Hydrogen peroxide sensing electrode, preparation method thereof, hydrogen peroxide electrochemical sensor and monitoring method

Abstract

The invention discloses a hydrogen peroxide sensing electrode, a preparation method thereof, a hydrogen peroxide electrochemical sensor and a monitoring method. The hydrogen peroxide sensing electrode comprises a tube body, the tube body is provided with a first end and a second end, the first end is provided with a first opening, the second end is a tip, the tip is provided with a second opening, the first opening is communicated with the second opening, and the caliber of the second opening is smaller than that of the first opening; the tip is filled with solid hydrogel embedded with catalase, and the aperture of the solid hydrogel is 20-50 nm; the tube body is filled with an electrolyte, and the electrolyte is a solution with ionic conductivity; the electrolyte is in contact with the solid hydrogel. According to the invention, nano confinement hydrogel and electrophoresis-like technologies are combined, oxygen nano bubbles are used as a signal transduction mechanism, ultrasensitive detection of intracellular H2O2 is realized through ionization sensing, and dynamic change of intracellular hydrogen peroxide can be monitored in situ in real time.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical sensors, and in particular relates to a hydrogen peroxide sensing electrode and a preparation method thereof, a hydrogen peroxide electrochemical sensor and a monitoring method. Background Art

[0002] Parkinson's disease (PD) is a degenerative disease that seriously harms the nervous system, and its pathogenesis is closely related to oxidative stress. Selective degeneration of dopaminergic neurons in the substantia nigra pars compacta is a typical pathological feature of PD. Levodopa (L-DOPA), as the gold standard drug for clinical treatment of PD, replenishes the missing dopamine in the brain and restores nerve conduction function, thereby improving the movement disorder symptoms of Parkinson's patients. However, with the progression of the disease and long-term medication, the therapeutic effect of the drug gradually weakens and may induce motor complications, which is directly related to the oxidative stress generated during drug metabolism. The core mechanism of oxidative stress involves the imbalance of reactive oxygen species, in which hydrogen peroxide (H2O2) plays a crucial regulatory role. Studies have shown that H2O2, as an important reactive oxygen species, exhibits a concentration-dependent dual function in cells: it participates in cell signal transduction at low concentrations (10-100nM), while it can cause severe oxidative damage at high concentrations (μM-mM). Its concentration dynamics are closely related to the progression of neurodegenerative diseases. Therefore, it is crucial to achieve dynamic real-time monitoring of intracellular H2O2.

[0003] At present, the existing H2O2 detection technology has obvious technical bottlenecks: fluorescence imaging is limited by photobleaching and probe permeability, making it difficult to conduct long-term in situ monitoring; conventional electrochemical sensors are susceptible to interference from endogenous substances such as ascorbic acid (0.1-1mM) and uric acid (0.2-2mM); existing nanopore sensors are not selective enough in distinguishing different reactive oxygen species in cells. These technical limitations have severely restricted in-depth research on the dynamic changes of intracellular hydrogen peroxide. Therefore, the development of an efficient, highly sensitive, and highly selective sensing and analysis method for intracellular H2O2 is of key significance for revealing the molecular mechanisms of neurodegenerative diseases, and is an important direction that urgently needs to be broken through in current bioanalytical chemistry. Summary of the invention

[0004] The object of the present invention is to provide a hydrogen peroxide sensing electrode, a preparation method thereof, a hydrogen peroxide electrochemical sensor and a monitoring method. The hydrogen peroxide sensor is a highly sensitive iontronic sensor based on dual signal amplification, which can be used for in-situ monitoring of intracellular hydrogen peroxide. By using the pores of the hydrogel as nano-confined ion channels to amplify the iontronic signal and enhancing the interfacial reaction efficiency through the electrophoretic-like enrichment of hydrogel enzyme molecules, combined with the dual signal mechanism of bubble-induced charge screening and volume exclusion, the nM-level dynamic monitoring of H2O2 is realized, providing a new sensing mechanism for cell-related biomolecular sensing and a new idea for the research related to the ultimate realization of real-time and in-situ dynamic monitoring of intracellular substances, with broad application prospects.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a hydrogen peroxide sensing electrode, including a tube body, the tube body having a first end and a second end, the first end having a first opening, the second end being a tip, the tip having a second opening, the first opening and the second opening being in communication, and the diameter of the second opening being smaller than that of the first opening;

