Multi-biomolecule detection MOF (Metal Organic Framework) probe widely applicable to pH (Potential of Hydrogen) as well as preparation method, detection method and application

Abstract

The invention provides a wide pH applicable multi-biomolecule detection MOF probe, a preparation method, a detection method and application, the probe is an MIL-53 (Fe) (at) AA (at) EZ probe, and comprises a metal organic framework MIL-53 (Fe), amino acid and enzyme; the preparation method comprises the following steps: weighing terephthalic acid and ferric chloride hexahydrate, adding into N, N-dimethylformamide, mixing and stirring, pouring the mixed solution into a stainless steel autoclave, heating and reacting, naturally cooling to room temperature, centrifugally separating a product, and washing with N, N-dimethylformamide and absolute ethyl alcohol to obtain the metal organic framework MIL-53 (Fe). The preparation method comprises the following steps: mixing a metal organic framework MIL-53 (Fe) with an amino acid solution, and carrying out ultrasonic treatment and stirring to obtain an MIL-53 (Fe) coated AA solution; the method comprises the following steps: dissolving enzyme in an MIL-53 (Fe) (at) AA solution, uniformly mixing, stirring, centrifugally separating a product, taking precipitate, and drying in vacuum to obtain the MIL-53 (Fe) (at) AA (at) EZ probe. The pH application range is wide, one-step detection can be achieved, and the method has the advantages of being rapid in reaction, high in sensitivity, low in detection limit and easy and convenient to operate.

Description

Technical Field

[0001] This invention belongs to the field of colorimetric sensing and detection technology, specifically relating to a widely pH-applicable MOF probe for detecting multiple biomolecules, its preparation method, detection method, and application. Background Technology

[0002] Abnormal levels of biomolecules such as glucose, cholesterol, and uric acid in the body have been proven to be closely related to the occurrence and development of diseases such as diabetes, cardiovascular disease, and hyperuricemia. Therefore, accurate monitoring of these biomolecules plays a crucial role in the diagnosis and treatment of diseases such as diabetes, cardiovascular disease, and hyperuricemia. Natural enzymes can specifically and efficiently catalyze various biochemical reactions under mild conditions, playing an important role in biochemistry. For example, horseradish peroxidase has good substrate specificity and high catalytic reaction efficiency, and is therefore widely used for blood glucose monitoring or other colorimetric sensing detection of biomolecules. However, natural enzymes have significant drawbacks, such as thermal instability, high cost, and limited sources, which severely limit their widespread application. Due to the similarity or higher catalytic activity and specificity of artificial enzymes, as well as their advantages such as low cost, good stability, tunable structure, and mature synthetic pathways, the research on various artificial enzyme mimics has become a research hotspot in recent years.

[0003] Nanozymes, as a novel type of artificial enzyme, have attracted widespread interest and attention in recent years. Compared with natural enzymes, nanozymes have significant advantages such as low cost, high stability, and convenient production. Furthermore, due to their abundant sites that can bind to various functional groups, they exhibit high catalytic activity and strong signal values, thus being widely used in the colorimetric sensing detection of biomolecules such as glucose, cholesterol, and uric acid. Colorimetric sensing does not require large-scale instruments and equipment, and it is easy to achieve accurate and rapid detection, obtaining intuitive results, which is beneficial for rapid clinical diagnosis.

