A zirconium-based metal-organic framework nanoscale enzyme and a preparation method and application thereof

Abstract

The application discloses a zirconium-based metal organic framework nano-enzyme as well as a preparation method and application thereof. The nano-enzyme takes Zr4+ as a metal node and takes ETTC as an organic ligand, is synthesized through an acetic acid-assisted hydrothermal reaction, has excellent phosphatase catalytic activity and stable optical performance. Based on a specific competitive inhibition mechanism of ATP hydrolysis product Pi on the phosphatase activity of the nano-enzyme, a phenol-4-AP color reaction is coupled, and colorimetric, fluorescent and electrochemiluminescence three-mode signal outputs are realized synchronously. The application constructs a multi-mode detection system through a single nano-enzyme material, three kinds of signals are verified with each other, the sensitivity, accuracy and anti-interference ability of detection are significantly improved, the detection limit is as low as 0.24 nM, and the application is suitable for quantitative detection of ATP in complex biological samples, and has important application value in the fields of disease diagnosis and biomedical analysis.

Description

Technical Field

[0001] This invention relates to a zirconium-based metal-organic framework nanozyme (Zr-ETTC-Ac) synthesized with acetic acid assistance, its preparation method and application, as well as a three-mode detection application of adenosine triphosphate (ATP) based on this nanozyme, which is suitable for highly sensitive and specific quantitative detection of ATP in biological samples and can be applied to fields such as early disease diagnosis and biomedical analysis. It belongs to the fields of fluorescent nanomaterials and biosensing detection technology. Background Technology

[0002] Adenosine triphosphate (ATP) is a core energy molecule in cellular metabolism. Abnormal ATP concentrations are closely related to the occurrence and development of various diseases such as malignant tumors, cardiovascular diseases, and Parkinson's disease. Establishing efficient, sensitive, and reliable ATP detection methods is of great significance for disease diagnosis and monitoring.

[0003] Nanozymes, as artificial mimics of natural enzymes, possess advantages such as simple synthesis, low cost, high stability, and functionalization potential, leading to their widespread application in biosensing. However, among existing nanozymes, those with hydrolytic activity are particularly scarce, especially those capable of efficiently mimicking phosphatase function. Their quantity and catalytic performance fall far short of practical application requirements, limiting the development of related biosensing technologies.

[0004] Metal-organic frameworks (MOFs) have become ideal candidate materials for mimicking hydrolases due to their tunable porosity, adjustable functionality, and stable structural properties. Zirconium-based MOFs exhibit unique advantages in constructing phosphatase mimics due to the strong Lewis acidity of zirconium ions. However, the phosphatase activity of traditional zirconium-based MOFs still needs improvement, and most sensing platforms employ single-mode detection, making them susceptible to interference from testing conditions and sample background effects, resulting in insufficient detection accuracy and anti-interference capabilities. Therefore, developing zirconium-based MOF nanozymes with high phosphatase activity and constructing multi-mode detection platforms is crucial for solving the problem of efficient ATP detection. Summary of the Invention

[0005] Objectives of the invention: One objective of the present invention is to provide a zirconium-based metal-organic framework nanozyme (Zr-ETTC-Ac) synthesized with acetic acid assistance. Another objective of the present invention is to provide a method for preparing the zirconium-based metal-organic framework nanozyme. A final objective of the present invention is to provide the application of the zirconium-based metal-organic framework nanozyme in three-mode ATP detection.

[0006] Technical solution: The present invention provides a zirconium-based metal-organic framework nanozyme (Zr-ETTC-Ac), wherein the zirconium-based metal-organic framework nanozyme uses Zr... 4⁺ is a metal node and tetra[4-(4'-carboxyphenyl)phenyl]ethylene (ETTC) is an organic ligand, which is synthesized via a hydrothermal reaction assisted by acetic acid and DMF.

[0007] Furthermore, the zirconium-based metal-organic framework nanozyme exhibits stable fluorescence emission at 508 nm under an excitation wavelength of 370 nm, and displays maximum electrochemiluminescence (ECL) emission intensity at 526 nm. Moreover, its optical properties are stable within the range of pH 5.0-9.0 and temperature 25-65℃.

[0008] The method for preparing the acetic acid-assisted zirconium-based metal-organic framework nanozyme of the present invention includes the following steps:

[0009] ZrCl4 and ETTC were dissolved in DMF, HAc was added, and the mixture was subjected to a hydrothermal reaction. The mixture was then washed and dried.

[0010] Furthermore, the mass-to-volume ratio of ZrCl4, ETTC, HAc, and DMF is 30:15:0.2:(4-8), the hydrothermal reaction temperature is 100-150℃, and the hydrothermal reaction time is more than 24 hours.