[0007] The interior of the tip is filled with a solid hydrogel embedding hydrogen peroxide enzyme, and the pore size of the solid hydrogel is 20 - 50 nm;

[0008] The tube body is filled with an electrolyte, and the electrolyte is a solution having ionic conductivity;

[0009] The electrolyte is in contact with the solid hydrogel.

[0010] In the above hydrogen peroxide sensing electrode, further, the hydrogel is 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium] propane-1-sulfonate inner salt hydrogel.

[0011] In the above hydrogen peroxide sensing electrode, further, the mass ratio of the hydrogen peroxide enzyme to the monomer of the solid hydrogel is (1 - 5):50.

[0012] In the above hydrogen peroxide sensing electrode, further, the inner diameter of the tube body between the first end and the second end of the tube body is 1.1 mm;

[0013] The length of the tip is 5 - 10 mm;

[0014] The gel filling length inside the tip is 5 - 10 μm;

[0015] The semi-cone angle of the tip is 5°;

[0016] The diameter of the second opening is 200 - 300 nm.

[0017] In the above hydrogen peroxide sensing electrode, further, the electrolyte is phosphate buffer solution, Tris-HCl buffer solution or HEPES buffer solution.

[0018] In a second aspect, the present invention provides a method for preparing the hydrogen peroxide sensing electrode described in any one of the above, comprising the following steps:

[0019] S1. Provide the tube body;

[0020] S2. Fill the solid hydrogel embedding catalase at the tip;

[0021] S3. Inject the electrolyte into the tube body from the first opening to obtain the hydrogen peroxide sensing electrode.

[0022] In the above method for preparing the hydrogen peroxide sensing electrode, further, the monomer for preparing the hydrogel is 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium] propane-1-sulfonate inner salt, and the step S2 includes: 1) adding a catalyst to a precursor solution composed of the monomer, the catalase, a crosslinking agent, an initiator and a buffer solution to obtain a mixed solution; 2) inserting the tip of the tube body into the mixed solution, enabling the mixed solution to enter the tip by capillary action and performing free radical polymerization at the tip to form the hydrogel embedding catalase.

[0023] In the above method for preparing the hydrogen peroxide sensing electrode, further, the crosslinking agent is N,N'-methylenebisacrylamide;

[0024] The initiator is ammonium persulfate;

[0025] The catalyst is tetramethylethylenediamine;

[0026] The buffer solution is phosphate buffer solution;

[0027] The ratio of the monomer, the crosslinking agent, the initiator, the catalyst and the buffer solution is (200 - 300) mg : (5 - 8) mg : (5 - 6) mg : (5 - 10) μL : (500 - 1000) μL;

[0028] The temperature of the crosslinking reaction is 20 - 40 °C, and the time is 1 - 3 minutes.

[0029] In a third aspect, the present invention provides an electrochemical hydrogen peroxide sensor, comprising:

[0030] An electrochemical workstation;

[0031] A hydrogen peroxide sensing electrode, which is the hydrogen peroxide sensing electrode described in any one of the above or the hydrogen peroxide sensing electrode prepared by the method described in any one of the above;

[0032] A working electrode, one end of which is in contact with the electrolyte solution inside the tube body of the hydrogen peroxide sensing electrode;

[0033] A counter electrode.

[0034] In the above-mentioned electrochemical hydrogen peroxide sensor, both the working electrode and the counter electrode are Ag / AgCl electrodes.

[0035] In a fourth aspect, the present invention provides an application of the hydrogen peroxide sensing electrode described in any one of the above, the hydrogen peroxide sensing electrode prepared by the method described in any one of the above, or the electrochemical hydrogen peroxide sensor described in any one of the above in any one of the following or in preparing a product having any one of the following functions:

[0036] A1) Monitoring the concentration of hydrogen peroxide in a solution;

[0037] A2) Monitoring the concentration of hydrogen peroxide in cells;

[0038] A3) Evaluating the oxidative stress effect of drugs related to Parkinson's disease.