[0004] Metal-organic frameworks (MOFs) are a novel class of porous crystalline polymers composed of metal nodes and organic linkers, exhibiting nanozyme activity. Due to their tunable structure and function, and their ever-expanding applications, MOF materials have become a research hotspot in the field of sensing. Fe MOFs, as a metal-based MOF, possess inherent characteristics of multiple active sites and high porosity, exhibiting good biocompatibility and high stability in biological environments. However, the catalytic activity of Fe MOFs is limited by factors such as pH, significantly affecting their detection sensitivity and accuracy, thus limiting their application in colorimetric techniques. Studies have found that the catalytic activity of nanozymes is pH-dependent, with optimal reaction pH mostly between 3 and 5. For example, the optimal catalytic pH for α-Fe₂O₃ is 2.5, and for the metal-organic framework MIL-53(Fe) it is 3, while the optimal reaction pH for natural enzymes is neutral. Therefore, the cascade catalytic reactions of nanozymes and natural enzymes show pH mismatch, affecting the detection sensitivity of the sensing system. Therefore, developing enhanced Fe MOFs that overcome pH limitations for colorimetric sensing detection is of great significance. Furthermore, current biomolecule detection technologies are limited to a reaction temperature of 37°C, requiring the addition of GOD enzymes to react with serum, followed by nanozyme reaction and UV measurement. This approach cannot achieve one-step detection, has a long reaction time, and only detects a single type of biological sample, resulting in a narrow detection range and insufficient sensitivity. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a widely pH-applicable MOF probe for detecting multiple biomolecules, along with its preparation, detection, and applications. This probe has a wide pH range, enabling the detection of biomolecules in various biological samples at different pH values. It allows for one-step detection without the need for additional pH adjustments. It exhibits excellent catalytic activity under neutral conditions, demonstrating advantages such as rapid reaction, high sensitivity, low detection limit, and ease of operation. It also possesses good anti-interference capabilities. In practical applications, particularly in the determination of biomolecules such as glucose, cholesterol, uric acid, and lactose, it shows broad application prospects.

[0006] Note that the description of these objectives does not preclude the existence of other objectives. One aspect of the invention does not require achieving all of the above objectives. Objectives other than those described above can be extracted from the description, drawings, and claims.

[0007] The present invention achieves the above-mentioned technical objectives through the following technical means.

[0008] A wide pH-suitable multi-biomolecule detection MOF probe, namely MIL-53(Fe)@AA@EZ probe, comprising metal-organic framework MIL-53(Fe), amino acid (AA) and enzyme (EZ), is obtained by reacting the metal-organic framework MIL-53(Fe), amino acid (AA) and enzyme (EZ) through a solvent method.

[0009] In the above scheme, the mass ratio of the metal-organic framework MIL-53 (Fe), amino acids, and enzymes is 1:(0.5-8.0):(0.5-6.0).

[0010] Preferably, the mass ratio of the metal-organic framework MIL-53 (Fe), amino acids, and enzymes is 1:(2.8-4.6):(2.2-3.5).

[0011] In the above scheme, the enzyme is glucose oxidase, cholesterol oxidase, galactose oxidase or uricase;

[0012] The amino acid in question is an acidic amino acid: glutamic acid or aspartic acid.

[0013] A method for preparing a wide-pH-suitable multi-biomolecule detection MOF probe includes the following steps:

[0014] Step S1, Preparation of MIL-53(Fe)@AA: Weigh terephthalic acid and ferric chloride hexahydrate (FeCl3·6H2O) and add them to N,N-dimethylformamide (DMF). Mix and stir. Pour the mixed solution into a stainless steel autoclave and heat to react. Allow it to cool naturally to room temperature. Centrifuge to separate the product. Wash with N,N-dimethylformamide (DMF) and anhydrous ethanol to obtain the metal-organic framework MIL-53(Fe). Mix the metal-organic framework MIL-53(Fe) with an amino acid (AA) solution, sonicate, and stir to obtain the MIL-53(Fe)@AA solution.

[0015] Step S2, Preparation of MIL-53(Fe)@AA@EZ: Dissolve the enzyme in the MIL-53(Fe)@AA solution obtained in step S1, mix well, stir, centrifuge to separate the product, take the precipitate and vacuum dry to obtain the MIL-53(Fe)@AA@EZ probe.

