Microneedle electrochemical sensor based on zinc-based metal framework (Zn-MOF)

Abstract

The invention discloses a micro-needle electrochemical sensor based on Zn-MOF as well as a preparation method and application of the micro-needle electrochemical sensor. The Zn-MOF is used for loading the biological enzyme and modifying the biological enzyme on the surface of the microneedle, so that the stability of the enzyme is enhanced, active sites are expanded, the biological enzyme is used for preparing a microneedle working electrode, and an Ag / AgCl microneedle reference electrode and a microneedle pair electrode are combined to construct a three-electrode system. The microneedle sensor realizes the detection of target molecules through the recognition / catalytic effect of enzyme after transdermal penetration. The preparation method of the Zn-MOF microneedle electrochemical sensor is convenient and easy to implement, the Zn-MOF microneedle electrochemical sensor has excellent stability and sensitivity, wearable sensing, real-time detection and continuous monitoring can be achieved, and the Zn-MOF microneedle electrochemical sensor has extremely high clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection and relates to a microneedle array, in particular to a sensing microneedle based on a zinc-based metal organic framework (Zn-MOF). Background Art

[0002] Interstitial fluid (ISF) is a bodily fluid found in the extracellular spaces, serving as a transporter of nutrients and metabolites. Because ISF resembles plasma in composition, it can be used to detect important biomarkers such as glucose and electrolytes. Microneedles are an emerging transdermal sampling technology that offers numerous advantages for ISF sampling and detection, including being painless, non-invasive, and easy to use. By penetrating the skin's surface, they enable rapid and accurate collection of ISF components.

[0003] Zinc-based metal-organic frameworks (Zn-MOFs) are a type of porous material composed of zinc ions and organic ligands connected by coordination bonds. They have highly ordered pores and ultra-high specific surface area, stable chemical properties, and excellent biocompatibility. Therefore, they have shown great potential in the fields of enzyme immobilization and biosensing. The present invention significantly improves the stability and activity of enzymes by loading biological enzymes on Zn-MOFs and modifying them on the surface of microneedles. The reasons are: 1) By encapsulating the biological enzymes in Zn-MOFs, the active centers of the enzymes are effectively protected, reducing the impact of microneedle interface contamination on the enzymes; 2) the high specific surface area of Zn-MOFs is fully utilized to increase the catalytic reaction interface and electrochemical active sites, further improving the response speed and detection sensitivity of electrochemical sensors. In addition, Zn-MOF sensing microneedles can detect target biomarkers by loading different biological enzymes, providing technical support for the construction of microneedle sensors with high sensitivity, high selectivity, and multi-target detection.

[0004] The present invention designs an efficient and precise ZnMOF microneedle electrochemical sensor for real-time detection of important biochemical indicators in ISF, providing scientific basis and technical support for personalized medical treatment and health management. Summary of the Invention

[0005] The present invention aims to provide a preparation method and related applications of a Zn-MOF-based microneedle sensor. By constructing Zn-MOF sensing microneedles, the transdermal ability of metal microneedles and the sensing performance of enzyme@Zn-MOF composite materials are integrated to achieve real-time detection of target molecules in ISF.

[0006] The technical solutions of the present invention are as follows:

[0007] A Zn-MOF electrochemical sensing microneedle array comprises a conductive microneedle matrix and comprises three components: a microneedle working electrode, a microneedle reference electrode, and a microneedle counter electrode. The size, shape, and array distribution of the microneedle array are adjustable; the microneedle working electrode is modified with an enzyme@Zn-MOF composite material. The Zn-MOF material is in the form of a powder or crystal and can load biological enzymes, including but not limited to glucose oxidase, cholesterol oxidase, and lactate oxidase. The Zn-MOF has an unlimited micromorphology and a size of 0.05-10 μm. The microneedle reference electrode is surface-modified with Ag / AgCl; the microneedle counter electrode is surface-modified with Pt and / or Au. The mechanical strength of the sensing microneedle array is 0.05-10 MPa, enabling effective epidermal penetration. The sensing microneedles have sampling properties and can collect subcutaneous interstitial fluid. The sensing microneedle array constitutes an electrochemical three-electrode detection system, enabling electrochemical detection of target biomarkers through the recognition / catalytic effect of biological enzymes.