[0039] In a fifth aspect, the present invention provides a method for monitoring the concentration of intracellular hydrogen peroxide or extracellular hydrogen peroxide, using the electrochemical hydrogen peroxide sensor described in any one of the above, comprising the following steps:

[0040] (1) Insert the hydrogen peroxide sensing electrode into the electrolyte solution containing the sample to be tested. The electrolyte solutions inside and outside the tube body are the same. The other end of the working electrode is connected to the electrochemical workstation;

[0041] Insert one end of the counter electrode into the electrolyte solution containing the sample to be tested, and the other end is connected to the electrochemical workstation;

[0042] (2) Apply a voltage between the working electrode and the counter electrode, measure the ionic current of the sample to be tested, and monitor the concentration of hydrogen peroxide in the sample to be tested according to the ionic current.

[0043] In the above method for monitoring intracellular hydrogen peroxide or extracellular hydrogen peroxide concentration, further, before monitoring the sample to be tested, the method includes the following steps: 1) Insert the hydrogen peroxide sensing electrode into the external electrolyte solution of the tube. The electrolyte solutions inside and outside the tube are the same. The other end of the working electrode is connected to the electrochemical workstation, and one end of the counter electrode is inserted into the external electrolyte solution of the tube, and the other end is connected to the electrochemical workstation; 2) Apply a voltage between the working electrode and the counter electrode and maintain it for a period of time to pre-concentrate the catalase at the tip of the tube. Preferably, the applied voltage is -100 to -300 mV, and the duration is 100 to 300 s.

[0044] In the above method for monitoring intracellular hydrogen peroxide or extracellular hydrogen peroxide concentration, further, in step (2), the applied voltage of the amperometry method is -100 to -300 mV.

[0045] The present invention has the following beneficial effects:

[0046] (1) The detection sensitivity is improved, and the real-time monitoring of H2O2 as low as 1 nM is realized, which is significantly better than the prior art.

[0047] (2) By combining the nano-confinement effects of nanotubes and hydrogels, the sensitivity of the sensor is improved, the off-electrical signal is amplified, and the in-situ real-time dynamic monitoring of intracellular H2O2 is realized, providing a new sensing mechanism for cell-related biomolecular sensing.

[0048] (3) The hydrogel used acts gently on the enzyme molecules, can maintain the normal activity of the enzyme, and ensures the accuracy and reliability of the detection.

[0049] (4) The system is easy to operate, can be applied to fields such as cellomics research, clinical diagnosis and treatment of tumor cells, etc., and has broad application prospects.

[0050] In summary, the hydrogen peroxide electrochemical sensor of the present invention combines nano-confined hydrogels and electrophoresis-like techniques, uses oxygen nanobubbles as a signal transduction mechanism, and realizes ultrasensitive detection of intracellular H2O2 through off-electrical sensing. The unique dual-signal amplification strategy amplifies the ionic signal by using the hydrogel pores as nano-confined ion channels and uses electrophoresis-like techniques to aggregate catalase at the tip of the nanopores, significantly improving the signal-to-noise ratio and sensitivity of the sensor. The sensor has a low detection limit (1 nM H2O2), high sensitivity, high selectivity, and real-time dynamic monitoring ability, providing a new analysis platform for in-depth research on the molecular mechanisms of oxidative stress-related diseases (such as Parkinson's disease) and drug screening. Description of the Drawings

[0051] Figure 1Shows the schematic structural diagram of the H2O2 sensing electrode prepared in Example 1 of the present invention; in the figure, the marks are as follows: 100: tube body; 110: tip; 120: electrolyte; 130: solid hydrogel.

