Electrode for monitoring reactive oxygen species of tumor cells in real time, detection system and preparation method of electrode

Abstract

The invention provides an electrode for monitoring active oxygen substances of tumor cells in real time, a detection system and a preparation method of the electrode. According to the electrode, an electrode array is constructed on a glass substrate through an MEMS technology, the electrode comprises a working electrode, a reference electrode and a counter electrode, the surface of the working electrode is modified with an AuNPs / ZIF-8 ZIF-67 nano-enzyme material so as to enhance the detection sensitivity of reactive oxygen species (ROS), and the detection system comprises the electrode, a PDMS cavity and an electrochemical detection device. The cell culture chamber is fixed on the electrode through plasma bonding and is provided with a through hole to be matched with the Transwell cell culture chamber, so that ROS released by incubated tumor cells can be directly diffused to the surface of the electrode, and efficient detection is realized. By optimizing the electrode modification material and the system structure, the detection sensitivity and stability are improved, and the sensor has real-time monitoring capability and is suitable for biomedical research and anti-cancer drug screening.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical detection, and more particularly, to an electrode for real-time monitoring of reactive oxygen species in tumor cells, a detection system and a preparation method thereof. Background Art

[0002] Reactive oxygen species (ROS) are a class of oxygen-containing free radicals or non-free radical compounds, including superoxide radical (O2 •− ), hydrogen peroxide (H2O2), hydroxyl radical (•OH) and singlet oxygen ( 1 O2), etc. Since these molecules contain unpaired electrons and have high chemical reactivity, they play an important role in cell biological processes. ROS are involved in cell signal transduction, cell growth, differentiation and immune response, and have important regulatory functions in both physiological and pathological processes.

[0003] Under normal physiological conditions, low concentrations of ROS act as signaling molecules to regulate cell proliferation, differentiation and apoptosis, and can oxidize redox-sensitive cysteine residues in target proteins to regulate enzyme activity, transcription factor function and protein interactions. However, within a moderate concentration range, ROS can promote the proliferation of certain cells. For example, in colon cancer cells (CT26) and hepatoma cells (Hepa1-6), a decrease in ROS levels inhibits cell proliferation, while an appropriate increase in ROS concentration can promote their growth. On the contrary, high concentrations of ROS can cause oxidative stress, damage lipid, protein and DNA molecules, and then induce cell cycle arrest, autophagy, apoptosis or ferroptosis. For example, in a melanoma cell model, increasing the ROS level by ascorbic acid can lead to an increase in DNA damage and promote cell death. Therefore, the dynamic changes in ROS levels play a key role in cancer development and treatment. Since the ROS level in tumor cells is generally higher than that in normal cells and is more sensitive to ROS, inducing apoptosis by further increasing the ROS level in tumor cells or disrupting their antioxidant system can achieve selective killing, providing a new strategy for tumor treatment.

[0004] Currently, the methods for real-time monitoring of hydrogen peroxide (H2O2) released by tumor cells mainly include fluorescence method and chemiluminescence method. Although these methods can provide ROS-related detection data to a certain extent, there are still many deficiencies, which affect the experimental accuracy and practical application, as follows: Fluorescence detection method: The fluorescence probe method is one of the most commonly used ROS detection methods. Among them, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) is a common fluorescence probe. DCFH-DA can freely penetrate cell membranes and, within cells, the acetate group is removed by the action of esterase to form non-fluorescent 2',7'-dichlorodihydrofluorescein (DCFH). When the ROS level increases, DCFH is oxidized to fluorescent 2',7'-dichlorofluorescein (DCF), and its fluorescence intensity is proportional to the ROS concentration. It can be detected by fluorescence microscopy, flow cytometry or fluorescence spectrophotometer. Although the advantage of the fluorescence method lies in its high detection sensitivity and the ability to achieve real-time monitoring of the ROS level in living cells, this method still has the following deficiencies:

[0005] Limited quantitative accuracy: The fluorescence intensity depends not only on the ROS level but also on the probe specificity and the resolution of the micro-fluorescence imaging instrument, making it difficult to achieve precise quantification of ROS; Influence of interference signals: Fluorescent dyes may non-specifically bind to non-target molecules in cells, resulting in increased background noise and affecting data accuracy; Insufficient stability of fluorescence probes: DCFH-DA may undergo auto-oxidation under light conditions, leading to enhanced fluorescence signals and affecting the specificity and reproducibility of detection results; Strong instrument dependence: Fluorescence detection relies on complex imaging equipment, restricting its application in portable or point-of-care testing.

[0006] Chemiluminescence method: The chemiluminescence method is a ROS detection technique that does not require an external light source. It uses specific chemiluminescent reagents to react with ROS to generate light signals and realizes quantitative analysis of ROS by measuring the light intensity. The main advantages of the chemiluminescence method include: No need for an excitation light source, low background signal, suitable for high-sensitivity detection; High signal detection sensitivity, capable of identifying changes in ROS levels at low concentrations.

[0007] However, the chemiluminescence method still has the following deficiencies in practical applications: Poor biocompatibility: Some chemiluminescent reagents have potential toxicity to biological samples, especially under high-concentration or long-time reaction conditions, which may affect cell viability and interfere with experimental results; Limited real-time monitoring ability: The decay rate of chemiluminescence signals is relatively fast, making it a great challenge for long-term and stable real-time monitoring; Inability to accurately detect low-concentration hydrogen peroxide: In a complex biological environment, the signals of the chemiluminescence method may be interfered with, resulting in inaccurate detection of low-concentration H2O2; Complicated operation and long reaction time: Some chemiluminescence methods have problems of complex operation and long experimental cycles, making it difficult to meet the needs of real-time monitoring.

[0008] In summary, the current mainstream ROS monitoring methods still have many deficiencies in terms of sensitivity, specificity, stability, real-time performance, and biocompatibility. There is an urgent need to develop a new type of ROS monitoring method with high sensitivity, strong specificity, real-time monitoring ability, simple operation, and excellent stability to improve its application value in biomedical research and disease diagnosis. Summary of the Invention

[0009] The first technical problem to be solved by the present invention is to provide a preparation method of an electrode for real-time monitoring of reactive oxygen species in tumor cells, so as to solve the problems of low stability, insufficient catalytic activity, and difficulty in batch preparation in the prior art.

[0010] To overcome the defects of the above prior art, the present invention provides a preparation method of an electrode for real-time monitoring of reactive oxygen species in tumor cells, comprising the following steps: S1. Prepare nanozyme material: Provide ZIF-8@ZIF-67 material and dissolve it in methanol to form a solution. Under stirring conditions, add chloroauric acid solution, sodium citrate solution, and sodium borohydride solution to the solution in sequence. After stirring and reacting, a core-shell structure composite material modified with gold nanoparticles is formed. Subsequently, centrifuge and wash, and dry under vacuum to obtain AuNPs / ZIF-8@ZIF-67 nanozyme material.

[0011] S2. Prepare MEMS electrode: Using the MEMS method of photolithography, development, sputtering, and etching, form a gold / chromium working electrode, a reference electrode, and a counter electrode on a glass substrate to obtain a MEMS electrode; S3. Modify the specific electrode: Dissolve the AuNPs / ZIF-8@ZIF-67 nanozyme material prepared in step S1 in a mixed solution of isopropanol and Nafion to obtain a mixed solution. Take the mixed solution and drop-coat it onto the surface of the working electrode of the MEMS chip prepared in step S2. After drying, a specific catalytic film is formed, that is, an electrode for real-time monitoring of reactive oxygen species in tumor cells is prepared.