[0016] A method for detecting biomolecule concentration, utilizing the aforementioned wide-pH-suitable multi-biomolecule detection MOF probe or the MIL-53(Fe)@AA@EZ probe prepared by the aforementioned method, includes the following steps:

[0017] Add the MIL-53(Fe)@AA@EZ probe loaded with the corresponding enzyme to water or buffer solution, then add biological sample (such as blood or saliva) and TMB solution, mix well and react, observe the color change, and measure the ultraviolet absorption peak at 652 nm wavelength. Determine the concentration of biological sample based on the established standard curve.

[0018] In the above scheme, the pH range of the water or buffer solution is 3.0 to 8.4, and the reaction temperature is 20 to 37°C.

[0019] Preferably, the pH range of the water or buffer solution is 4.0 to 7.4.

[0020] Application of a MIL-53(Fe)@AA@EZ probe prepared according to the broad pH applicable multi-biomolecule detection MOF probe or the preparation method described herein in the quantitative detection of biomolecule concentration.

[0021] A detection kit for quantitative detection of biomolecule concentration, comprising the wide pH applicable multi-biomolecule detection MOF probe or the MIL-53(Fe)@AA@EZ probe prepared by the preparation method.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] 1. The MIL-53Fe@AA@EZ nanozyme of the present invention utilizes an EZ enzyme loaded in a MOF material to catalyze the production of H2O2 from corresponding biological samples. Under neutral conditions, MIL-53Fe@AA catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) into a blue chromogenic product using H2O2, thereby enabling colorimetric biosensing. By loading different types of enzymes, detection of corresponding biological samples can be achieved, resulting in a rich variety of detectable substances. It can be used for the detection of biomolecules in various biological samples, such as cholesterol, galactose, and uric acid.

[0024] 2. The MIL-53Fe@AA@EZ nanozyme of the present invention, through amino acid modification, regulates the electron transfer between the nanozyme and the substrate in the microenvironment, significantly improving the reaction sensitivity. For example, the widely pH-suitable multi-biomolecule detection MOF probe of the present invention exhibits a broad linear range (3μM-500μM) and a low detection limit (0.322μM) when used for the detection of glucose in saliva, which is superior to commercially available glucose kits (300μM-1mM), and can meet the needs of glucose detection in saliva.

[0025] 3. The method of this invention modifies the microscopic H near the active metal sites of MOFs through amino acid modification. +The concentration expands the pH range of MOF nanozymes. Compared with MIL-53Fe (optimal catalytic pH is 3-4, with almost no catalytic activity under neutral conditions), the amino acid-modified nanozyme system can efficiently catalyze the TMB reaction under neutral conditions. The reaction conditions are highly compatible with natural enzymes, eliminating the need to adjust the pH in the cascade reaction. It also enables one-step detection, making it simple and easy to use.

[0026] 4. This invention can be used at 20-37℃, and the applicable reaction temperature range for detection is wider.

[0027] Note that the description of these effects does not preclude the existence of other effects. One aspect of the invention does not necessarily have all the aforementioned effects. Effects other than those described above can be readily observed and extracted from the description, drawings, claims, etc. Attached Figure Description

[0028] Figure 1 Zeta potential maps of MIL-53Fe and MIL-53Fe@Asp were obtained in Example 1.

[0029] Figure 2 The scanning electron microscope (SEM) images of MIL-53Fe, MIL-53Fe@Asp, and MIL-53Fe@Asp@GOX prepared in Example 1 are shown below. Figure 2 (a) is a scanning electron microscope (SEM) image of MIL-53Fe. Figure 2 (b) is a scanning electron microscope (SEM) image of MIL-53Fe@Asp. Figure 2 (c) is a scanning electron microscope (SEM) image of MIL-53Fe@Asp@GOX.

[0030] Figure 3 The standard curve of blood glucose concentration versus absorbance was prepared using MIL-53Fe and MIL-53Fe@Asp as shown in Example 1.

[0031] Figure 4 This is a comparison chart of the absorbance of MIL-53Fe and MIL-53Fe@Asp prepared in Example 1 for other sugars and glucose.