[0052] Figure 2 Shows the test principle diagram of the H2O2 electrochemical sensor in Example 1 of the present invention;

[0053] Figure 3 Shows the SEM micrograph of the glass nanotube probe and its tip in Example 1 of the present invention;

[0054] Figure 4 Shows the SEM image of the SBMA hydrogel in the H2O2 sensing electrode prepared in Example 1 of the present invention;

[0055] Figure 5 Shows the alternating appearance characteristics of current rise and current drop during the test of hydrogen peroxide in Example 1 of the present invention;

[0056] Figure 6 Shows the sensing performance test diagram of the H2O2 sensor prepared in Example 1 of the present invention - the signal frequency values corresponding to each concentration of ①②③;

[0057] Figure 7 Shows the selectivity test diagram of the H2O2 sensor prepared in Example 1 of the present invention;

[0058] Figure 8 Shows the intracellular H2O2 sensing result diagram of HeLa cells in Example 2 of the present invention incubated with different concentrations of PMA;

[0059] Figure 9 Shows the intracellular H2O2 sensing result diagram of PC12 cells in Example 2 of the present invention incubated with different concentrations of MPTP;

[0060] Figure 10 Shows the intracellular H2O2 sensing result diagram of SH-SY5Y cells in Example 2 of the present invention incubated with different concentrations of L-DOPA;

[0061] Figure 11 Shows the experimental result diagram of the ultra-sensitive ionized sensor for real-time monitoring of the dynamic changes of H2O2 in cells in Example 2 of the present invention. Detailed implementation manners

[0062] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0063] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by terms such as "upper", "lower", etc. is based on the orientation or positional relationship shown in the accompanying drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the system or component referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus cannot be construed as a limitation to the present invention. In addition, the use of terms such as "first", "second", etc. to limit components is only for the convenience of distinguishing the above-mentioned components. Without further statement, the above terms have no special meaning and cannot be construed as indicating or implying relative importance.

[0064] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "assembly", "installation", "connection" 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, and it can be the communication inside two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0065] The present invention will be further described below with reference to the accompanying drawings of the specification.

[0066] As Figure 1 shown, in the first part, the hydrogen peroxide sensing electrode provided by the present invention includes a tube body 100. The tube body 100 has a first end and a second end. The first end has a first opening, and the second end is a tip 110. The tip 110 has a second opening. The first opening and the second opening are in communication. The diameter of the second opening is smaller than that of the first opening. The inside of the tip 110 is filled with a solid hydrogel 130 embedding hydrogen peroxide enzyme. The pore diameter of the solid hydrogel 130 is 20-50 nm. The tube body 100 is filled with an electrolyte 120, and the electrolyte 120 is a solution with ionic conductivity. The electrolyte 120 is in contact with the solid hydrogel 130.

[0067] Based on the above technical solutions, as one of the dual-signal amplification strategies, the present invention uses the pores of the hydrogel as a closed space for ion flow, so that the signal generated by the nanobubbles in the pores is amplified by the smaller gel pores, thereby amplifying the ion change signal and improving the sensitivity.

[0068] In the present invention, the hydrogel needs to maintain a good shape and not swell in a buffer solution. In addition, it has biocompatibility and has no effect on enzyme activity. In some embodiments of the present invention, the hydrogel is a 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium] propane-1-sulfonate inner salt (methacryloylethylsulfobetaine, SBMA) hydrogel, which has sulfonate anions and quaternary ammonium cations, a net charge of zero, strong water solubility, and is gentle on enzyme molecules.

[0069] In at least one embodiment of the present invention, the mass ratio of the catalase to the monomer of the hydrogel is (1-5):50, such as 1:50.

[0070] In at least one embodiment of the present invention, the inner diameter of the tube between the first end and the second end of the tube body 100 is 1.1 mm; the length of the tip is 5-10 mm, such as 5 mm; the gel filling length in the tip is 5-10 μm, such as 8.4 μm; the half-cone angle of the tip is 5°; the diameter of the second opening is 200-300 nm, such as 300 nm. The term "half-cone angle" refers to half of the cone apex angle (i.e., the angle formed by the cone axis and the side). In the present invention, the cone is the cone formed by the tip.