[0008] The preparation method of the Zn-MOF microneedle electrochemical sensor of the present invention comprises the following steps:

[0009] (1) Preparation of enzyme@Zn-MOF composite material: Zn-based metal salt, ligand and biological enzyme are dissolved in deionized water and / or organic solvent, reacted at a certain temperature for a certain time, and then the product is centrifuged and washed.

[0010] Preferably, the metal salt described in step (1) above can be any soluble zinc salt, such as Zn(CH3COO)2, ZnCl2, ZnSO4, Zn(NO3)2, etc.

[0011] Preferably, the biological enzyme in step (1) above can be any biological enzyme with catalytic effect, such as glucose oxidase, lactate oxidase, ethanol oxidase, etc.

[0012] Preferably, the concentration of the biological enzyme in step (1) is 0.01-10 mg / mL.

[0013] Preferably, the ligand described in step (1) above is capable of reacting with Zn 2+ Organic ligands that effectively coordinate ions to form three-dimensional crystal structures, such as dimethylimidazole, terephthalic acid (H2BDC), 4,4′,4″-s-triazine-1,3,5-triyltri-p-aminobenzoic acid (H3TATB), etc.

[0014] Preferably, the mass ratio of the metal salt to the solvent in step (1) is 1:(1-1000), and the mass ratio of the organic ligand to the solvent is 1:(0.1-1000), and the solvent includes but is not limited to water, ethanol, methanol, N,N-dimethylformamide, etc.

[0015] Preferably, the centrifugal speed in step (1) can be 1000-20000 rpm, and the centrifugal time can be 1-30 min.

[0016] (2) Preparation of microneedle matrix. Design the sensing microneedle matrix and the tip needle structure, prepare the metal microneedle array using micro-nano processing technology, and the end connection interface is used to connect to the signal analysis device.

[0017] Preferably, the microneedle matrix material in the above step (2) can be any conductive metal or alloy, such as copper, stainless steel, etc.

[0018] Preferably, the microneedle array preparation process in step (2) above can be any metal micro-nano processing process, such as electroforming, laser cutting, photolithography, electron beam etching, laser etching, etc.

[0019] Preferably, the microneedle substrate end connection interface in the above step (2) can be any device for connecting to a circuit board, such as a pin header, a cable, a connector, an FPC (flexible printed circuit), a ZIF (zero insertion force) connector, etc.

[0020] Preferably, the microneedle base in step (2) has a length of 5 mm to 30 mm and a width of 5 mm to 30 mm.

[0021] Preferably, the needle structure in the above step (2) is a needle-like structure, specifically a triangular pyramid, a quadrangular pyramid or a cone.

[0022] Preferably, the length of the needle structure in the above step (2) is 100 μm-2000 μm.

[0023] (3) Preparation of Zn-MOF microneedle sensor working electrode: The Zn-MOF composite material suspension obtained in step (1) is dropwise applied to the microneedle substrate obtained in step (2), and then placed in a vacuum drying oven to uniformly modify the Zn-MOF composite material on the surface of the microneedle array.

[0024] Preferably, the amount of the Zn-MOF material added dropwise in step (3) is 10-1000 μL.

[0025] Preferably, the vacuuming time in step (3) is 1-360 min.

[0026] Preferably, the Zn-MOF microneedle sensor working electrodes prepared in the above step (3) can be used in parallel in multiple groups to achieve multi-target detection.

[0027] (4) Preparation of microneedle reference electrode: Ag / AgCl slurry was evenly coated on the surface of the microneedle substrate obtained in step (2), and then dried in a vacuum drying oven. The Ag / AgCl was adhered to the microneedle array repeatedly.