[0032] Figure 5 Fourier transform infrared (FT-IR) spectra of MIL-53Fe and MIL-53Fe@Asp obtained in Example 2.

[0033] Figure 6 Example 2 shows a standard curve of blood cholesterol concentration versus absorbance constructed using MIL-53Fe@Asp@COD.

[0034] Figure 7The X-ray diffraction (XRD) patterns of MIL-53Fe and MIL-53Fe@Asp prepared in Example 3 are shown.

[0035] Figure 8 Example 3 shows the standard curve of galactose concentration versus absorbance in blood constructed using MIL-53Fe and MIL-53Fe@Asp.

[0036] Figure 9 This is a standard curve of uric acid concentration versus absorbance in urine constructed using MIL-53Fe@Glu@UAO, as shown in Example 4. Detailed Implementation

[0037] The embodiments of the present invention are described in detail below with reference to the accompanying drawings. These embodiments are intended to explain the present invention and should not be construed as limiting it. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the reagents and materials used in the following embodiments are commercially available unless otherwise specified.

[0038] The preparation method of the widely pH-applicable multi-biomolecule detection MOF probe of the present invention includes the following steps:

[0039] Step S1, Preparation of MIL-53(Fe)@AA: Weigh terephthalic acid and ferric chloride hexahydrate FeCl3·6H2O and add them to N,N-dimethylformamide DMF. Mix and stir. Pour the mixed solution into a stainless steel autoclave and heat to react. Let it cool naturally to room temperature. Centrifuge to separate the product. Wash with N,N-dimethylformamide DMF and anhydrous ethanol to obtain the metal-organic framework MIL-53(Fe). Mix the metal-organic framework MIL-53(Fe) with an amino acid solution, sonicate and stir to obtain the MIL-53(Fe)@AA solution.

[0040] Step S2, Preparation of MIL-53(Fe)@AA@EZ: Dissolve the enzyme in the MIL-53(Fe)@AA solution obtained in step S1, mix well, stir, centrifuge to separate the product, take the precipitate and vacuum dry to obtain the MIL-53(Fe)@AA@EZ probe.

[0041] Example 1:

[0042] A wide pH-suitable multi-biomolecule detection MOF probe, the MIL-53(Fe)@Asp@GOX probe, comprising the metal-organic framework MIL-53(Fe), aspartic acid Asp, and glucose oxidase GOX.

[0043] The preparation method of the MIL-53(Fe)@Asp@GOX probe includes the following steps:

[0044] Dissolve 6 mg of metal-organic framework MIL-53(Fe) and 3 mg of aspartic acid (Asp) in 1 ml of water, sonicate, and stir. Dissolve 3 mg of glucose oxidase (GOX) in the above 1 ml MIL-53(Fe)@Asp solution, stir at room temperature for 1 hour, centrifuge at 15000 rpm / min for 10 minutes, collect the precipitate and dry it under vacuum to obtain MIL-53(Fe)@Asp@GOX.

[0045] A method for detecting biomolecule concentrations using the MIL-53(Fe)@Asp@GOX probe includes the following steps:

[0046] The MIL-53(Fe)@Asp@GOX nanozyme was prepared into a 3 mg / ml solution. 100 μl of the MIL-53(Fe)@Asp@GOX solution was added to 2700 μl of pure water, followed by 120 μl of TMB solution and 100 μl of a saliva sample solution with an unknown glucose concentration. The mixture was thoroughly mixed and reacted at 20°C for 10 minutes. Color changes were observed, and UV absorbance was measured. The glucose concentration in the saliva sample solution was determined based on the established standard curve.