[0011] The application of the zirconium-based metal-organic framework nanozyme described in this invention in the quantitative detection of ATP.

[0012] A three-mode detection method for quantitative detection of ATP using zirconium-based metal-organic framework nanozymes as described in this invention includes the following steps:

[0013] (1) Construction of reaction system: ATP standard solution of different concentrations, zirconium-based metal-organic framework nanozyme dispersion, disodium phenyl phosphate (PPDS) solution, and Tris-HCl buffer solution were mixed and incubated;

[0014] (2) Add potassium ferricyanide solution and 4-aminoantipyrine (4-AP) solution, vortex, and react in the dark;

[0015] (3) Perform colorimetric, fluorescence and / or electrochemiluminescence detection in three modes respectively, and establish standard curves for absorbance value and ATP concentration, fluorescence intensity value and ATP concentration and / or ECL intensity value and ATP concentration respectively;

[0016] (4) Replace the ATP standard solution with the ATP to be tested according to steps (1)-(2), measure the absorbance value, fluorescence intensity value and / or ECL intensity value of the ATP to be tested, and use the standard curve of step (3) to realize the quantitative detection of the ATP to be tested.

[0017] In step (1), the concentration of the ATP standard solution is 1-50000 nM. When performing UV analysis to measure absorbance, a 200-50000 nM ATP standard solution is used. When performing fluorescence intensity testing, a 2-1000 nM ATP standard solution is used. When performing ECL intensity testing, a 1-1000 nM MATP standard solution is used. The concentration of the zirconium-based metal-organic framework nanozyme dispersion is approximately 1 mg / mL. The volume ratio of the ATP standard solution, zirconium-based metal-organic framework nanozyme dispersion, disodium phenyl phosphate solution, and Tris-HCl (20 mM, pH=8.0) buffer solution is 1:2:1:94. The incubation temperature is 32-42℃, and the incubation time is 20-30 min.

[0018] In step (2), the volume ratio of ATP standard solution, potassium cyanide solution (concentration of 0.5 mM), and 4-aminoantipyrine (4-AP) solution (concentration of 1 mM) is 1:1:1. The temperature of the reaction in the dark is about 37°C, and the reaction time in the dark is 20-30 min.

[0019] In step (3), during the three-mode signal detection:

[0020] Colorimetric detection: The absorbance of the solution after the reaction was measured at 506 nm using a UV-Vis spectrophotometer. The absorbance value was negatively correlated with the ATP concentration.

[0021] Fluorescence detection: The fluorescence intensity at 508 nm of the solution after reaction was recorded using a fluorescence spectrophotometer with 370 nm as the excitation wavelength. The fluorescence intensity value was positively correlated with the ATP concentration.

[0022] Electrochemiluminescence detection: The reaction solution was drop-coated onto the surface of a pretreated glassy carbon electrode. The electrode was then placed in a PBS buffer solution (pH 7.5) containing triethylamine (TEA). An electrochemical workstation coupled with a photomultiplier tube was used to record the ECL intensity-time curve within a specific voltage range. The ECL intensity value was positively correlated with the ATP concentration. The specific voltage for electrochemiluminescence detection was 300V.