[0028] Preferably, the Ag / AgCl slurry is applied 1-10 times.

[0029] (5) Preparation of microneedle electrodes: The microneedle substrate obtained in step (2) is plated with gold or platinum using an electroplating method.

[0030] (6) Assemble the Zn-MOF microneedle sensor working electrode, Ag / AgCl microneedle reference electrode and microneedle counter electrode prepared in steps (3) (4) (5) to form a three-electrode electrochemical detection system. Insert it into the skin tissue and leave it under the skin for a certain period of time. Then use the expansion device to connect to the external sensor data receiver to achieve quantitative detection of the target molecule.

[0031] Preferably, the target molecules in step (6) above include but are not limited to glucose, lactic acid, uric acid, cholesterol, etc.

[0032] This paper proposes a preparation strategy and application method for a Zn-MOF microneedle sensor. By constructing a microneedle three-electrode system suitable for electrochemical analysis, the microneedle working electrode is modified with an enzyme@ZnMOF composite material tailored to the characteristics of different target molecules, enabling precise detection. Furthermore, multiple working electrode groups can be combined to enable simultaneous detection of multiple targets. The terminal extension device can be used to output and analyze sensor signals, enabling in situ detection and real-time sensing of target biomarkers. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the microneedle sensor prepared in Example 1 of the present invention. Figure 1 is the microneedle substrate; 2 is the microneedle interface; 3 is the working electrode; 4 is the reference electrode; 5 is the counter electrode; 6 is an enlarged schematic diagram of the working electrode; 7 is the enzyme@Zn-MOF material modified with the working electrode; 8 is an enlarged schematic diagram of the enzyme@Zn-MOF material; 9 is an enlarged schematic diagram of the reference electrode; 10 is the Ag / AgCl surface-modified reference electrode; 11 is an enlarged schematic diagram of the counter electrode; and 12 is the Au surface-modified counter electrode.

[0034] Figure 2 This is an electron micrograph of the lactate oxidase@Zn-MOF material prepared in Example 1 of the present invention.

[0035] Figure 3 This is a data graph of the detection of lactic acid by cyclic voltammetry using the microneedle sensor prepared in Example 1 of the present invention.

[0036] Figure 4 This is a physical picture of the microneedle sensor array prepared in Example 2 of the present invention.

[0037] Figure 5This is a data graph of the quantitative detection of glucose in subcutaneous ISF by the microneedle sensor prepared in Example 2 of the present invention.

[0038] Figure 6 This is a photo of the transdermal microneedle sensor prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below through examples in conjunction with the accompanying drawings, but the protection scope of the present application is not limited by the specific conditions of these examples.

[0040] Example 1:

[0041] In Example 1, the present invention prepares a microneedle sensor based on Zn-MOF material for real-time detection of lactic acid concentration. Figure 1 As shown in the figure, the microneedle sensor consists of a microneedle working electrode, a microneedle reference electrode, and a microneedle counter electrode. Figures 1, 2, and 3 show the three parts of a microneedle electrode: 1 is the microneedle substrate; 2 is the microneedle interface; and 3 is the microneedle electrode body. The working electrode, reference electrode, and counter electrode all have a microneedle substrate and microneedle interface; the main difference lies in the surface modification of the microneedle body. An enlarged schematic diagram of the working electrode is shown in Figure 6, which is modified with the enzyme@Zn-MOF material shown in Figure 7. An enlarged diagram of the enzyme@Zn-MOF material is shown in Figure 8, where the ZnMOF is loaded with lactate oxidase for lactate detection. The microneedle reference electrode is shown in Figure 4, and an enlarged schematic diagram of its microneedle body is shown in Figure 9, which is modified with a layer of Ag / AgCl thin film shown in Figure 10. The microneedle electrode is shown in Figure 5, and an enlarged schematic diagram of its microneedle body is shown in Figure 11, which is electroplated with a layer of Au thin film shown in Figure 12.