[0047] Figure 1 The Zeta potential diagrams of MIL-53Fe and MIL-53Fe@Asp obtained in Example 1 show that the potential of MIL-53Fe@Asp increased after Asp modification, indicating that the MIL-53(Fe)@Asp nanozyme has been successfully synthesized. Figure 2 The image shown is a scanning electron microscope (SEM) image of MIL-53Fe, MIL-53Fe@Asp, and MIL-53Fe@Asp@GOX prepared in Example 1. Figure 2 (a) is a scanning electron microscope (SEM) image of MIL-53Fe. Figure 2 (b) is a scanning electron microscope (SEM) image of MIL-53Fe@Asp. Figure 2 (c) is a scanning electron microscope (SEM) image of MIL-53Fe@Asp@GOX. From Figure 2 As can be seen, both MIL-53(Fe)@Asp and MIL-53Fe@Asp@GOX exhibit plate-like particles, with a structure basically identical to MIL-53(Fe), indicating that the morphology of MIL-53(Fe) did not change significantly after modification with glucose oxidase and aspartic acid. Figure 3 As can be seen, there is a good linear relationship between glucose concentration and absorbance in saliva (R0). 2 =0.9987), the detection range is 3μM-500μM, and the detection limit is 0.322μM.

[0048] Figure 4To compare the absorbance of the reaction after adding 100 mM of other sugars with that after adding 1 mM of glucose. Figure 4 As shown, only the addition of glucose caused a significant color change, with a high absorption peak appearing at 652 nm. Even when the concentrations of sucrose and lactose were 100 times that of glucose, the absorbance at 652 nm did not change significantly, indicating that MIL-53(Fe)@Asp@GOX has good anti-interference ability for glucose detection.

[0049] Example 2:

[0050] A wide pH-suitable multi-biomolecule detection MOF probe, the MIL-53(Fe)@Asp@COD probe, comprising the metal-organic framework MIL-53(Fe), aspartic acid Asp, and cholesterol oxidase COD.

[0051] The preparation method of the MIL-53(Fe)@Asp@COD probe includes the following steps:

[0052] Dissolve 4 mg of metal-organic framework MIL-53(Fe) and 32 mg of aspartic acid (Asp) in 4 ml of water, sonicate, and stir. Dissolve 24 mg of cholesterol oxidase (COD) in the above 4 ml MIL-53(Fe)@Asp solution, stir at room temperature for 1 hour, centrifuge at 15000 rpm / min for 10 minutes, collect the precipitate and vacuum dry to obtain MIL-53(Fe)@Asp@COD.

[0053] A method for detecting biomolecule concentrations using the MIL-53(Fe)@Asp@COD probe includes the following steps:

[0054] The MIL-53(Fe)@Asp@COD nanozyme was prepared into a 3 mg / ml solution. 100 μl of the MIL-53(Fe)@Asp@COD solution was added to 2700 μl of pH 6.0 acetate buffer, followed by 120 μl of TMB solution and 100 μl of a blood sample solution with an unknown cholesterol concentration. The mixture was thoroughly mixed and reacted at 25°C for 10 minutes. Color changes were observed, and UV absorbance was measured. The cholesterol concentration in the blood sample solution was determined based on the established standard curve.

[0055] Figure 5 Fourier transform infrared (FT-IR) spectra of MIL-53Fe and MIL-53Fe@Asp obtained in Example 2. From... Figure 5 It can be seen that at 1595 and 1387cm -1 The left and right sides show absorption peaks, which are characteristic vibrational bands of the framework-(OCO)- group in MIL-53(Fe). Simultaneously, at 2090 cm⁻¹... -1Absorption peaks appeared on the left and right sides, which are characteristic of α-amino acid bonds. This proves that MIL-53(Fe) was successfully modified by aspartic acid. Figure 6 This is a standard curve of blood cholesterol concentration versus absorbance constructed using MIL-53Fe@Asp@COD, as shown in Example 2. Figure 6 As can be seen, there is a good linear relationship between blood cholesterol concentration and absorbance (R0). 2 =0.9832), the detection range is 10μM-400μM, and the detection limit is 0.225μM.