[0023] To overcome the limitations of traditional single-mode sensing methods that rely on colorimetric or fluorescence signals and are easily affected by testing conditions, equipment performance, or sample background effects, leading to false positives or false negatives, and to address issues with high-sensitivity ATP detection, this invention utilizes a hydrothermal synthesis method to prepare a zirconium-based metal-organic framework nanozyme (Zr-ETTC-Ac) with phosphatase activity and fluorescence properties. The Zr-ETTC-Ac nanozyme consists of uniformly sized cubic monodisperse particles with a uniform distribution of C, O, and Zr elements, exhibiting a topological structure isomorphic to PCN-94. It specifically catalyzes the hydrolysis of disodium phthalate phosphate (PPDS) to phenol. Zr-ETTC-Ac possesses phosphatase activity and exhibits strong yellow fluorescence at 508 nm. Based on its catalytic properties, a multimode biosensing platform using disodium phthalate phosphate (PPDS) as a substrate was constructed. Specifically, the sensor uses phenylenediamine disodium phosphate (PPDS) as a substrate. Under the catalysis of Zr-ETTC-Ac, the phosphorus-oxygen bond of PPDS breaks to generate phenol. Phenol is then oxidized by potassium ferricyanide under alkaline conditions to an active intermediate, which in turn complexes with the chromogenic agent 4-aminoantipyrine (4-AP) to form a red quinone imine with a characteristic absorption peak at 506 nm, enabling colorimetric detection. Simultaneously, this red product effectively quenches the fluorescence and electrochemiluminescence signals of Zr-ETTC-Ac itself through the fluorescence internal filtration effect (IFE), establishing two supplementary detection modes: fluorescence and ECL. Subsequent studies revealed that the hydrolysis product of adenosine triphosphate (ATP) (i.e., inorganic phosphate, Pi) competitively binds to the active site of the nanozyme and may induce conformational changes, thereby effectively inhibiting its phosphatase activity, ultimately leading to a decrease in the colorimetric signal and an increase in the fluorescence and electrochemiluminescence signals. Therefore, this invention achieves a three-mode sensing based on a single nanozyme material, namely colorimetric-fluorescence-electrochemiluminescence, significantly improving the reliability and applicability of detection and providing a novel multifunctional platform for enzyme activity analysis and biosensing. This invention's three-mode detection method for adenosine triphosphate (ATP) based on the aforementioned nanozyme utilizes the specific competitive inhibition mechanism of inorganic phosphate (Pi), an ATP hydrolysis product, on Zr-ETTC-Ac phosphatase activity, coupled with a phenol-4-AP colorimetric reaction. Quantitative detection of ATP is achieved by regulating the output of three signals: colorimetric, fluorescence, and electrochemiluminescence.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0025] This invention utilizes a hydrothermal synthesis method, with the regulatory effect of acetic acid effectively optimizing the nucleation and growth process of MOFs, inducing the formation of more unsaturated coordination sites and structural defects, significantly enhancing the phosphatase catalytic activity of Zr-ETTC-Ac, exhibiting good substrate affinity and catalytic efficiency. A single nanozyme material and a single reaction system are constructed to simultaneously achieve three detection modes: colorimetric, fluorescence, and electrochemiluminescence. The colorimetric mode has a detection limit of 61.4 nM and a linear range of 200-50000 nM; the fluorescence mode has a detection limit of 0.57 nM and a linear range of 2-1000 nM; and the ECL mode has a detection limit of 0.24 nM and a linear range of 1-10000 nM. This method is effective for Na... + K + Ca 2+ Mg 2+ Cl - SO4 2- HCO3 - Common biomolecules and interfering substances such as maltose, glucose, glycine, arginine, and lysine show no significant response, demonstrating high specificity. The signals from the three modes mutually validate each other, effectively avoiding false positive / false negative results from single-mode detection, and significantly improving the accuracy, reliability, and anti-interference ability of ATP detection. Attached Figure Description

[0026] Figure 1 The images shown are scanning electron microscope (SEM) and transmission electron microscope (TEM) images of Zr-ETTC-Ac from Example 1, where A is the SEM image and B is the TEM image.

[0027] Transmission electron microscope image;

[0028] Figure 2 X-ray photoelectron spectrum of Zr-ETTC-Ac in Example 1;

[0029] Figure 3 The fluorescence excitation and emission spectra of Zr-ETTC-Ac in Example 1 are shown below.

[0030] Figure 4 This is a feasibility verification diagram of the phosphatase-like activity of Zr-ETTC-Ac in Example 1; where A is the UV spectrum of different reaction systems, B is the FL spectrum of different reaction systems, and C is the ECL intensity diagram of different reaction systems.

[0031] Figure 5The graphs show the UV, FL, and ECL intensities of the solutions catalyzed by different concentrations of PPDS (0.1-200 μM) in Example 1, as well as the linear relationships between PPDS concentration and relative absorbance ((A-A0) / A0), relative fluorescence intensity ((F-F0) / F0), and relative electrochemiluminescence intensity ((I-I0) / I0). Wherein, A represents the UV spectrum, B represents the FL spectrum, C represents the ECL intensity, D represents the linear relationship between different PPDS concentrations and relative absorbance ((A-A0) / A0), E represents the linear relationship between different PPDS concentrations and relative fluorescence intensity ((F-F0) / F0), and F represents the linear relationship between different PPDS concentrations and relative electrochemiluminescence intensity ((I-I0) / I0).

[0032] Figure 6 The images show SEM images of different phosphatases in Example 2. In the images, a, b, and c are Zr-ETTC-Ac prepared with DMF contents of 4 mL, 6 mL, and 8 mL (Example 1), respectively; d and e are Zr-ETTC-Ac-H2O prepared by adding 0.05 mL and 0.2 mL of water, respectively, in the synthesis step of c.

[0033] Figure 7 The diagram shows a comparison of the catalytic activity of nanomaterials under different synthesis conditions in Example 2. In the diagram, a, b, and c are Zr-ETTC-Ac prepared with DMF contents of 4 mL, 6 mL, and 8 mL (Example 1), respectively; d and e are Zr-ETTC-Ac-H2O prepared by adding 0.05 mL and 0.2 mL of water, respectively, in the synthesis step c.