[0012] Compared with the prior art, the preparation method of an electrode for real-time monitoring of reactive oxygen species in tumor cells of the present application effectively solves the deficiencies of the existing electrode preparation methods in terms of stability, catalytic activity, durability, and batch preparation through technical means such as nanozyme catalysis, electrode microfabrication, and modification layer optimization, and provides an efficient, accurate, and reusable electrode preparation method for real-time monitoring of reactive oxygen species (ROS) released by tumor cells, having the following advantages: Improved stability: Using AuNPs / ZIF-8@ZIF-67 nanozyme material to replace traditional natural enzymes overcomes the problem of easy inactivation of natural enzymes, improves the long-term stability and anti-interference ability of the electrode, and can adapt to different detection environments.

[0013] High efficiency catalysis and high sensitivity: The porous structure of the ZIF-8@ZIF-67 material combined with the high catalytic activity of nano-gold enables a larger specific surface area and stronger electron transfer ability on the electrode surface, effectively enhancing the catalytic effect on reactive oxygen species (ROS) and improving the detection sensitivity and signal intensity. Enhanced adhesion and improved electrode durability: In the preparation method of the present invention, a Nafion solution is used to optimize the electrode modification layer, improving the binding force of the nanozyme material on the electrode surface, ensuring that the electrode does not fall off during long-term use, improving the reusability, and being suitable for long-term monitoring of ROS. Standardized preparation by MEMS process: The MEMS microfabrication technology is used to construct a high-precision electrode array on a glass substrate, ensuring consistent electrode morphology and batch production, meeting the actual application requirements, and improving the detection reproducibility and reliability. Optimized preparation process and improved production efficiency: The preparation method of the present invention optimizes the nanozyme synthesis parameters, reasonably adjusts the composition of the modification solution, and combines with the efficient MEMS microfabrication technology to make the electrode preparation process more controllable and scalable, enhancing the practicality and industrial value of the electrode.

[0014] In a possible implementation manner, in the step S1, the mass-volume ratio of the ZIF-8@ZIF-67 material to methanol is (0.020 - 0.030) g : 5 mL; the volume ratio of methanol, chloroauric acid solution, sodium citrate solution, and sodium borohydride solution is 5 : 2 : 3 : 3; the mass concentration of the chloroauric acid solution is (0.0004–0.0006) g·mL -1 , the mass concentration of the sodium citrate solution is (0.002–0.003) g·mL -1 , the mass concentration of the sodium borohydride solution is (0.0008–0.0012) g·mL -1 .

[0015] Compared with the prior art, adopting the above technical solution, by controlling the mass-volume ratio of the ZIF-8@ZIF-67 material to methanol at (0.020 - 0.030) g : 5 mL, it ensures that the precursor material is fully dissolved to form a uniform precursor solution, providing a stable reaction environment for the subsequent deposition of gold nanoparticles. Further controlling the volume ratio of methanol, chloroauric acid solution, sodium citrate solution, and sodium borohydride solution at 5:2:3:3 ensures that the reduction process of chloroauric acid is controllable, preventing excessive aggregation or sedimentation of gold nanoparticles, improving the uniformity and stability of the catalytic layer. By optimizing the synthesis ratio of the nanozyme, it ensures the stable existence of the catalytic layer on the electrode surface, improves the catalytic activity of the gold nanoparticles, enhances the detection sensitivity and stability of the electrode, and enables accurate and real-time monitoring of reactive oxygen species (ROS) released by tumor cells.

[0016] In a possible implementation manner, in the step S1, the stirring reaction time is 24 hours, and the centrifugation and washing conditions are centrifugation at a frequency of 9000 r·min -1 Then, the composite material is washed with methanol more than 3 times, and the vacuum drying conditions are vacuum drying in an oven at 65 - 70 °C for 12 h.

[0017] Compared with the prior art, adopting the above technical solution, by optimizing the stirring, centrifugation and washing, and drying conditions, the purity, catalytic activity, and long-term stability of the nanozyme material are improved, it is evenly distributed on the electrode surface, and it is ensured that it is not easy to fall off during the electrochemical detection process, thereby improving the detection sensitivity and reusability performance, and realizing efficient and accurate real-time monitoring of tumor cell reactive oxygen species.

[0018] In a possible implementation manner, in the step S2, in the gold / chromium working electrode, the thickness of Au is 200 nm, and the thickness of Cr is 30 nm.

[0019] Compared with the prior art, adopting the above technical solution, through reasonable design of the electrode layer thickness, the working electrode not only has excellent electrical conductivity, but also ensures stability and corrosion resistance during long-term use. At the same time, it effectively improves the sensitivity and reproducibility of the detection system, which helps to improve the real-time monitoring effect of tumor cell reactive oxygen species.

[0020] In a possible implementation manner, in the step S3, in the mixed solution, the volume ratio of isopropanol to Nafion is 99:1, and the added mass of the AuNPs / ZIF-8@ZIF-67 nanozyme material is 0.8 - 1.2% of the mixed solution.

[0021] Compared with the prior art, by adopting the above technical solution, with the volume ratio of isopropanol to Nafion being 99:1, where isopropanol serves as a solvent, it can effectively reduce the aggregation phenomenon of the nanozyme material, enabling the AuNPs / ZIF-8@ZIF-67 nanozyme material to be uniformly dispersed in the solution. Since Nafion is an ion-exchange polymer, at a low concentration (1%), it can provide appropriate adhesion force to stably immobilize the nanozyme material on the electrode surface without affecting its catalytic activity; by controlling the added mass of the nanozyme material to be 0.8 - 1.2% of the mixed solution, it can ensure the formation of a uniform and stable catalytic film of the nanozyme material on the electrode surface.

[0022] The second technical problem to be solved by the present invention is to provide an electrode for real-time monitoring of reactive oxygen species in tumor cells, so as to solve the problems of low detection sensitivity, poor stability, easy shedding of the modification layer, and insufficient long-term monitoring ability existing in the prior art.

[0023] To overcome the defects of the above prior art, the present invention provides an electrode for real-time monitoring of reactive oxygen species in tumor cells. The electrode is prepared by the above preparation method, and the electrode includes: A substrate, the material of the substrate being glass; A counter electrode, a working electrode, and a reference electrode, and the counter electrode, the working electrode, and the reference electrode are adjacent to each other and fixedly spaced on the surface of the substrate.