[0056] Example 3:

[0057] A wide pH-suitable multi-biomolecule detection MOF probe, the MIL-53(Fe)@Asp@GLOX probe, comprising the metal-organic framework MIL-53(Fe), aspartic acid Asp, and galactose oxidase GLOX.

[0058] The preparation method of the MIL-53(Fe)@Asp@GLOX probe includes the following steps:

[0059] Dissolve 10 mg of metal-organic framework MIL-53(Fe) and 28 mg of aspartic acid (Asp) in 4 ml of water, sonicate, and stir. Dissolve 35 mg of galactose oxidase (GLOX) in the above 4 ml MIL-53(Fe)@Asp solution, stir at room temperature for 1 hour, centrifuge at 15000 rpm / min for 10 minutes, collect the precipitate and dry it under vacuum to obtain MIL-53(Fe)@Asp@GLOX.

[0060] A method for detecting biomolecule concentrations using the MIL-53(Fe)@Asp@GLOX probe includes the following steps:

[0061] The MIL-53(Fe)@Asp@GLOX nanozyme was prepared into a 3 mg / ml solution. 100 μl of the MIL-53(Fe)@Asp@GLOX solution was added to 2700 μl of pH 3 acetate buffer, followed by 120 μl of TMB solution and 100 μl of a biological sample solution with an unknown galactose concentration. The mixture was thoroughly mixed and reacted at 37°C for 10 minutes. Color changes were observed, and UV absorbance was measured. The concentration of galactose in the biological sample solution was determined based on the established standard curve.

[0062] Figure 7 The X-ray diffraction (XRD) patterns of MIL-53Fe and MIL-53Fe@Asp prepared in Example 3 are shown below. Figure 7As can be seen, MIL-53(Fe)@Asp exhibits similar diffraction peaks to MIL-53(Fe). After adding Asp to MIL-53(Fe), the typical characteristic peaks of MIL-53(Fe) still exist, indicating that the addition of Asp did not change the crystal structure of MIL-53(Fe).

[0063] Figure 8 This is a standard curve of galactose concentration versus absorbance constructed using MIL-53(Fe)@Asp@GLOX, prepared in Example 3. From... Figure 8 As can be seen, there is a good linear relationship between galactose concentration and absorbance in biological sample solutions (R2 = 0.9843), the detection range is 5 μM-500 μM, and the detection limit is 0.053 μM.

[0064] Example 4:

[0065] A wide pH-suitable multi-biomolecule detection MOF probe, the MIL-53(Fe)@Glu@UAO probe, comprising the metal-organic framework MIL-53(Fe), aspartate Asp, and uricase UAO.

[0066] The preparation method of the MIL-53(Fe)@Glu@UAO probe includes the following steps:

[0067] Dissolve 10 mg of metal-organic framework MIL-53(Fe) and 50 mg of glutamate Glu in 5 ml of water, sonicate and stir. Dissolve 27 mg of uricase UAO in the above 5 ml MIL-53(Fe)@Glu solution, stir at room temperature for 1 hour, centrifuge at 15000 rpm / min for 10 minutes, take the precipitate and dry it under vacuum to obtain MIL-53(Fe)@Glu@UAO.

[0068] A method for detecting biomolecule concentrations using the MIL-53(Fe)@Glu@UAO probe includes the following steps:

[0069] The MIL-53(Fe)@Glu@UAO nanozyme was prepared into a 3 mg / ml solution. 100 μl of the MIL-53(Fe)@Glu@UAO solution was added to 2700 μl of water adjusted to pH 8.4 with NaOH. Then, 120 μl of TMB solution and 100 μl of a urine sample solution with an unknown uric acid concentration were added. The mixture was thoroughly mixed and reacted at 37°C for 10 minutes. Color changes were observed, and UV absorbance was measured. The concentration of uric acid in the urine sample solution was determined based on the established standard curve.