[0034] Figure 8 This is a schematic diagram of the principle of the quantitative detection method for ATP of the present invention;

[0035] Figure 9 The following are performance characterization diagrams for the three-mode ATP detection in Example 2: A is the UV spectrum of ATP at different concentrations (200-50000nM); B is the linear relationship between relative absorbance ((A-A0) / A0) and the logarithm of ATP concentration; C is the FL spectrum of ATP at different concentrations (2-1000nM); D is the linear relationship between relative fluorescence intensity ((F-F0) / F0) and the logarithm of ATP concentration; E is the ECL intensity of ATP at different concentrations (1-1000nM); and F is the linear relationship between relative ECL intensity ((I-I0) / I0) and the logarithm of ATP concentration.

[0036] Figure 10 This is a selective experimental diagram of ATP detection in Example 2. Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0038] Example 1

[0039] (1) Preparation of Zr-ETTC-Ac nanozyme

[0040] 30 mg of anhydrous ZrCl4 and 15 mg of tetrakis[4-(4'-carboxyphenyl)phenyl]ethylene (ETTC) were dissolved in 8 mL of DMF in a 10 mL vial. After sonication for 5 min, 0.2 mL of HAC was added to the mixture, and the mixture was then reacted in an oven at 120 °C for 24 hours. Finally, the white precipitate was collected by centrifugation, washed repeatedly with water and ethanol, and the resulting Zr-ETTC-Ac was dried at room temperature for later use.

[0041] Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses were performed on the unspecified Zr-ETTC-Ac in this embodiment, and the results are as follows: Figure 1 As shown. By Figure 1 It can be seen that Zr-ETTC-Ac has a uniform cubic structure and good dispersibility.

[0042] X-ray photoelectron spectroscopy analysis was performed on the unspecified Zr-ETTC-Ac in this embodiment, and the results are as follows: Figure 2 As shown; Figure 2 XPS characterization results confirmed the successful preparation of Zr-ETTC-Ac. The full spectrum showed characteristic signal peaks of O 1s, C 1s, and Zr 3d at 531 eV, 285 eV, and 183 eV, respectively, confirming that only three intrinsic elements—Zr, O, and C—exist on the material surface, with no obvious impurities remaining. This composition is completely consistent with the theoretical elemental composition of Zr-MOF, proving the successful preparation of the material.

[0043] Fluorescence excitation and emission spectroscopy analysis was performed on the unspecified Zr-ETTC-Ac in this embodiment, and the results are as follows: Figure 3 As shown. By Figure 3 It is known that the optimal excitation wavelength of Zr-ETTC-Ac is 370 nm and the optimal emission wavelength is 508 nm.

[0044] (2) Phosphatase activity of Zr-ETTC-Ac nanozyme

[0045] like Figure 4 As shown, to verify the phosphatase-mimicking activity of the Zr-ETTC-Ac zirconium-based metal-organic framework nanozyme prepared in this invention, multiple control reaction systems were constructed based on the multi-mode sensing principle of this invention. A represents the UV spectrum of different reaction systems, B represents the FL spectrum of different reaction systems, and C represents the ECL intensity map of different reaction systems. The colorimetric response of each system was characterized by UV-Vis absorption spectroscopy. The experimental principle and system construction are as follows:

[0046] Core sensing principle: The Zr-ETTC-Ac nanozyme possesses phosphatase-like activity, specifically catalyzing the hydrolysis of the phosphate ester substrate disodium phenyl phosphate (PPDS), breaking the phosphorus-oxygen bond to generate phenol. The generated phenol is oxidized in an alkaline Tris-HCl buffer system by potassium ferricyanide (K3[Fe(CN)6]) to an intermediate, which then reacts with the chromogenic agent 4-aminoantipyrine (4-AP) to generate a red quinone imine with a characteristic absorption peak at 506 nm. This colorimetric reaction directly verifies the phosphatase catalytic activity of the nanozyme. Simultaneously, this experiment included a 2,4-dichlorophenol (2,4-DP) positive control system (using a synthetically produced phenol directly in the colorimetric reaction) and an acetic acid (CH3COOH) degradation control system (decomposing the red quinone imine product) to fully verify the reaction mechanism and specificity of the sensing system.

[0047] Different reaction systems were constructed: all systems used 1.5 mL centrifuge tubes as reaction vessels. The concentrations of the different solutions added to the systems were uniform: 20 μL 1 mg / mL Zr-ETTC-Ac dispersion, 10 μL PPDS solution (200 μM), 940 μL Tris-HCl buffer solution (20 mM, pH=8.0), 10 μL K3[Fe(CN)6] (0.5 mM), 10 μL 4-AP (1 mM), 10 μL 2,4-DP (1 mM), and 10 μL CH3COOH. The reaction conditions were uniform: constant temperature incubation at 37℃, reaction in the dark, vortex mixing, and reaction time set at 20-30 min.