[0042] The specific preparation steps are as follows:

[0043] (1) Synthesis of lactate oxidase@Zn-MOF material. 0.5 mL Zn(CH3COO)2 solution (1.0 mmol / L) and 0.5 mL lactate oxidase (1 mg / mL) were dissolved in deionized water, and terephthalic acid (4.3 mmol / L) was added as a ligand. After reacting at 25°C for 10 minutes, the mixture was centrifuged at 10,000 rpm for 5 minutes to obtain the following product: Figure 2 Lactate oxidase-Zn-MOF composite material shown.

[0044] (2) Preparation of microneedle electrode substrates. Using mechanical processing, stainless steel was used to prepare a microneedle substrate with a length of 15 mm, a width of 5 mm, a needle body length of 1000 μm, and a conical needle body structure.

[0045] (3) Preparation of microneedle electrode system. The Zn-MOF composite material (1 mg / mL, 20 μL) obtained in step (1) was added dropwise to the microneedle electrode substrate and vacuumed for 20 minutes to obtain a working electrode. Ag / AgCl slurry was added dropwise to the microneedle electrode substrate and vacuumed for 5 times to obtain a reference electrode. The microneedle electrode substrate was placed in a gold thiocyanate solution and electrochemically plated with gold to obtain a gold counter electrode.

[0046] (4) The three-electrode system was assembled and connected to an electrochemical workstation. Cyclic voltammetry was used to measure the performance of the microneedle in detecting lactic acid. The results were as follows: Figure 3 shown.

[0047] Example 2:

[0048] In Example 2, the present invention prepares a Zn-MOF microneedle sensor capable of detecting subcutaneous glucose concentration in real time, and the required steps are as follows:

[0049] (1) Synthesis of glucose oxidase@Zn-MOF material. 0.5 mL of ZnCl2 solution (1.0 mmol / L) and glucose oxidase (5 mg / mL) were dissolved in deionized water. 0.5 mL of dimethylimidazole (4.3 mmol / L) was added as a ligand. The mixture was reacted at 25°C for 1 minute and then centrifuged at 10,000 rpm for 25 minutes to obtain the glucose oxidase@Zn-MOF composite material.

[0050] (2) Preparation of microneedle electrode substrate. Using brass as raw material, a microneedle substrate with a length of 15 mm, a width of 15 mm, a needle body length of 1200 μm, and a needle body structure of a quadrangular pyramid was prepared by cutting ( Figure 4 ).

[0051] (3) Preparation of a microneedle three-electrode system. The Zn-MOF composite material (0.5 mg / mL, 100 μL) obtained in step (1) was added dropwise to the microneedle electrode substrate and vacuumed for 20 minutes to obtain a microneedle working electrode. Ag / AgCl slurry was added dropwise to the microneedle electrode substrate and vacuumed for 5 times to obtain a microneedle reference electrode. The microneedle electrode substrate was placed in a potassium hexahydroxyplatinate solution and electrochemically plated with platinum to prepare a microneedle pair electrode.

[0052] (4) Assemble the microneedle three-electrode system and connect it to a wearable electrochemical detection chip, and use the constant potential method to achieve quantitative detection of glucose in subcutaneous ISF ( Figure 5 ).

[0053] Example 3:

[0054] In Example 3, the present invention prepares a Zn-MOF microneedle sensor capable of simultaneously detecting multiple target biomarkers, and the required steps are as follows:

[0055] (1) Synthesis of various Zn-MOF materials. 0.5 mL of Zn(NO3)2 solution (1.0 mmol / L) was dissolved in deionized water with glucose oxidase (2 mg / mL), cholesterol oxidase (2 mg / mL), and urate oxidase (2 mg / mL), respectively. 0.5 mL of dimethylimidazole (4.3 mmol / L) was added as a ligand. The mixture was reacted at 25°C for 30 seconds and then centrifuged at high speed (12000 r / min) for 2 minutes to prepare glucose oxidase@Zn-MOF composite materials, cholesterol oxidase@Zn-MOF composite materials, and urate oxidase@Zn-MOF composite materials, respectively.