[0024] Compared with the prior art, an electrode for real-time monitoring of reactive oxygen species (ROS) in tumor cells of the present application has significantly superior detection sensitivity, signal stability, durability, and long-term monitoring ability through multiple innovative designs such as optimizing nanomaterial modification, MEMS fine manufacturing, and electrode layout optimization. It realizes highly sensitive, long-term, and stable detection of ROS in tumor cells, providing a more accurate tool for tumor microenvironment research and real-time dynamic monitoring. Specifically, it has the following advantages: The electrode of the present invention uses AuNPs / ZIF-8@ZIF-67 nanozyme material to modify the working electrode. This material has a high specific surface area and excellent catalytic activity, which can effectively promote the electrochemical oxidation reaction of hydrogen peroxide, thereby improving the detection sensitivity of the electrode to ROS. By optimizing the modification amount and dispersibility of the nanozyme material, the electrode has a lower detection limit and a wider linear detection range, suitable for real-time monitoring of low-concentration ROS; and the electrode is prepared on a glass substrate by MEMS technology, ensuring the structural stability and reproducibility of the electrode. By introducing a Nafion polymer protective layer, the adhesion of the nanomaterial is improved, making the modification layer more stable and avoiding the problem of nanozyme shedding or inactivation during long-term monitoring; and the structural design of surrounding the working electrode with a reference electrode can provide a more stable potential reference compared with the traditional parallel electrode layout, improving the detection accuracy. The counter electrode is arranged on the periphery, optimizing the electron transfer channel and reducing the charge transfer impedance, making the electrochemical signal more stable and reliable.

[0025] The third technical problem to be solved by the present invention is to provide a detection system for real-time monitoring of reactive oxygen species in tumor cells to solve the problems in the prior art such as the inability to achieve synchronous monitoring of cell real-time incubation and ROS, unstable structure of the detection system, poor detection repeatability, and complex operation.

[0026] To overcome the defects of the above prior art, the present invention provides a detection system for real-time monitoring of reactive oxygen species in tumor cells. The detection system includes: The electrode for real-time monitoring of reactive oxygen species in tumor cells, which is used for electrochemically detecting reactive oxygen species, a Transwell cell culture chamber, and: A PDMS chamber is arranged above the working electrode and is in close contact with it. A through hole is opened at the top of the PDMS chamber, and the diameter of the through hole matches the outer diameter of the Transwell cell culture chamber. It is used to fix the Transwell cell culture chamber and make the bottom permeable layer face the surface of the working electrode, forming a detection chamber to incubate tumor cells and allow ROS to diffuse to the electrode surface; An electrochemical detection device is electrically connected to the working electrode and is used to collect and analyze electrochemical signals to real-time detect the concentration of ROS released by tumor cells.

[0027] Compared with the prior art, a detection system for real-time monitoring of reactive oxygen species (ROS) in tumor cells integrates innovative technologies such as cell culture, real-time ROS detection, and optimized electrode design. It is superior to the prior art in terms of real-time performance, sensitivity, stability, and operational convenience, achieving long-term dynamic monitoring of the ROS level released by tumor cells. It can be used in multiple application fields such as tumor microenvironment research, drug screening, and cell biology experiments, providing an efficient and accurate experimental tool for related biomedical research. It has the following advantages: The combination of a PDMS chamber and a Transwell cell culture insert enables electrochemical detection while cells are incubated in situ, avoiding interference with the ROS level during cell processing in traditional detection methods. Secondly, the PDMS chamber and the MEMS electrode are bonded after plasma cleaning to achieve a tight fit, ensuring a stable ROS diffusion path and avoiding signal fluctuations caused by a loose structure of the culture chamber. A through-hole with a fixed diameter is used to ensure that the Transwell cell culture insert can be stably placed, improving the repeatability of the experiment and data consistency. Additionally, an electrode modified with AuNPs / ZIF-8@ZIF-67 nanozymes is combined to provide efficient catalytic detection ability, making the electrochemical signal response of ROS more sensitive, improving the sensitivity and detection limit of the detection. By optimizing the electrode layout and electrochemical detection parameters, background noise interference is reduced, signal stability is improved, and accurate detection of low-concentration ROS is ensured. Moreover, the detection system of the present invention uses a detachable Transwell cell culture insert. For each experiment, only the cell culture insert needs to be replaced to start a new round of experiment, avoiding the cumbersome operation of replacing the entire electrode system. Since the electrode uses a stable nanozyme modification layer + MEMS manufacturing process, the detection performance can be maintained for a long time, giving the detection system a long service life and reducing the cost of repeated manufacturing.

[0028] In a possible implementation, the diameter of the working electrode is the same as the diameter of the Transwell cell culture insert.

[0029] Compared with the prior art, adopting the above technical solution can optimize the matching degree between the cell culture environment and the electrochemical detection area, ensuring that the reactive oxygen species (ROS) released by tumor cells can uniformly diffuse to the electrode surface, thereby improving the sensitivity and data stability of the detection.

[0030] The fourth technical problem to be solved by the present invention is to provide a preparation method for the detection system to solve the problems of complex assembly, insufficient stability, and limited detection sensitivity in the prior art.

[0031] To overcome the defects of the above prior art, the present invention provides a preparation method for the detection system, including the following steps: A1. Prepare the PDMS chamber: Prepare a PDMS chamber using PDMS material, and open a through-hole on the PDMS chamber, the diameter of the through-hole being matched with the outer diameter of the Transwell cell culture insert; obtain the PDMS chamber by curing and peeling. A2: Assemble the detection system: Use plasma to clean and bond the PDMS chamber obtained in step A1 to the working electrode to make them closely fit; then place the Transwell cell culture insert in the through-hole of the PDMS chamber with its bottom liquid-permeable layer facing the surface of the working electrode to form a detection chamber for incubating tumor cells so that ROS can diffuse towards the surface of the working electrode; connect the electrode to an electrochemical detection device to ensure the transmission of electrical signals and achieve real-time detection of the concentration of ROS released by tumor cells, thus completing the preparation.

[0032] Compared with the prior art, the preparation method of a detection system in this application has the following advantages: The preparation method of the present invention uses plasma cleaning technology to closely bond the PDMS chamber to the electrode, avoiding signal interference caused by uneven adhesion, and improving the stability and reliability of the detection system. By precisely designing the diameter of the through-hole of the PDMS chamber, the Transwell cell culture insert can be firmly fixed, avoiding the influence on the diffusion and detection of ROS due to position offset; secondly, through the optimized design of the PDMS chamber, the incubation environment of tumor cells is more stable, and the combination with the electrode is closer, ensuring that the ROS signal can effectively diffuse to the working electrode and improving the detection sensitivity. The selection of PDMS material ensures good biocompatibility and will not interfere with the growth of tumor cells and the release of reactive oxygen species; moreover, the preparation method of the present invention manufactures the electrode using a standardized MEMS process and modularly assembles it with the PDMS chamber to ensure that the size and performance of each detection system are consistent, reducing the fluctuation of experimental data and improving data repeatability. The use of plasma cleaning and bonding technology greatly reduces additional modification steps, making the assembly of the system more efficient and reliable.

[0033] In a possible implementation manner, step A1 includes: (a) Prepare a PDMS prepolymer solution: Weigh the PDMS prepolymer and the curing agent, mix them in a weight ratio of 10:(0.8 - 1.2), and stir evenly. (b) Degassing treatment: Place the obtained mixture in a vacuum dryer and let it stand for 20 - 40 min under a vacuum of 10 - 15 psi to remove air bubbles. (c) Mold casting: Pour the degassed PDMS mixture into a 3D printed mold and place it on a horizontal workbench to make the PDMS mixture evenly fill the microstructure of the mold. (d) Curing and forming: After standing at room temperature for 20 - 40 min, place the mold in an oven at 75 - 85 °C and heat for 0.8 - 1.2 h to completely cure the PDMS; (e) Peeling and forming: After the PDMS is cured, carefully peel the PDMS chamber from the mold to obtain the formed PDMS chamber, and drill a through-hole with a diameter of 6.0 - 7.0 mm at its top to match the outer diameter of the Transwell cell culture insert.