[0071] According to the present invention, the electrolyte solution is phosphate buffer solution, Tris-HCl buffer solution or HEPES buffer solution. Preferably, in some embodiments of the present invention, the electrolyte solution is selected from phosphate buffer solution (PBS) to maintain the normal activity of CAT.

[0072] Second, the present invention provides a method for preparing the hydrogen peroxide sensing electrode described in any one of the above, including the following steps:

[0073] S1. Provide the tube body;

[0074] S2. Fill the solid hydrogel embedding catalase in the tip;

[0075] S3. Inject the electrolyte solution into the tube body from the first opening to obtain the hydrogen peroxide sensing electrode.

[0076] In at least one embodiment of the present invention, the monomer for preparing the hydrogel is 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium] propane-1-sulfonate inner salt, and the step S2 includes: 1) adding a catalyst to a precursor solution composed of the monomer, the catalase, a crosslinking agent, an initiator, and a buffer solution to obtain a mixed solution; 2) inserting the tip of the tube body into the mixed solution, enabling the mixed solution to enter the tip through capillary action and undergoing free radical polymerization at the tip to form the hydrogel embedding catalase. Optionally, the crosslinking agent is N,N'-methylenebisacrylamide; the initiator is ammonium persulfate; the catalyst is tetramethylethylenediamine; the buffer solution is phosphate buffer solution; the ratio of the monomer, the crosslinking agent, the initiator, the catalyst, and the buffer solution is (200-300) mg: (5-8) mg: (5-6) mg: (5-10) μL: (500-1000) μL, such as 200 mg: 5 mg: 5 mg: 5 μL: 500 μL; the temperature of the crosslinking reaction is 20-40 °C, and the time is 1-3 minutes, such as crosslinking for 5-10 s at 25 °C.

[0077] As Figure 2 shown, in the third part, the present invention provides an electrochemical hydrogen peroxide sensor, including:

[0078] An electrochemical workstation;

[0079] A hydrogen peroxide sensing electrode, which is the hydrogen peroxide sensing electrode described in any one of the above or the hydrogen peroxide sensing electrode prepared by the method described in any one of the above;

[0080] A working electrode, one end of which is in contact with the electrolyte solution inside the tube body of the hydrogen peroxide sensing electrode;

[0081] A counter electrode.

[0082] In at least one embodiment of the present invention, both the working electrode and the counter electrode are Ag / AgCl electrodes.

[0083] In the fourth part, the present invention provides the application of the hydrogen peroxide sensing electrode described in any one of the above, the hydrogen peroxide sensing electrode prepared by the method described in any one of the above, or the electrochemical hydrogen peroxide sensor described in any one of the above in any one of the following or in preparing a product with any one of the following functions:

[0084] A1) Monitoring the concentration of hydrogen peroxide in a solution;

[0085] A2) Monitoring the concentration of hydrogen peroxide in cells;

[0086] A3) Evaluating the oxidative stress effect of drugs related to Parkinson's disease.

[0087] Part V. The present invention provides a method for monitoring the concentration of hydrogen peroxide in cells or in vitro, using the electrochemical hydrogen peroxide sensor described in any one of the above, comprising the following steps:

[0088] (1) Insert the hydrogen peroxide sensing electrode into the electrolyte solution containing the sample to be tested. The electrolyte solutions inside and outside the tube are the same. The other end of the working electrode is connected to the electrochemical workstation;

[0089] Insert one end of the counter electrode into the electrolyte solution containing the sample to be tested, and the other end is connected to the electrochemical workstation;

[0090] (2) Apply a voltage between the working electrode and the counter electrode, measure the ionic current of the sample to be tested, and monitor the concentration of hydrogen peroxide in the sample to be tested according to the ionic current.