[0056] (2) Preparation of microneedle electrode substrate: Using brass as raw material, a microneedle substrate with a length of 15 mm, a width of 15 mm, a needle body length of 1200 μm, and a needle body structure of a quadrangular pyramid was prepared.

[0057] (3) Preparation of microneedle working electrodes: The three Zn-MOF composite materials (1 mg / mL, 200 μL) obtained in step (1) were added dropwise to the microneedle electrode substrate, and vacuum was applied for 20 minutes to obtain three working electrodes.

[0058] (4) Preparation of microneedle working electrode: Add Ag / AgCl slurry dropwise onto the microneedle electrode substrate and evacuate the sample. Repeat this process 5 times to obtain a reference electrode.

[0059] (5) The microneedle electrode substrate is placed in a potassium hexahydroxyplatinate solution and electrochemically plated with platinum to prepare a microneedle pair electrode.

[0060] (6) Assemble the microneedle three-electrode system and connect it to the electrochemical detection system, insert it into the subcutaneous tissue ( Figure 6 ), thereby achieving the quantitative detection of glucose, cholesterol and uric acid in living ISF.

Claims

1. A zinc-based metal organic framework (Zn-MOF) microneedle electrochemical sensor, characterized in that It includes a microneedle working electrode, a microneedle reference electrode and a microneedle counter electrode. The microneedle matrix material is metal. The microneedle array is distributed on the base and has transdermal properties. The target molecule can be detected by loading the enzyme-Zn-MOF composite material.

2. The Zn-MOF microneedle electrochemical sensor according to claim 1, characterized in that The microneedle working electrode, microneedle reference electrode, and microneedle pairing electrodes have physical interfaces that can be connected to external electrochemical detection modules to achieve signal transmission and target molecule detection. External electrochemical detection modules include, but are not limited to, electrochemical workstations and electrochemical detection chips. Detection methods include, but are not limited to, current analysis, differential pulse amperometry, differential pulse voltammetry, cyclic voltammetry, linear sweep voltammetry, impedance analysis, and the like.

3. The microneedle working electrode according to claim 1, characterized in that The surface of the microneedle electrode is modified with an enzyme-Zn-MOF composite material. The biological enzymes include but are not limited to glucose oxidase, cholesterol oxidase, lactate oxidase, urate oxidase, ethanol oxidase, etc. Zn-MOF detects the corresponding target molecules by loading the biological enzymes.

4. The microneedle reference electrode according to claim 1, wherein The surface of the microneedle electrode is uniformly modified with Ag / AgCl, and the modification methods include but are not limited to drop coating, spin coating, sputtering, etc.

5. The microneedle electrode according to claim 1, wherein The surface of the microneedle electrode is electroplated with Au and / or Pt.

6. The Zn-MOF microneedle electrochemical sensor according to claim 1, characterized in that The base size is 0.5-3.5 cm, the microneedle array height is 500-1500 μm, and the needle body morphology includes but is not limited to triangular pyramids, square pyramids, cones, etc.

7. The Zn-MOF microneedle electrochemical sensor according to claim 1, characterized in that Microneedle working electrodes can detect multiple target molecules by combining multiple groups.

8. The method for using the Zn-MOF microneedle electrochemical sensor according to claim 1 is as follows: (1) Insert the Zn-MOF microneedle electrochemical sensor into the skin and leave it in place or remove it after a certain period of time; (2) During the retention period, the electrochemical detection module is connected to detect the concentration of the target analyte online or offline based on the electrochemical analysis principle.

9. The Zn-MOF according to claims 1 to 8, characterized in that Central ion Zn 2+ Zn-MOF is formed by coordination with organic ligands, which include but are not limited to 2-methylimidazole, terephthalic acid, etc.

10. The Zn-MOF according to claims 1 to 9, characterized in that It can load biological enzymes and still maintain the activity of the enzymes after loading.

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