[0034] Compared with the prior art, adopting the above technical solution, by optimizing the ratio of the PDMS prepolymer to the curing agent (10:(0.8 - 1.2)), the flexibility and mechanical strength of the PDMS material are ensured, enabling it to form a stable culture chamber without affecting the experimental operation due to being too fragile or too soft; adopting vacuum degassing (10 - 15 psi, 20 - 40 min) can effectively remove the bubbles in the mixture, ensuring no bubble defects inside the PDMS culture chamber and avoiding structural damage or uneven cell culture environment caused by the rupture of micro-bubbles; through 3D printing the mold and horizontal casting, the uniform distribution of the PDMS mixture is ensured, making the sizes of the culture chambers consistent, and guaranteeing the repeatability and reliability of the experiment; curing in an oven at 75 - 85 °C (0.8 - 1.2 h) enables the PDMS to be fully cross-linked, improving its mechanical strength and ensuring no deformation during peeling, thus improving the preparation yield. Description of the Drawings

[0035] Figure 1 It is a side view structure diagram of the PDMS chamber and the electrode chip; Figure 2 It is a diagram of the MEMS electrode chip; Figure 3 It is a top view structure diagram of the PDMS chamber and the electrode chip; Figure 4 It is a result diagram of the fabricated electrode for monitoring the reactive oxygen species of tumor cells used to measure ROS at different concentrations; Figure 5 It is a corresponding calibration diagram of the electrode response and the ROS concentration (n = 3); Figure 6 It is an SEM diagram of AuNPs / ZIF-8@ZIF-67.

[0036] Description of the Reference Numerals: 1. Counter electrode; 2. Working electrode; 3. Reference electrode; 4. Substrate; 5. PDMS chamber; 6. Transwell cell culture insert; 7. Incubated tumor cells. Detailed Embodiments

[0037] First of all, those skilled in the art should understand that these embodiments are only used to explain the technical principles of the embodiments of the present application, and are not intended to limit the protection scope of the embodiments of the present application. Those skilled in the art can make adjustments according to needs to adapt to specific application scenarios.

[0038] In the description of the embodiments of the present application, it should be noted that unless otherwise clearly specified and limited, the terms "connected" and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of the present application can be understood according to specific situations.

[0039] The present invention provides a method for preparing an electrode for real-time monitoring of reactive oxygen species in tumor cells, including the following steps: S1. Prepare nanozyme material: Provide ZIF-8@ZIF-67 material and dissolve it in methanol to form a solution. Under stirring conditions, add chloroauric acid solution, sodium citrate solution and sodium borohydride solution to the solution in sequence. After stirring and reacting, a core-shell structure composite material modified with gold nanoparticles is formed, and then centrifuged and washed, and dried in vacuum to obtain AuNPs / ZIF-8@ZIF-67 nanozyme material.

[0040] S2. Prepare MEMS electrode: Using the MEMS method of photolithography, development, sputtering, and etching, form a gold / chromium working electrode 2, a reference electrode 3, and a counter electrode 1 on the glass substrate 4 to obtain a MEMS electrode; S3. Modify the specific electrode: Dissolve the AuNPs / ZIF-8@ZIF-67 nanozyme material prepared in step S1 in a mixed solution of isopropanol and Nafion to obtain a mixed solution. Take the mixed solution and drop it onto the surface of the working electrode 2 of the MEMS chip prepared in step S2, and dry it to form a specific catalytic film, that is, an electrode for real-time monitoring of reactive oxygen species in tumor cells is prepared.

[0041] As a preferred solution, in the step S1, the mass-volume ratio of the ZIF-8@ZIF-67 material to methanol is (0.020 - 0.030) g : 5 mL; the volume ratio of methanol, chloroauric acid solution, sodium citrate solution, and sodium borohydride solution is 5 : 2 : 3 : 3; the mass concentration of the chloroauric acid solution is (0.0004–0.0006) g•mL-1, the mass concentration of the sodium citrate solution is (0.002–0.003) g•mL-1, and the mass concentration of the sodium borohydride solution is (0.0008–0.0012) g•mL-1.

[0042] As a preferred solution, in the step S1, the stirring reaction time is 24 hours, the centrifugation and washing conditions are centrifugation at a frequency of 9000 r•min-1, and then the composite material is washed with methanol more than 3 times. The vacuum drying conditions are vacuum drying in an oven at 65 - 70°C for 12 h.

[0043] As a preferred solution, in the step S2, in the gold / chromium working electrode 2, the thickness of Au is 200 nm and the thickness of Cr is 30 nm.

[0044] As a preferred solution, in the step S3, in the mixed solution, the volume ratio of isopropanol to Nafion is 99 : 1, and the added mass of the AuNPs / ZIF-8@ZIF-67 nanozyme material is 0.8 - 1.2% of the mixed solution.

[0045] The present invention also provides an electrode for real-time monitoring of reactive oxygen species in tumor cells, and the electrode is prepared by the above preparation method, as Figure 1-3 shown. The present invention provides an electrode for real-time monitoring of reactive oxygen species in tumor cells, and the electrode is prepared by the above preparation method, and the electrode includes: A substrate 4, and the material of the substrate 4 is glass; A counter electrode 1, a working electrode 2, and a reference electrode 3, and the counter electrode 1, the working electrode 2, and the reference electrode 3 are adjacent to each other and are fixedly spaced on the surface of the substrate.

[0046] The present invention also provides a detection system for real-time monitoring of reactive oxygen species in tumor cells, and the detection system includes: The electrode for real-time monitoring of reactive oxygen species in tumor cells, which is used for electrochemically detecting reactive oxygen species; A PDMS chamber 5 is provided above the working electrode and is in close contact with it. A through hole is opened at the top of the PDMS chamber 5. The diameter of the through hole matches the outer diameter of the Transwell cell culture chamber 6, which is used to fix the Transwell cell culture chamber 6 and make the bottom liquid-permeable layer face the surface of the working electrode, forming a detection chamber for incubating tumor cells and allowing ROS to diffuse towards the electrode surface. An electrochemical detection device (not marked in the figure) is electrically connected to the working electrode and is used to collect and analyze electrochemical signals to detect the concentration of ROS released by tumor cells in real time.

[0047] As a preferred solution, the diameter of the working electrode 2 is the same as the diameter of the Transwell cell culture chamber 6.

[0048] The present invention also provides a preparation method of the detection system, including the following steps: A1. Prepare the PDMS chamber 5: Use PDMS material to prepare the PDMS chamber 5. Open a through hole in the PDMS chamber 5, and the diameter of the through hole matches the outer diameter of the Transwell cell culture chamber 6. Through curing and forming and peeling, the PDMS chamber 5 is obtained. A2: Assemble the detection system: Use plasma cleaning and bonding to bond the PDMS chamber 5 obtained in step A1 to the working electrode to make it in close contact. Subsequently, place the Transwell cell culture chamber 6 in the through hole of the PDMS chamber 5 so that the bottom liquid-permeable layer faces the surface of the working electrode 2 to form a detection chamber for incubating tumor cells and allowing ROS to diffuse towards the surface of the working electrode 2. Connect the electrode to the electrochemical detection device to ensure the transmission of electrical signals and realize the real-time detection of the concentration of ROS released by tumor cells, thus completing the preparation.