[0091] Based on the above technical solution, H2O2 molecules in the solution diffuse into the tip of the nanotube and contact and react with the electrophoretically enriched catalase (CAT), generating oxygen nanobubbles and producing a current change (rise / fall) signal. Based on this signal parameter, real-time detection of the H2O2 concentration is achieved. Specifically, after applying a negative electrokinetic voltage to the enzyme in the gel, the enzyme migrates to the tip interface under the drive of the voltage due to its negative charge, causing it to react with the H2O2 to be tested outside the tube and generate oxygen nanobubbles. There are usually a large number of anionic OH- adsorbed on the surface of the bubbles near the surface of the bubbles, making the bubbles as a whole negatively charged. When the bubbles are generated, the ion accumulation induced by the surface charge plays a dominant role, forming a current rise signal; when the volume of the bubbles grows large enough, the generated oxygen nanobubbles can be regarded as insulating nanoparticles, occupying the electrolyte volume at the nanotube orifice and blocking the ionic current at the orifice, forming a current fall signal. By statistically analyzing the signal frequency of the above current signals, the frequency of the current signals has a good linear relationship with the logarithm of the H2O2 concentration. Therefore, the detection of nanobubble events can effectively realize the sensing of the H2O2 concentration. Specifically, the current-time curve is used to record the events of H2O2 decomposition outside the tube to generate oxygen bubbles, and the H2O2 signal is detected by analyzing the current rise / fall events caused by the increase or blockage of the ionic current when the bubbles approach the orifice. The detection time for one time is usually 1000 s. Through statistical calculation, the frequency of the peak signal is proportional to the H2O2 concentration.

[0092] In some embodiments of the present invention, before monitoring the sample to be tested, the method includes the following steps: 1) Insert the hydrogen peroxide sensing electrode into the external electrolyte solution of the tube. The electrolyte solutions inside and outside the tube are the same. The other end of the working electrode is connected to the electrochemical workstation, and one end of the counter electrode is inserted into the external electrolyte solution of the tube, and the other end is connected to the electrochemical workstation; 2) Apply a voltage between the working electrode and the counter electrode and maintain it for a period of time to pre-concentrate the catalase at the tip of the tube. Preferably, the applied voltage is -100 to -300 mV, and the duration is 100 to 300 s. More preferably, the applied voltage is -300 mV, and the duration is 100 s. As the second dual-signal amplification strategy, the present invention further adopts the mimetic-electrophoresis method (MEP) to effectively electrophorese CAT to the gel interface at the tip of the tube through the hydrogel, thereby improving the signal-to-noise ratio and signal frequency and realizing the real-time monitoring of H2O2 as low as 1 nM.

[0093] In some embodiments of the present invention, in step (2), the applied voltage of the amperometry method is -100 to -300 mV, such as -100 mV.

[0094] In some embodiments of the present invention, the sample is a cell sample. The nanotube of the sensing electrode is inserted into the liquid storage space for storing the incubation solution containing cells and can be inserted into the cells. The other end of the working electrode is connected to the electrochemical workstation. One end of the counter electrode is inserted into the liquid storage space for storing the incubation solution containing cells and can be inserted into the cells, and the other end is connected to the working electrode through a patch clamp. Observe the nerve cells through a microscope to insert the nanotube into the cells. When a voltage is applied to the working electrode and the counter electrode to form a current path, H2O2 molecules move from the bulk solution to the vicinity of the nanotube mouth by diffusion and contact-react with CAT at the hydrogel interface at the tip of the tube under the action of the electric field in the sensitive area outside the tube, generating oxygen bubbles that cause changes in the ionic current, realizing single-cell H2O2 analysis.

[0095] In some embodiments of the present invention, when this sensor is implanted into cells, for cells stimulated with different concentrations of drugs, the peak signals generated by single-cell events show different peak frequencies. By statistically analyzing the change frequency of the ionic current, the change in the intracellular H2O2 release after the cells are externally stimulated by drugs can be monitored in real time. In this method, the change in the ionic current signal caused by the bubbles during the H2O2 release process is used as the basis for judging the change in H2O2, which can be used to analyze the changes in oxidative stress in different physiological nerve cells and the impact of such changes on the functions of nerve cells under pathological conditions. This non-invasive and non-labeled single-cell detection and recognition method provides a new platform for future clinical diagnosis, medicine and other fields.