[0048] Experimental Group 1: Zr-ETTC-Ac+4-AP+PPDS+K3[Fe(CN)6] (core catalytic system). System composition: 20 μL 1mg / mL Zr-ETTC-Ac dispersion + 10 μL PPDS solution + 940 μL 20 mM pH=8.0 Tris-HCl buffer solution. Specific operation steps: ① After vortex mixing, incubate at 37℃ for 30 min to allow Zr-ETTC-Ac to fully catalyze the hydrolysis of PPDS to generate phenol; ② Add equal volumes (10 μL each) of 0.5 mM K3[Fe(CN)6] solution and 1 mM 4-AP solution to the system, and vortex mix; ③ React at 37℃ in the dark for 20 min to ensure the complete colorimetric reaction of phenol-potassium ferricyanide-4-AP, and the solution turns a distinct red; ④ Measure the UV-Vis absorption spectrum at 506 nm. This system is a complete catalytic colorimetric system, exhibiting a significant characteristic absorption peak at 506 nm, directly demonstrating that Zr-ETTC-Ac has highly efficient phosphatase activity, which can catalyze the hydrolysis of PPDS and trigger subsequent colorimetric reactions.

[0049] Experimental Group 2: Zr-ETTC-Ac+4-AP+2,4-DP+ K3[Fe(CN)6] (phenolic positive control system). 2,4-DP is a synthetic phenolic substance that can directly participate in the colorimetric reaction without catalytic hydrolysis. This system serves as a positive control, verifying the feasibility of the "phenol-potassium ferricyanide-4-AP" colorimetric reaction. It also demonstrates that Zr-ETTC-Ac itself does not interfere with the colorimetric process, but only provides the phenolic substrate by catalyzing the hydrolysis of PPDS.

[0050] Control group 1: Zr-ETTC-Ac+4-AP+PPDS+ K3[Fe(CN)6]]+ CH3COOH (product destroys control system). Acetic acid can destroy the structure of red quinone imine products. The absorption signal of this system is significantly reduced, which verifies that the absorption peak at 506 nm does indeed come from the quinone imine product generated by the colorimetric reaction, rather than other side reactions. At the same time, it clarifies the alkaline reaction conditions required for the system.

[0051] Control group 2: Zr-ETTC-Ac, 4-AP, 2,4-DP, or PPDS alone showed no obvious characteristic absorption peaks, proving that they do not trigger a colorimetric reaction when present alone, thus verifying the necessity of nanozyme catalysis. This experiment fully verified the phosphatase catalytic activity of Zr-ETTC-Ac, laying a core experimental foundation for the subsequent construction of a colorimetric-fluorescence-electrochemiluminescence three-mode ATP sensing platform based on this nanozyme.

[0052] The signal response mechanism of the system was further verified using fluorescence spectroscopy (FL) and electrochemiluminescence spectroscopy (ECL), clarifying the regulatory role of the internal filtration effect (IFE) of the red quinone imine product on the intrinsic signal of Zr-ETTC-Ac. Using a fluorescence spectrometer, equal volumes of the reaction solution were placed in fluorescent cuvettes, and the fluorescence spectra of the solutions were recorded at an excitation wavelength of 370 nm. Figure 4 As can be seen in Figure B, the fluorescence of Zr-ETTC-Ac is significantly quenched in the presence of Zr-ETTC-Ac, PPDS, and 4-AP, indicating that the fluorescence signal can be used as a second output signal in the enzyme catalytic reaction.