[0049] As a preferred solution, step A1 includes: (a) Prepare the PDMS prepolymer solution: Weigh the PDMS prepolymer and the curing agent, mix them in a weight ratio of 10:(0.8 - 1.2), and stir evenly. (b) Defoaming treatment: Place the obtained mixture in a vacuum dryer and let it stand for 20 - 40 min under a vacuum of 10 - 15 psi to remove air bubbles. (c) Mold casting: Pour the defoamed PDMS mixture into a 3D printed mold and place it on a horizontal workbench to make the PDMS mixture evenly fill the microstructure of the mold. (d) Curing and forming: After standing at room temperature for 20 - 40 min, place the mold in an oven at 75 - 85 °C and heat it for 0.8 - 1.2 h to completely cure the PDMS. (e) Peeling and forming: After the PDMS is cured, carefully peel the PDMS chamber 5 from the mold to obtain the formed PDMS chamber 5, and open a through-hole with a diameter of 6.0 - 7.0 mm at its top to match the outer diameter of the Transwell cell culture chamber 6.

[0050] Compared with the prior art, for the electrode and detection system of the present invention, tumor cells are incubated in real time in the designed cell culture chamber, and the cell culture chamber adopted by the present invention can have different sizes to incubate different numbers of tumor cells as detection objects. The working electrode 2 of the MEMS electrode chip layer adopted will increase proportionally according to the different cell culture chambers. The detection system of the present invention can detect the ROS released by tumor cells, aiming to realize long-term monitoring of the ROS released by tumor cells. The specific advantages are as follows: Using specific materials as artificial nanozymes to replace the disadvantages of natural enzymes and maintain stability: Although enzyme-based electrochemical sensors have good selectivity and sensitivity, they have serious disadvantages such as cumbersome immobilization operations, susceptibility to environmental influences, high costs, and low reproducibility. Therefore, many non-enzymatic sensors based on nanomaterials have been developed for the detection of cellular ROS. Metal-Organic Frameworks (MOFs) are a class of porous nanomaterials with a periodic network structure formed by the self-assembly of metal ions or metal clusters with organic ligands. Compared with other inorganic materials, MOFs have the advantages of diverse and adjustable topological structures, large specific surface areas, uniform pore channels, and adjustable structures. Moreover, there are a large number of unsaturated metal potentials in MOFs that can change physical or chemical properties by combining with other materials, and it has rapidly developed into a hot research direction in the field of materials science. Zeolitic Imidazolate Frameworks (ZIFs), as an important branch of MOFs materials, combine the characteristics of MOFs and zeolites. It has a sodalite topological structure similar to aluminosilicate zeolite materials. ZIFs have a variety of tetrahedral topological structures (BCT, DFT, GIS, MER, RHO, CAG, CRB, and SOD) designated by the International Zeolite Association. It is a coordination compound formed with metal as the central ion and imidazole or imidazole derivatives as ligands, showing a highly porous structure. It not only has a large surface area and active catalytic active sites but also has high chemical stability and thermal stability. Due to its unique pore structure, rich active sites, and chemical stability, ZIFs materials show broad application potential in many fields such as optics, catalysis, biomedicine, gas storage and separation, energy storage and conversion, and electrochemical sensing. Especially in the detection of ROS, ZIFs and their derivatives have shown great potential.

[0051] ZIFs have broad application prospects in the detection of hydrogen peroxide. Its unique structure and function make it of great significance and application value in the field of biosensing. With the in-depth research, the application of ZIFs and their composites in the field of electrochemical sensing and other fields will be further expanded, providing more accurate and efficient tools for biomedical research and clinical diagnosis.

[0052] Cells can be cultured in real time, and the ROS levels released by the incubated tumor cells 7 can be detected in real time: Real-time monitoring technology allows researchers to dynamically observe and monitor the change process of intracellular ROS in real time, enabling researchers to observe cell behavior in an environment closer to physiological conditions, thereby obtaining more accurate and comprehensive experimental results. The real-time incubation of tumor cells in the present invention can reduce interference, that is, the real-time monitoring technology allows for non-destructive monitoring without destroying cells. Cells can be observed and monitored without staining, reducing interference to cells.

[0053] High sensitivity and accurate quantification of ROS level: In the present invention, by using characteristic materials as nanozymes for detection, AuNP / ZIF-8@ZIF-67 synthesizes composite materials by in-situ assembling gold nanoparticles in a porous core-shell, endowing the catalyst with a large surface area, abundant catalytic centers, good conductivity, and good dispersion of components. Therefore, AuNP / ZIF-8@ZIF67 shows significant catalytic activity to reduce ROS. This modified material not only has a wide linear range and low detection limit, but also has strong selectivity, stability, and reproducibility. It can successfully detect ROS released by cancer live cells under drug stimulation.

[0054] The present invention has developed an integrated chip for real-time detection of H2O2 based on a MEMS chip combined with a cell culture chamber, which is convenient, repeatable, and reduces the operation process: In the present invention, by culturing tumor cells in real time and directly detecting with the lower electrode chip, compared with traditional methods such as fluorescence method and chemiluminescence method, it can be reused. Each time of detection only needs to replace the cell culture chamber and re-culture cells for detection. In addition, compared with traditional methods, it reduces troublesome operation steps such as preparing reagents and reduces the interference of background signals.

[0055] Achieve long-term monitoring: Compared with traditional methods, the chemiluminescence process of the chemiluminescence method is short, the chemiluminescence reaction duration is short, continuous excitation may be required to maintain detection, and the stability of chemical reagents is not as stable as that of electrochemical sensors. The fluorescence method usually uses fluorescence probes for staining and requires quantitative or semi-quantitative analysis of cells through a control group, and it cannot accurately achieve long-term detection. The electrochemical sensor of the present invention solves the pain points of traditional methods and realizes long-term detection of the change of ROS level in tumor cells.

[0056] The following provides examples combined with specific data to further expand and explain the above technical solutions of the present invention: Example 1: This example provides an electrode, a detection system, and a preparation method for real-time monitoring of reactive oxygen species in tumor cells, specifically including: S1. Prepare nanozyme materials Weigh 0.025 g of ZIF-8@ZIF-67 material, dissolve it in 5 mL of methanol, and stir evenly to form a solution. Under magnetic stirring, sequentially add 2 mL of chloroauric acid solution with a mass concentration of 0.0005 g•mL-1, 3 mL of sodium citrate solution with a mass concentration of 0.0025 g•mL-1, and 3 mL of sodium borohydride solution with a mass concentration of 0.001 g•mL-1. Continuously stir and react for 24 h to form a core-shell structure composite material modified with gold nanoparticles. Subsequently, centrifuge the obtained precipitate at a speed of 9000 r•min-1, wash it 3 times with methanol, and then dry it in vacuum at 65-70 °C for 12 h to obtain the AuNPs / ZIF-8@ZIF-67 nanozyme material.

[0057] S2. Preparation of MEMS electrode Using MEMS microfabrication technology, construct an electrode array on the glass substrate 4. Specifically, it includes:

[0058] Coat the surface of the glass substrate 4 with negative photoresist (ROL7133), and sequentially perform pre-baking (110 °C, 180 s), photolithography (exposure for 15 s), post-baking (135 °C, 3 min), and development (FHD-320 developer, development time 40 s).