[0096] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0097] The methods used in the following embodiments, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0098] The sources of the various materials in the following embodiments are as follows:

[0099] Catalase is a product of Sigma-Aldrich, with the product catalog number C9322 and an enzyme activity of 2,000 - 5,000 units / mg protein. HeLa cells, SH-SY5Y cells, and PC12 cells are all provided by the "National Stem Cell Bank of China (NSTI-BMCR)".

[0100] The composition of 1×PBS is as follows: 15.5 mM NaCl, 0.3 mM Na2HPO4, 0.1 mM KH2PO4, without CaCl2 and MgCl2, with a pH of 7.4, purchased from Gibco.

[0101] Example 1: Preparation and performance testing of an ultrasensitive hydrogen peroxide electrochemical sensing electrode

[0102] 1. Preparation of the hydrogen peroxide sensing electrode

[0103] (1) Preparation of nanotubes

[0104] Borosilicate glass with an outer diameter of 1.50 mm and an inner diameter of 1.1 mm was drawn into a nano-glass tube with a tube diameter of about 300 nm and a half-cone angle of 5° at the tube opening by a CO2 laser puller (P-2000, Sutter Instrument Co., Ltd.), which is the core component of the detection sensor. The specific pulling procedure is as follows:

[0105] (Cycle 1) Temperature = 325, focus range = 5, speed = 20, delay = 128, pull force = 50;

[0106] (Cycle 2) Temperature = 350, focus range = 4, speed = 15, delay = 130, pull force = 175.

[0107] The following-sized glass nanotubes were obtained: total length 5 cm, tip length 5 mm, inner diameter of the tube body 1.1 mm, and tip opening diameter 300 nm.

[0108] (2) Hydrogel filling

[0109] Dissolve 0.2 g of 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium] propane-1-sulfonate (methacryloylethylsulfobetaine, SBMA) and 2 mg of catalase in 500 μL of phosphate buffer (the concentration of the catalase solution is 4 mg / mL). Add 5 mg of N,N'-methylenebisacrylamide (MBAA) as a crosslinking agent and 5 mg of ammonium persulfate (APS) as an initiator, and vortex for 1 minute to obtain a precursor solution for gel-embedded enzyme. Subsequently, add 5 μL of N,N,N',N'-tetramethylethylenediamine (TEMED) as a catalyst. The mixed solution quickly enters the tip of the nanotube by capillary action and is further crosslinked at 25 °C for 5 - 10 s through free radical polymerization to form a solid hydrogel, fixing the enzyme molecules at the tip of the nanotube. The scanning electron microscope (SEM) characterization diagram of the hydrogel-filled nanotube is as shown in the appendix Figure 3 As shown, the SEM diagram of the SBMA hydrogel is as shown in the appendix Figure 4 As shown, the filling length of the gel is 8.4 μm.

[0110] (3) Quasi-electrophoresis application

[0111] Inject the electrolyte solution into the glass nanotube from the upper opening with a syringe. Insert an Ag / AgCl electrode as the working electrode into the nanotube filled with the electrolyte solution, and another Ag / AgCl electrode as the counter electrode is immersed in the electrolyte solution outside the tube. The electrolyte solution is PBS. Apply a voltage between the two electrodes to form an ionic current path, and the application of the voltage and the recording of the ionic current are completed by an Axopatch 200B patch clamp recording system. Insert the nanotube into the electrode, apply a voltage of -300 mV for 100 s to pre-concentrate CAT at the tip of the nanotube to enhance the enrichment of the enzyme at the mouth of the nanotube and improve the sensing performance.

[0112] (4) H2O2 sensing performance test

[0113] Using the above system, insert the Ag / AgCl working electrode into the nanotube in the sensing electrode filled with PBS. The above-mentioned CAT-preconcentrated sensing electrode and another Ag / AgCl counter electrode are placed in a PBS solution of H2O2 with a concentration of 100 μM. Use an electrochemical workstation and adopt the amperometric method to apply a voltage of -100 mV between the working electrode and the counter electrode. As shown in the appendix Figure 5 As shown, after H2O2 contacts with CAT in the hydrogel, it quickly decomposes to produce oxygen nanobubbles. These bubbles will cause instantaneous ionic current changes near the nanotube, generating observable current spikes (current rise / fall changes) at a negative applied potential.

