig.23 As shown, the relative laccase mimetic activity of the Tyr-Mn based colorimetric sensor remained above 90% within 30 days.

[0071] In order to expand the sensor strategy to a wider range of application scenarios and achieve simpler and more portable on-site detection, a smartphone-assisted colorimetric sensing platform based on Tyr-Mn oxidase-active TCH was designed. Fig.24 As shown in a, TCH significantly inhibits the oxidase activity of Tyr-Mn, resulting in a lighter blue color. Based on the precise color recognition function of smartphones, a TCH detection platform for on-site and visual monitoring was constructed. First, a series of samples with different concentrations were immediately captured by the smartphone camera. Then, the captured images were color recognized by the software and converted into readings of red (R), green (G), and blue (B) values. Finally, to ensure the accuracy of the method, a linear relationship for the quantitative detection of TCH was established with the ratio of R / B as the vertical coordinate and the TCH concentration as the horizontal coordinate. R / B and TCH concentration (5-150μM) showed good linearity, with LOD = 1.48μM.

[0072] At the same time, if Fig.24 As shown in b, INH significantly inhibited the laccase activity of Tyr-Mn, resulting in a lighter pink color. Based on the accurate color recognition capability of smartphones, we constructed an INH detection platform that allows on-site and visual monitoring. Based on the same method described above, a series of RGB values ​​were obtained, with G / R as the ordinate and INH concentration as the abscissa, to establish a linear relationship for INH quantitative detection. G / R and INH concentration (0.5-50 μM) showed good linearity, with LOD = 151.4 nM.

[0073] 2.5 Analysis of TCH and INH in real samples

[0074] In order to further study the applicability of the colorimetric sensing method based on Tyr-Mn in the analysis of actual samples, the method provided in this embodiment and high performance liquid chromatography (HPLC) were used to determine the TCH content in fish pond water, Jialing River water and rabbit blood in actual samples. As shown in Table 3, although TCH was not detected in the actual samples, the sensor had a good recovery rate (95.67% to 102.98%) and relative standard deviation (RSD, 0.31% to 3.64%). Compared with the standard HPLC method, its detection ability and stability are not inferior. This shows that the colorimetric sensor based on Tyr-Mn oxidase has potential application value in TCH detection.

[0075] At the same time, in order to further study the applicability of the colorimetric sensing method based on Tyr-Mn laccase in the analysis of actual samples, the method provided in this embodiment and high performance liquid chromatography (HPLC) were used to determine the urine INH content in actual samples. As shown in Table 4, although we did not detect INH in the actual sample, the sensor had a good recovery rate (99.7% to 102.6%) and relative standard deviation (RSD, 0.124% to 2.84%), and its detection ability and stability were not inferior to those of the standard HPLC method.

[0076] This shows that the colorimetric sensor based on Tyr-Mn laccase has potential application value in INH detection. At the same time, in order to further study the applicability of the smartphone-assisted colorimetric sensing method based on Tyr-Mn oxidase and laccase in the analysis of actual samples, the content of TCH and INH in the actual samples was determined by the method provided in this embodiment and high performance liquid chromatography (HPLC). As shown in Tables 5 and 6, they all have good recoveries and relative standard deviations. Compared with the standard HPLC method, its detection ability and stability are not inferior, indicating that the smartphone-assisted colorimetric sensor based on Tyr-Mn oxidase and laccase has potential application value in the detection of TCH and INH.

[0077] Table 3 Determination results of TCH in real samples based on oxidase activity colorimetric method and comparison with HPLC standard method

[0078]

[0079] Table 4 Determination results of isoniazid in real samples based on laccase activity colorimetric method and comparison with HPLC standard method

[0080]

[0081] Table 5 Determination results of tetracycline hydrochloride in real samples based on smartphone RGB-assisted method and comparison with HPLC standard method

[0082]

[0083] Table 6 Determination results of isoniazid in real samples based on smartphone RGB-assisted method and comparison with HPLC standard method

[0084]

[0085] In summary, this example successfully constructed a TCH and INH colorimetric sensor based on Tyr-Mn dual enzyme mimetic activity. Steady-state kinetics showed that Tyr-Mn had a high affinity for TMB and 2,4-DCP and had good oxidase and laccase enzyme mimetic activity. In addition, the excellent oxidase-like catalytic performance of Tyr-Mn was used to detect the TCH and laccase activities of INH. The sensor has good sensitivity and specificity, a wide linear range and a low detection limit. This opens up new horizons for the development of transition metal bionic nanozymes based on dual enzyme activity in the field of analytical detection.

[0086] The above embodiment is only one of the preferred implementation modes of the present invention and should not be used to limit the protection scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention and have no substantive significance, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention.

Claims

1. A method for synthesizing a Tyr-Mn biomimetic nanozyme with multi-enzyme activity, characterized in that: Mn 2+ The solution is mixed with a mixed solution of NaOH and L-tyrosine, stirred, washed and dried to obtain the Tyr-Mn biomimetic nanozyme.

2. The method for synthesizing the Tyr-Mn biomimetic nanozyme with multi-enzyme activity according to claim 1, characterized in that: Mn 2+ The solution was prepared by dissolving 5 mmol of MnCl2·4H2O in 5 mL of deionized water and ultrasonically treating the solution for 5 min.

3. The method for synthesizing the Tyr-Mn biomimetic nanozyme with multi-enzyme activity according to claim 1 or 2, characterized in that: The preparation method of the mixed solution of NaOH and L-tyrosine is as follows: 5.2 mmol NaOH and 2.5 mmol L-tyrosine are dissolved in 25 mL deionized water.

4. The method for synthesizing the Tyr-Mn biomimetic nanozyme with multi-enzyme activity according to claim 3, characterized in that: Mn 2+ The solution and the mixed solution of NaOH and L-tyrosine were mixed, stirred at 700 rpm for 90 min, then washed three times with deionized water and centrifuged, and dried in a vacuum oven at 60° C. overnight after centrifugation.

5. Use of the Tyr-Mn biomimetic nanozyme synthesized according to any one of claims 1 to 4 in catalyzing oxidase substrates.

6. The use of the Tyr-Mn biomimetic nanozyme according to claim 5, characterized in that: The catalytic conditions were as follows: pH 4.0, temperature 25°C, volume ratio of Tyr-Mn bionic nanozyme to oxidase substrate of 2:5, reaction time 10 min; the concentration of Tyr-Mn bionic nanozyme was 2 mg / mL, and the concentration of oxidase substrate was 7.5 mM.

7. Use of the Tyr-Mn biomimetic nanozyme synthesized according to any one of claims 1 to 4 in catalyzing laccase substrates.

8. The use of the Tyr-Mn biomimetic nanozyme according to claim 7, characterized in that: The catalytic conditions were as follows: pH 6.0, temperature 25°C, volume ratio of Tyr-Mn bionic nanozyme and laccase substrate added was 1:2, and reaction time was 30 min; the concentration of Tyr-Mn bionic nanozyme was 2 mg / mL, and the concentration of laccase substrate was 20 mM.

9. Use of the Tyr-Mn biomimetic nanozyme synthesized according to any one of claims 1 to 4 in detecting TCH and INH.

10. The use of the Tyr-Mn biomimetic nanozyme according to claim 9, characterized in that: A colorimetric sensor based on Tyr-Mn biomimetic nanozyme was constructed and combined with the precise color recognition function of a smartphone to establish a smartphone-assisted colorimetric sensing platform for on-site visual detection of TCH and INH.

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