[0053] Using an electrochemical workstation and an ECL analyzer, 5 μL of the reaction solution was drop-coated onto the surface of a pretreated glassy carbon electrode (the glassy carbon electrode was first polished to a mirror finish on chamois leather with 0.3 μm and 0.05 μm Al2O3 polishing powder, rinsed with ultrapure water, and then sonicated in ultrapure water and ethanol for 30 s each; subsequently, cyclic voltammetry was performed in 0.5 mol / L H2SO4 within a potential range of −1.0 to +1.0 V until the curve stabilized, and then rinsed with ultrapure water for later use). The ECL intensity was measured in a Tris-HCl buffer solution (20 mM, pH=8.0) containing 40 mM co-reactant TEA. Similarly, from... Figure 4 As shown in C, when Zr-ETTC-Ac, PPDS, and 4-AP coexist, the generated red quinone imine quenches the electrochemiluminescence signal of Zr-ETTC-Ac, indicating that the electrochemiluminescence signal can be used as the third output signal in the enzyme catalytic reaction. PPDS and 4-AP do not have ultraviolet peaks, and the presence of PPDS and 4-AP alone has no effect on the fluorescence and ECL of Zr-ETTC-Ac. (3) Following the experimental procedure in step (2), different concentrations of PPDS (0.1-200 μM) were used, and the results are as follows. Figure 5 As shown. Figure 5 The graphs show the UV, FL, and ECL intensities of solutions catalyzed with different concentrations of PPDS (0.1-200 μM) in Example 1, as well as the linear relationships between PPDS concentration and relative absorbance ((A-A0) / A0) (A: absorbance with PPDS, A0: absorbance without PPDS), relative fluorescence intensity ((F-F0) / F0) (F: fluorescence intensity with PPDS, F0: fluorescence intensity without PPDS), and relative electrochemiluminescence intensity ((I-I0) / I0) (I: ECL intensity with PPDS, I0: ECL intensity without PPDS). Figure 5 As shown in Figure A, the absorbance at 506 nm gradually increases with increasing PPDS concentration, and the color gradient of the reaction solution from light red to dark red is clearly visible in the inset. Meanwhile, the fluorescence intensity at 508 nm shows a decreasing trend. Figure 5 (See Figure B). The blue fluorescence of the corresponding sample under ultraviolet light also gradually weakened, a phenomenon visually verified by the illustration. Correspondingly, the electrochemiluminescence signal of the sample also showed a decreasing trend. Figure 5 (C). Furthermore, the PPDS response based on the triple outputs of colorimetry, fluorescence, and electrochemiluminescence showed good linearity in the concentration range of 0.1–200 μM (C). Figure 4 The linear equations are y = 0.089x - 0.17, RF. 2 =0.996; y=-0.022-0.0044x, R 2=0.997; y=-0.023-0.0036x, R 2 =0.995.

[0054] Example 2: Effect of synthesis parameters on Zr-ETTC-Ac phosphatase activity

[0055] (1) Effect of DMF dosage on Zr-ETTC-Ac phosphatase activity

[0056] The preparation process of Zr-ETTC-Ac is the same as in Example 1, except that 4 mL and 6 mL of DMF were used to obtain two groups of Zr-ETTC-Ac nanozyme powders.

[0057] (2) Effect of water on Zr-ETTC-Ac phosphatase activity

[0058] The preparation process of Zr-ETTC-Ac is the same as in Example 1, except that 4 mL of DMF was added during the experiment, followed by ultrasonic treatment, and then 0.05 mL and 0.2 mL of water were added respectively. Acetic acid was then added to obtain two groups of Zr-ETTC-Ac-H2O phosphatases.

[0059] The four groups of phosphatases prepared in this embodiment were subjected to scanning electrophoresis analysis, and the comparison results with Zr-ETTC-Ac prepared in Example 1 are as follows: Figure 6 As shown. From Figure 6 It can be seen that as the amount of DMF increases, the particle size gradually decreases and the shape becomes more uniform, indicating that the amount of DMF affects the growth state and morphological regularity of the particles. Figure 6 The effects of water introduction and dosage were investigated in the A and D cases. Water contents were 0.05 mL and 0.2 mL, while DMF and acetic acid were 4 mL and 0.2 mL, respectively. The particle morphology changed from relatively regular lumps to a more prismatic shape. With the introduction of water and a decrease in DMF, the particles exhibited a prismatic morphology and increased in size. As the water dosage further increased, they became elliptical with a slightly larger size. This indicates that the introduction and dosage of water promote the transformation of particles from lumps to prismatic shapes, and the more water used, the longer the prismatic shape becomes, eventually transforming into an ellipsoidal shape. The above analysis shows that without water, using 0.2 mL HAC and 8 mL DMF as regulators, and ZrCl4 and ETTC at masses of 30 mg and 15 mg, respectively, monodisperse nanozymes with uniform size, regular morphology, and optimal AIE and catalytic performance are produced.

[0060] (3) The catalytic activity of the above material was verified according to step (2) in Example 1, wherein the substrate consisted of PPDS at concentrations of 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1 mM, and then the kinetic parameter K was determined.m and V max Calculated using the Michaelis-Menten model. Based on the fit to the Michaelis-Menten equation, V... max They are 5.7532×10 -8 M·s -1 2.9924×10 -8 M·s -1 12.1371×10 -8 M·s -1 1.9555 ×10 -8 M·s -1 3.7043×10 -8 M·s -1 K m The concentrations were 0.4464 mM, 0.3159 mM, 0.2862 mM, 0.3098 mM, and 0.4170 mM (ae), respectively. The results are as follows... Figure 7 As shown. Figure 7 Kinetic results showed that the Zr-ETTC-Ac(c) synthesized in Example 1 exhibited excellent substrate affinity and reaction rate, and could be used as a phosphatase-like material with excellent enzyme kinetics. Therefore, a third ratio was selected to prepare Zr-ETTC-Ac nanozyme for subsequent experiments.