[0059] Adopt the sputtering process, sequentially sputter 30 nm of Cr as an adhesion layer, and then sputter 200 nm of Au to form an electrode layer.

[0060] Perform a de-glue treatment to remove the photoresist and etch the pattern to obtain the MEMS electrode structure including the working electrode 2, the reference electrode 3, and the counter electrode 1.

[0061] S3. Modify the specific electrode Take 1 mg of the AuNPs / ZIF-8@ZIF-67 nanozyme material prepared in S1, dissolve it in a mixed solution of 99 μL of isopropanol and 1 μL of Nafion, and disperse it evenly by ultrasonic wave. Subsequently, take 2 μL of the obtained mixed solution and drop-coat it on the surface of the working electrode 2 of the MEMS chip, and dry it at room temperature to form a specific catalytic film, obtaining the final specific electrode.

[0062] A1. Preparation of PDMS chamber 5 Prepare a PDMS prepolymer solution: Weigh the PDMS prepolymer and the curing agent, mix them in a weight ratio of 10:(0.8-1.2), and stir evenly.

[0063] Defoaming treatment: Place the obtained mixture in a vacuum dryer and leave it standing for 20 - 40 min under a vacuum of 10 - 15 psi to remove air bubbles.

[0064] Mold casting: Pour the defoamed PDMS mixture into a 3D printing mold and place it on a horizontal workbench to evenly fill the microstructures of the mold with the PDMS mixture.

[0065] Curing and forming: After leaving it standing at room temperature for 20 - 40 min, place the mold in an oven at 75 - 85 °C and heat it for 0.8 - 1.2 h to completely cure the PDMS.

[0066] Peeling and forming: After the PDMS is cured, carefully peel off the PDMS cell culture chamber and drill a through - hole with a diameter of 6.0 - 7.0 mm at its top to match the outer diameter of the Transwell cell culture chamber.

[0067] A2. Assemble the detection system Plasma cleaning and bonding: Use a plasma cleaner to clean the PDMS cell culture chamber and the MEMS electrode, and then bond them to make them fit tightly.

[0068] Place the Transwell cell culture chamber: Insert the Transwell cell culture chamber into the through - hole of the PDMS cell culture chamber, with its bottom liquid - permeable layer facing the surface of working electrode 2 to form a detection chamber.

[0069] Electrical connection: Use wires to connect the MEMS electrode to an electrochemical detection device to ensure the transmission of electrical signals for real - time detection.

[0070] Example 2: This example provides an electrode, a detection system and a preparation method for real - time monitoring of reactive oxygen species in tumor cells, specifically including: S1. Prepare the nano - enzyme material Weigh 0.020 g of the ZIF-8@ZIF-67 material, dissolve it in 5 mL of methanol, and stir evenly to form a solution. Under the condition of magnetic stirring, sequentially add 2 mL of chloroauric acid solution with a mass concentration of 0.0004 g•mL⁻¹, 3 mL of sodium citrate solution with a mass concentration of 0.002 g•mL⁻¹, and 3 mL of sodium borohydride solution with a mass concentration of 0.0008 g•mL⁻¹. Continuously stir and react for 24 h to form a core-shell structure composite material modified with gold nanoparticles. Subsequently, centrifuge the obtained precipitate at a speed of 9000 r•min⁻¹, wash it 3 times with methanol, and then dry it in vacuum at 65 °C for 12 h to obtain the AuNPs / ZIF-8@ZIF-67 nanozyme material.

[0071] S2. Preparation of MEMS electrodes Using MEMS micromachining technology, construct an electrode array on the glass substrate 4, specifically including: Coat the surface of the glass substrate 4 with a negative photoresist (ROL7133), and sequentially perform pre-baking (110 °C, 180 s), photolithography (exposure for 15 s), post-baking (135 °C, 3 min), and development (FHD-320 developer, development time 40 s).

[0072] Adopt a sputtering process, sequentially sputter 30 nm of Cr as an adhesion layer, and then sputter 200 nm of Au to form an electrode layer.

[0073] Perform a degluing process to remove the photoresist and etch the pattern to obtain the MEMS electrode structure including the working electrode 2, the reference electrode 3, and the counter electrode 1.

[0074] S3. Modify the specific electrode Take 0.8% of the AuNPs / ZIF-8@ZIF-67 nanozyme material prepared in S1, dissolve it in a mixed solution of 99 μL of isopropanol and 1 μL of Nafion, and ultrasonically disperse it evenly. Subsequently, take 2 μL of the obtained mixed solution and drop-coat it on the surface of the working electrode 2 of the MEMS chip, and dry it at room temperature to form a specific catalytic film to obtain the final specific electrode.

[0075] A1. Preparation of the PDMS chamber 5 Prepare the PDMS prepolymer solution: Weigh the PDMS prepolymer and the curing agent, mix them in a weight ratio of 10:0.8, and stir evenly.

[0076] Perform a defoaming treatment: Place the obtained mixture in a vacuum dryer and let it stand for 20 min at a vacuum degree of 10 psi to remove bubbles.

[0077] Mold casting: Pour the degassed PDMS mixture into a 3D printed mold and place it on a horizontal workbench to evenly fill the mold microstructure with the PDMS mixture.

[0078] Curing and forming: After standing at room temperature for 20 min, place the mold in an oven at 75 °C and heat for 0.8 h to completely cure the PDMS.

[0079] Peeling and forming: After the PDMS is cured, carefully peel off the PDMS cell culture chamber and drill a through-hole with a diameter of 6.0 mm at its top to match the outer diameter of the Transwell cell culture chamber.

[0080] A2. Assemble the detection system Plasma cleaning and bonding: Use a plasma cleaner to clean the PDMS cell culture chamber and the MEMS electrode, and bond them to make them fit tightly.

[0081] Place the Transwell cell culture chamber: Insert the Transwell cell culture chamber into the through-hole of the PDMS cell culture chamber, with its bottom liquid-permeable layer facing the surface of working electrode 2 to form a detection chamber.

[0082] Electrical connection: Use wires to connect the MEMS electrode to an electrochemical detection device to ensure the transmission of electrical signals and achieve real-time detection.

[0083] Example 3: This example provides an electrode for real-time monitoring of reactive oxygen species in tumor cells, a detection system and a preparation method thereof, specifically including: S1. Prepare the nanozyme material Weigh 0.030 g of ZIF-8@ZIF-67 material, dissolve it in 5 mL of methanol, and stir evenly to form a solution. Under magnetic stirring, sequentially add 2 mL of chloroauric acid solution with a mass concentration of 0.0006 g•mL⁻¹, 3 mL of sodium citrate solution with a mass concentration of 0.003 g•mL⁻¹, and 3 mL of sodium borohydride solution with a mass concentration of 0.0012 g•mL⁻¹. Continuously stir and react for 24 h to form a core-shell structure composite material modified with gold nanoparticles. Subsequently, centrifuge the obtained precipitate at a speed of 9000 r•min⁻¹, wash it 3 times with methanol, and then dry it in vacuum at 70 °C for 12 h to obtain the AuNPs / ZIF-8@ZIF-67 nanozyme material.

[0084] S2. Prepare the MEMS electrode Using MEMS microfabrication technology, an electrode array was constructed on the glass substrate 4 in the same way as in Example 2.