[0070] Figure 9This is a standard curve of uric acid concentration versus absorbance in urine constructed using MIL-53Fe@Glu@UAO, as shown in Example 4. Figure 9 As can be seen, there is a good linear relationship between the concentration of uric acid in urine and the absorbance (R0). 2 =0.9968), the detection range is 2μM-500μM, and the detection limit is 0.12μM.

[0071] It should be understood that although this specification is described according to various embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0072] The detailed descriptions listed above are merely specific illustrations of feasible embodiments of the present invention and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A wide-pH applicable MOF probe for detecting multiple biomolecules, characterized in that, The probe is a MIL-53(Fe)@AA@EZ probe, which includes the metal-organic framework MIL-53(Fe), amino acids, and enzymes.

2. The wide pH-suitable multi-biomolecule detection MOF probe according to claim 1, characterized in that, The mass ratio of the metal-organic framework MIL-53 (Fe), amino acids, and enzymes is 1:(0.5–8.0):(0.5–6.0).

3. The wide pH-applicable multi-biomolecule detection MOF probe according to claim 2, characterized in that, The mass ratio of the metal-organic framework MIL-53 (Fe), amino acids, and enzymes is 1:(2.8–4.6):(2.2–3.5).

4. The wide pH-suitable multi-biomolecule detection MOF probe according to claim 1, characterized in that, The enzyme is glucose oxidase, cholesterol oxidase, galactose oxidase, or uricase; The amino acid in question is an acidic amino acid: glutamic acid or aspartic acid.

5. A method for preparing a wide-pH applicable multi-biomolecule detection MOF probe according to any one of claims 1 to 4, characterized in that, Includes the following steps: Step S1, Preparation of MIL-53(Fe)@AA: Weigh terephthalic acid and ferric chloride hexahydrate FeCl3·6H2O and add them to N,N-dimethylformamide DMF. Mix and stir. Pour the mixed solution into a stainless steel autoclave and heat to react. Let it cool naturally to room temperature. Centrifuge to separate the product. Wash with N,N-dimethylformamide DMF and anhydrous ethanol to obtain the metal-organic framework MIL-53(Fe). Mix the metal-organic framework MIL-53(Fe) with an amino acid solution, sonicate and stir to obtain the MIL-53(Fe)@AA solution. Step S2, Preparation of MIL-53(Fe)@AA@EZ: Dissolve the enzyme in the MIL-53(Fe)@AA solution obtained in step S1, mix well, stir, centrifuge to separate the product, take the precipitate and vacuum dry to obtain the MIL-53(Fe)@AA@EZ probe.

6. A method for detecting the concentration of biomolecules, characterized in that, The detection of biomolecule concentration using the broad pH-suitable multi-biomolecule detection MOF probe according to any one of claims 1 to 4 or the MIL-53(Fe)@AA@EZ probe prepared by the preparation method according to claim 5 includes the following steps: Add the MIL-53(Fe)@AA@EZ probe loaded with the corresponding enzyme to water or buffer solution, then add biological sample and TMB solution, mix well and react, observe the color change, and measure the ultraviolet absorption peak at 652 nm wavelength. Determine the concentration of biological sample based on the established standard curve.

7. The method for detecting biomolecule concentration according to claim 6, characterized in that, The pH range of the water or buffer solution is 3.0 to 8.4, and the reaction temperature is 20 to 37°C.

8. The method for detecting biomolecule concentration according to claim 7, characterized in that, The pH range of the water or buffer solution is 4.0 to 7.

4.

9. The application of a wide pH-suitable multi-biomolecule detection MOF probe according to any one of claims 1 to 4, or the MIL-53(Fe)@AA@EZ probe prepared by the preparation method according to claim 5, in the quantitative detection of biomolecule concentrations.

10. A detection kit for quantitative detection of biomolecule concentration, characterized in that, Includes the wide pH applicable multi-biomolecule detection MOF probe according to any one of claims 1 to 4 or the MIL-53(Fe)@AA@EZ probe prepared by the preparation method of claim 5.

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