[0061] Example 3: Tri-mode ATP detection based on Zr-ETTC-Ac

[0062] The experimental procedure was the same as step (2) in Example 1. Different concentrations of ATP standard solution, 20 μL of 1 mg / mL Zr-ETTC-Ac dispersion prepared in Example 1, 10 μL of PPDS solution, and 940 μL of Tris-HCl buffer solution (20 mM, pH=8.0) were added sequentially to a 1.5 mL centrifuge tube. After vortexing, the mixture was incubated at 37 °C for 30 min. Subsequently, potassium ferricyanide solution (0.5 mM) and 4-aminoantipyrine solution (1 mM) were added to the system. The mixture was stirred again and reacted at 37 °C in the dark for 15 minutes to ensure complete phenol color development, resulting in a red solution. After the reaction, the absorbance of the solution was measured at 506 nm using a UV-Vis spectrophotometer. Using a fluorescence spectrometer, the same volume of the reaction solution was placed in a fluorescence cuvette, and the fluorescence spectrum of the solution was recorded at an excitation wavelength of 370 nm. Using an electrochemical workstation and ECL analyzer, 5 μL of the reaction solution was dropped onto the pretreated glassy carbon electrode surface. The ECL intensity was measured in Tris-HCl buffer (20 mM, pH=8.0) containing 40 mM co-reactant TEA. The principle of ATP detection is as follows: Figure 8As shown, the test results are as follows Figure 9 As shown.

[0063] Figure 8 This is a schematic diagram of the quantitative ATP detection method of this invention. The synthesized nanozyme (Zr-ETTC-Ac) has phosphatase activity and can catalyze the hydrolysis of phosphate ester substrates. Using disodium phenyl phosphate (PPDS) as a model substrate, Zr-ETTC-Ac first hydrolyzes the PO bond of PPDS to generate phenol. Subsequently, under alkaline conditions, phenol is oxidized by potassium ferricyanide to an active intermediate, which then undergoes a specific coupling reaction with the chromogenic reagent 4-aminoantipyrine (4-AP) to generate a red quinone imine with a characteristic absorption peak at 506 nm, thereby achieving colorimetric detection. Simultaneously, this red product can effectively quench the fluorescence and electrochemiluminescence signals of Zr-ETTC-Ac via IFE, thus establishing two detection modes: fluorescence and ECL, thereby constructing a three-mode detection method.

[0064] ATP detection is achieved through a delicate competitive inhibition pathway. In the presence of ATP, it is hydrolyzed, releasing a large amount of inorganic phosphate (Pi). This Pi reacts with Zr in MOFs... 4+ The active site possesses extremely high affinity, acting as a potent competitive inhibitor, occupying the catalytic center and severely hindering the hydrolysis of PPDS. As a result, the yield of phenol decreases sharply, leading to a reduction in the final yield of the quinone imine dye. This change is translated into three distinct signal outputs: a decrease in absorbance at 506 nm (colorimetric signal off), the recovery of MOF fluorescence (fluorescence signal on), and an increase in the intensity of the MOF's ECL (ECL signal on). Therefore, the concentration of ATP can be quantitatively correlated with the weakening of the colorimetric signal and the enhancement of the fluorescence and ECL signals, thereby achieving a three-mode detection with built-in cross-validation, significantly improving its reliability and anti-interference capability.

[0065] Figure 9 The image shows the performance characterization of ATP detection in the three modes in Example 2; where A represents the UV spectra of different ATP concentrations (200-50000 nM); B represents the linear relationship between relative absorbance ((A-A0) / A0) and the logarithm of ATP concentration, with the linear equation being y=0.62-0.27x, R 2 =0.997; C represents the FL spectra of ATP at different concentrations (2-1000 nM); D represents the linear relationship between fluorescence intensity ((F-F0) / F0) and the logarithm of ATP concentration, with the linear equation being y = -0.35 + 0.87x, R 2 =0.994; E represents the ECL intensity of different ATP concentrations (1-1000 nM); F represents the linear relationship between relative ECL intensity ((I-I0) / I0) and the logarithm of ATP concentration, with the linear equation being y=0.04+0.39x, R2 =0.996. The detection limits were 61.4 nM, 0.57 nM, and 0.24 nM, respectively. H is a summary graph of the linear range and detection limits for ATP detection based on colorimetric, fluorescence, and ECL detection modes (red dots indicate detection limits).