[0085] S3. Modify the specific electrode Take 1.2% of the AuNPs / ZIF-8@ZIF-67 nanozyme material prepared in S1, dissolve it in a mixed solution of 99 μL of isopropanol and 1 μL of Nafion, and disperse it evenly by ultrasonic treatment. Subsequently, take 2 μL of the obtained mixture and drop-coat it onto the surface of the working electrode 2 of the MEMS chip, and dry it at room temperature to form a specific catalytic film, thus obtaining the final specific electrode.

[0086] A1. Prepare the PDMS chamber 5 Prepare the PDMS prepolymer solution: Weigh the PDMS prepolymer and the curing agent, mix them in a weight ratio of 10:1.2, and stir evenly.

[0087] Defoaming treatment: Place the obtained mixture in a vacuum dryer and let it stand for 40 min under a vacuum of 15 psi to remove air bubbles.

[0088] Mold casting: Pour the defoamed PDMS mixture into a 3D printing mold and place it on a horizontal workbench to make the PDMS mixture evenly fill the microstructures of the mold.

[0089] Curing and forming: After standing at room temperature for 40 min, place the mold in an oven at 85°C and heat it for 1.2 h to completely cure the PDMS.

[0090] Peeling and forming: After the PDMS is cured, carefully peel off the PDMS cell culture chamber and drill a through-hole with a diameter of 7.0 mm at its top to match the outer diameter of the Transwell cell culture chamber.

[0091] A2. Assemble the detection system Plasma cleaning and bonding: Use a plasma cleaner to clean the PDMS cell culture chamber and the MEMS electrode, and perform bonding to make them closely fit.

[0092] Place the Transwell cell culture chamber: Insert the Transwell cell culture chamber into the through-hole of the PDMS cell culture chamber, with its bottom liquid-permeable layer facing the surface of the working electrode 2 to form a detection chamber.

[0093] Electrical connection: Use wires to connect the MEMS electrode to an electrochemical detection device to ensure the transmission of electrical signals and achieve real-time detection.

[0094] Further experiments were carried out on the detection system prepared in the above Example 1.Figure 6 SEM image of the AuNPs / ZIF-8@ZIF-67 nanozyme material prepared in Example 1 Figure 4 、 Figure 5 Results graph of the electrode for monitoring the reactive oxygen species of tumor cells prepared in Example 1 for measuring ROS at different concentrations and the corresponding calibration graph of the electrode response vs. ROS concentration (n = 3) Figure 4 Shows the electrochemical response curves of the electrode of the present invention for detecting different concentrations of ROS (represented by hydrogen peroxide), further illustrating that the electrode of the present invention has good catalytic activity towards ROS and can achieve efficient electrochemical signal conversion, thus meeting the requirement for real-time monitoring of ROS release from tumor cells; Figure 5 Shows the calibration curve between the current response value of the electrode and the ROS concentration (n = 3) Figure 5 It also further reflects that there is a good linear relationship between the electrode response signal and the ROS concentration of the present invention, indicating that the electrode has good quantitative detection ability when detecting different concentrations of ROS. This linear relationship further proves the stability and reliability of the electrode, showing that it can be used for quantitative analysis of ROS changes in the cell microenvironment and providing an accurate electrochemical detection tool for the study of tumor cell oxidative stress.

[0095] During the preparation of the PDMS chamber 5, the height of the chamber is set to 1.5 cm, and the length and width dimensions are 1.7 cm × 1.6 cm respectively to ensure that it can stably support the Transwell cell culture insert 6 and form a tight bond with the MEMS electrode. At the same time, through holes are preset at the top of the PDMS chamber 5, and the diameter is controlled within 6.0 - 7.0 mm to ensure the stable placement of the Transwell cell culture insert 6 and enable the liquid-permeable layer at the bottom of the cell culture insert to effectively adhere to the electrode surface to ensure the smooth diffusion of ROS molecules. The specific experiment is as follows:

[0096] Based on the detection system, real-time monitoring of ROS concentration is carried out, taking MCF-7 breast cancer cells as an example: (1) Cell culture and pre-experiment treatment Cell selection: Select MCF-7 breast cancer cells as the experimental object. As Figure 1 shown, the incubated tumor cells 7 are placed in the transwell cell culture insert 6. This cell is an adherent growth type tumor cell, suitable for culturing in the Transwell cell culture insert 6 and monitoring ROS release.

[0097] Cell culture: MCF-7 breast cancer cells were seeded in Transwell cell culture inserts 6, and the culture medium contained 10% fetal bovine serum and 2% antibiotics. The culture environment was set at 37°C and 5% CO2 to ensure the normal growth and metabolism of the cells.

[0098] Cell culture density: The cell seeding density was set at 1×106 cells / well. When the MCF-7 cells reached 80% confluence, the cells were rinsed with PBS solution and replaced with serum-free PBS solution to reduce background interference.

[0099] (2) Cell incubation and real-time monitoring of ROS Incubation: The MCF-7 breast cancer cells in PBS solution were incubated in the detection system for 30 min to stabilize the cell state and allow the release of ROS.

[0100] DPV electrochemical detection: An electrochemical workstation was connected to the detection system, and differential pulse voltammetry (DPV) was used to collect electrical signals. The peak current density at this time was recorded and converted into the basal ROS concentration level of MCF-7 cells; Stimulation experiment: AA (ascorbic acid) or PMA (phorbol ester) was added to the PBS culture medium as ROS stimulants respectively to induce different levels of ROS production in MCF-7 breast cancer cells; Second DPV detection: After 30 min of stimulation, the second measurement was performed using the DPV method. The ROS content produced by MCF-7 breast cancer cells after stimulation was calculated, and compared with the basal state to analyze the change of ROS level.

[0101] Analysis of experimental results: Determination of initial ROS level: In the absence of stimulation, the ROS release amount of MCF-7 breast cancer cells was low, and the measured peak current density was relatively stable.

[0102] Change of ROS level: After adding AA or PMA, the ROS level increased significantly, and the peak current density increased significantly, indicating that AA or PMA can be used as oxidative stress inducers to increase the intracellular ROS concentration.

[0103] Data comparison and analysis: Through the comparison and analysis of the measured electrochemical signals, the influence trend of AA or PMA on the ROS release of cells was obvious, indicating that the detection system of the present invention can effectively monitor the dynamic changes of the reactive oxygen species level in tumor cells and can be used for the ROS detection research under different experimental conditions.

[0104] The results of this experiment show that the constructed detection system for real-time monitoring of reactive oxygen species (ROS) in tumor cells can directly perform highly sensitive, real-time, and long-term monitoring of ROS released by MCF-7 breast cancer cells without fluorescence staining or chemiluminescent probes, and can accurately quantify the changes in ROS under different stimulation conditions. This also indicates that the system of the present invention has broad application prospects in the fields of research on tumor cell oxidative stress response, anti-cancer drug screening, and tumor microenvironment regulation.

[0105] In summary, the present invention provides an electrode, a detection system, and a preparation method thereof for real-time monitoring of reactive oxygen species in tumor cells. By using MEMS technology to prepare highly sensitive electrodes and modifying the surface thereof with AuNPs / ZIF-8@ZIF-67 nanozyme materials, a highly selective and stable electrochemical sensor is constructed. Combining the integrated design of the PDMS chamber 5 and the Transwell cell culture chamber 6, this system can achieve real-time and highly sensitive detection of reactive oxygen species (ROS) released by tumor cells without fluorescence staining or chemiluminescent probes. The present invention mainly solves the problems of non-specific interference, high background signal, poor quantitative accuracy, and complex operation in ROS detection by traditional fluorescence probes and chemiluminescent methods. Through the design of the MEMS chip-based detection system, cell incubation, culture, and detection are integrated, greatly improving the experimental repeatability and detection efficiency, and realizing long-term and dynamic monitoring of the oxidative stress level of tumor cells. Moreover, the detection system of the present invention can be widely applied in the fields of mechanism research of reactive oxygen species in tumor cells, anti-cancer drug screening, tumor microenvironment regulation, and personalized precision medicine, providing an efficient, convenient, and reusable new technical platform for label-free and real-time monitoring of the metabolic state of tumor cells.

[0106] In the description of the present application, the description referring to terms such as "one embodiment", "some embodiments", "in this embodiment", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0107] As described above, it is only the specific implementation manner of the present application. However, the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present application should be covered within the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. A method for preparing an electrode for real-time monitoring of reactive oxygen species in tumor cells, characterized in that, It includes the following steps: S1. Prepare the nanozyme material: Provide ZIF-8@ZIF-67 material and dissolve it in methanol to form a solution. Under stirring conditions, add chloroauric acid solution, sodium citrate solution and sodium borohydride solution to the solution in sequence. After stirring and reacting, a core-shell structure composite material modified with gold nanoparticles is formed. Subsequently, centrifugal washing is carried out and vacuum drying is performed to obtain the AuNPs / ZIF-8@ZIF-67 nanozyme material; S2. Prepare the MEMS electrode: Adopt the MEMS method of photolithography, development, sputtering and etching to form a gold / chromium working electrode, a reference electrode and a counter electrode on a glass substrate to obtain the MEMS electrode; S3. Modify the specific electrode: Dissolve the AuNPs / ZIF-8@ZIF-67 nanozyme material prepared in step S1 in a mixed solution of isopropanol and Nafion to obtain a mixed solution. Take the mixed solution and drop-coat it on the surface of the working electrode of the MEMS chip prepared in step S2. After drying, a specific catalytic film is formed, that is, an electrode for real-time monitoring of reactive oxygen species in tumor cells is prepared.

2. The preparation method of the electrode according to claim 1, characterized in that, In the step S1, the mass-volume ratio of the ZIF-8@ZIF-67 material to methanol is (0.020 - 0.030) g : 5 mL; the volume ratio of methanol, chloroauric acid solution, sodium citrate solution, and sodium borohydride solution is 5 : 2 : 3 : 3; the mass concentration of the chloroauric acid solution is (0.0004–0.0006) g·mL -1 , the mass concentration of the sodium citrate solution is (0.002–0.003) g·mL -1 , the mass concentration of the sodium borohydride solution is (0.0008–0.0012) g·mL -1 .

3. The preparation method of the electrode according to claim 1, wherein In the step S1, the time of the stirring reaction is 24 hours, and the conditions of the centrifugal washing are centrifugation at a frequency of 9000 r·min -1 , and then the composite material is washed with methanol more than 3 times. The conditions of the vacuum drying are vacuum drying in an oven at 65-70 °C for 12 h.

4. The preparation method of the electrode according to claim 1, wherein, In step S2, in the gold / chromium working electrode, the thickness of Au is 200 nm and the thickness of Cr is 30 nm.

5. The preparation method of the electrode according to claim 1, characterized in that, In step S3, in the mixed solution, the volume ratio of isopropanol to Nafion is 99:1, and the added mass of the AuNPs / ZIF-8@ZIF-67 nanozyme material is 0.8-1.2% of the mixed solution.

6. An electrode for real-time monitoring of reactive oxygen species in tumor cells, characterized in that, The electrode is prepared by the preparation method of any one of claims 1-5, and the electrode includes: A substrate (4), the material of the substrate (4) is glass; A counter electrode (1), a working electrode (2), and a reference electrode (3), and the counter electrode (1), the working electrode (2), and the reference electrode (3) are adjacent to each other and fixedly spaced on the surface of the substrate (4).

7. A detection system for real-time monitoring of reactive oxygen species in tumor cells, characterized in that, The detection system includes the electrode for real-time monitoring of reactive oxygen species in tumor cells according to claim 6, a Transwell cell culture chamber (6), and: A PDMS chamber (5), which is arranged above the working electrode (2) and is closely attached to the working electrode (2). A through hole is opened on the PDMS chamber (5), and the diameter of the through hole matches the outer diameter of the Transwell cell culture chamber (6) for fixing the Transwell cell culture chamber (6) and making its bottom permeable layer face the surface of the working electrode (2) to form a detection chamber for incubating tumor cells and allowing ROS to diffuse to the electrode surface; An electrochemical detection device, which is electrically connected to the working electrode (2) and is used for collecting and analyzing electrochemical signals to real-time detect the concentration of ROS released by tumor cells.

8. The detection system for real-time monitoring of reactive oxygen species in tumor cells according to claim 7, characterized in that, The diameter of the working electrode (2) is the same as the diameter of the Transwell cell culture chamber (6).

9. A method for preparing the detection system according to any one of claims 7-8, characterized in that, It includes the following steps: A1. Prepare the PDMS chamber (5): Prepare a PDMS chamber (5) using PDMS material. Open a through-hole in the PDMS chamber (5), and the diameter of the through-hole matches the outer diameter of the Transwell cell culture insert (6); obtain the PDMS chamber (5) through curing and forming and then peeling. A2: Assemble the detection system: Use plasma cleaning and bond the PDMS chamber (5) obtained in step A1 to the working electrode (2) to make them closely fit; then place the Transwell cell culture insert (6) in the through-hole of the PDMS chamber (5) with its bottom liquid-permeable layer facing the surface of the working electrode (2) to form a detection chamber and use it to incubate tumor cells so that ROS can diffuse towards the surface of the working electrode (2). Connect the electrode to an electrochemical detection device to ensure the transmission of electrical signals and achieve real-time detection of the concentration of ROS released by tumor cells, thus completing the preparation.

10. The preparation method of the detection system according to claim 9, characterized in that, The step A1 includes: (a) Prepare a PDMS prepolymer solution: Weigh the PDMS prepolymer and the curing agent, mix them in a weight ratio of 10:(0.8 - 1.2), and stir evenly. (b) Degassing treatment: Place the obtained mixture in a vacuum dryer and let it stand for 20 - 40 min under a vacuum of 10 - 15 psi to remove air bubbles. (c) Mold casting: Pour the degassed PDMS mixture into a 3D-printed mold and place it on a horizontal workbench to make the PDMS mixture evenly fill the microstructure of the mold. (d) Curing and forming: After standing at room temperature for 20 - 40 min, place the mold in an oven at 75 - 85 °C and heat it for 0.8 - 1.2 h to completely cure the PDMS. (e) Peeling and forming: After the PDMS is cured, carefully peel the PDMS chamber (5) from the mold to obtain the formed PDMS chamber (5), and open a through-hole with a diameter of 6.0 - 7.0 mm at its top to match the outer diameter of the Transwell cell culture insert (6).

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