[0066] Example 3: Selectivity Experiment for ATP Detection

[0067] Choose Na + K + Ca 2+ Mg 2+ Cl - SO4 2- HCO3 - Maltose, glucose, glycine, arginine, and lysine were studied as potential interfering agents. The experimental procedure was the same as in Example 2. The experiment was conducted in a system containing 500 nM ATP, and each of the above-mentioned interfering agents was added at a concentration of 1000 nM. The results are as follows. Figure 10 As shown. Figure 10 As shown, apart from ATP, the effects of other test substances on the changes in absorbance, fluorescence intensity, and electrochemiluminescence intensity of the system are negligible, indicating that the established method has high selectivity.

Claims

1. A zirconium-based metal-organic framework nanozyme, characterized in that, The zirconium-based metal-organic framework nanozyme uses Zr 4 ⁺ is a metal node and tetrakis[4-(4'-carboxyphenyl)phenyl]ethylene is an organic ligand, which is synthesized via a hydrothermal reaction assisted by acetic acid and DMF.

2. The acetic acid-assisted zirconium-based metal-organic framework nanozyme according to claim 1, characterized in that, The zirconium-based metal-organic framework nanozyme exhibits stable fluorescence emission at 508 nm under an excitation wavelength of 370 nm.

3. The method for preparing the acetic acid-assisted zirconium-based metal-organic framework nanozyme according to claim 1 or 2, characterized in that, Includes the following steps: ZrCl4 and ETTC were dissolved in DMF, HAc was added, and the mixture was subjected to a hydrothermal reaction. The mixture was then washed and dried.

4. The preparation method according to claim 3, characterized in that, The mass-to-volume ratio of ZrCl4, ETTC, HAc, and DMF is 30:15:0.2:(4-8). The hydrothermal reaction temperature is 100-150℃, and the hydrothermal reaction time is more than 24 hours.

5. The application of the zirconium-based metal-organic framework nanozyme according to claim 1 or 2 in the quantitative detection of ATP.

6. A three-mode detection method for quantitative detection of ATP using the zirconium-based metal-organic framework nanozyme according to claim 1 or 2, characterized in that, Includes the following steps: (1) Construction of reaction system: ATP standard solution of different concentrations, zirconium-based metal-organic framework nanozyme dispersion, disodium phenyl phosphate solution, and Tris-HCl buffer solution were mixed and incubated; (2) Add potassium ferricyanide solution and 4-aminoantipyrine solution, vortex mix, and react in the dark; (3) Perform colorimetric, fluorescence and / or electrochemiluminescence detection in three modes respectively, and establish standard curves for absorbance value and ATP concentration, fluorescence intensity value and ATP concentration and / or ECL intensity value and ATP concentration respectively; (4) Replace the ATP standard solution with the ATP to be tested according to steps (1)-(2), measure the absorbance value, fluorescence intensity value and / or ECL intensity value of the ATP to be tested, and use the standard curve of step (3) to realize the quantitative detection of the ATP to be tested.

7. The detection method according to claim 6, characterized in that, In step (1), the concentration of the ATP standard solution is 1-50000 nM, the concentration of the zirconium-based metal-organic framework nanozyme dispersion is 1 mg / mL, and the volume ratio of the ATP standard solution, zirconium-based metal-organic framework nanozyme dispersion, disodium phenyl phosphate solution, and Tris-HCl buffer solution is 1:2:1:

94. The incubation temperature is 32-42℃, and the incubation time is set to 20-30 min.

8. The detection method according to claim 6, characterized in that, In step (2), the volume ratio of ATP standard solution, potassium cyanide solution and 4-aminoantipyrine solution is 1:1:1, the temperature of the reaction in the dark is 32-42℃, and the reaction time in the dark is 20-30min.

9. The detection method according to claim 6, characterized in that, In step (3), during the three-mode signal detection: Colorimetric detection: The absorbance of the solution after the reaction was measured at 506 nm using a UV-Vis spectrophotometer. The absorbance value was negatively correlated with the ATP concentration. Fluorescence detection: The fluorescence intensity at 508 nm of the solution after reaction was recorded using a fluorescence spectrophotometer with 370 nm as the excitation wavelength. The fluorescence intensity value was positively correlated with the ATP concentration. Electrochemiluminescence detection: The reaction solution was drop-coated onto the surface of a pretreated glassy carbon electrode. The electrode was placed in a PBS buffer containing triethylamine. An electrochemical workstation coupled with a photomultiplier tube was used to record the ECL intensity-time curve within a specific voltage range. The ECL intensity value was positively correlated with the ATP concentration.

10. The detection method according to claim 9, characterized in that, The specific voltage for electrochemiluminescence detection is 300V.

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