[0114] Perform the same operations as above, and adjust the mass of catalase to 4 mg / mL, 4 mg / mL, and 0 mg / mL respectively. Set the pre-concentration electrophoresis voltage of CAT to -300 mV, 0 mV, and 0 mV respectively, and observe the changes in the ionic current of the sensing electrode embedded with different catalases and applied with different electrophoresis voltages, as shown in the appendix Figure 6 ① CAT-SBMA-MEP nanotubes, ② CAT-SBMA nanotubes, and ③ CAT nanotubes. The increase in the enzyme loading on the interface of CAT-SBMA-MEP nanotubes improves the reaction efficiency, resulting in a significant amplification of the current in the nanotubes. The peak signal frequency increases from 0.067 s -1 to 0.846 s -1 , which is better than the anhydrous gel CAT nanotubes and the CAT-SBMA nanotubes lacking MEP. Among them, the preparation steps of the CAT nanotubes are the same as those of the above-mentioned sensing electrode, only inhaling the hydrogel into the tip of the nanotube after preparation, without pre-embedding catalase; the preparation steps of the CAT-SBMA nanotubes lacking MEP are the same as those of the above-mentioned sensing electrode, only omitting the step of pre-concentrating catalase, i.e., the electrophoresis (MEP) step.

[0115] Perform the same operations as above, only adjust the concentrations of H2O2 in the substrate to 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM respectively, and observe the changes in the ionic current at different substrate concentrations. The experimental results are as shown in Figure 6 the signal frequency values corresponding to each concentration of ①②③. As the substrate concentration increases, the signal frequency also increases proportionally, confirming the positive correlation between the signal frequency and the H2O2 concentration.

[0116] As shown in the appendix Figure 7 Perform a selectivity test on the H2O2 sensing electrode. Apply a voltage of -100 mV and add 200 μM H2O2, 200 μM 5-HT, 15 μM AA, 200 μM DOPAC, 200 μM DA, 200 μM E, 200 μM NE, and 200 μM UA respectively. The results show that except for H2O2, the ion sensor has no obvious current response to other substances, proving that this sensor has good specificity for H2O2.

[0117] Example 2: Real-time monitoring of intracellular H2O2

[0118] (1) Cell culture

[0119] HeLa cells and SH-SY5Y cells: Culture in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin.

[0120] PC12 cells were cultured in RPMI 1640 medium supplemented with 10% horse serum, 5% FBS and 1% penicillin / streptomycin.

[0121] All cells were cultured in a humidified incubator at 37°C, 95% air and 5% CO2, and the culture medium was refreshed every two days. For single-cell experiments, cells were seeded in 60 mm tissue culture dishes at 5-10% confluence 24 hours before the experiment to obtain isolated single cells.

[0122] (2) Drug treatment

[0123] HeLa cells: incubated with different concentrations of PMA for 2 hours, the concentrations were 0, 100, 300, 500 nM respectively.

[0124] PC12 cells were incubated with different concentrations of MPTP for 2 hours, with the concentrations being 0, 100, and 300 μM, respectively.

[0125] SH-SY5Y cells: treated with different concentrations of L-DOPA for 2 hours, with concentrations of 0, 50, and 100 μM, respectively. The cell seeding density was 2×10 5 cells / 20 mm confocal dish.

[0126] (3) Ionization sensor Intracellular H2O2 sensing

[0127] The prepared ionization sensor was inserted into the cell, and a voltage of -100mV was applied between the probe inserted into the cell and the counter electrode to record the current rise / fall signal generated by the oxygen nanobubbles in the cell. The ion current data was collected and analyzed using the Axon Digidata 1440A digital-to-analog converter and pCLAMP 10 electrophysiology software. The frequency parameter was extracted from the current signal to achieve real-time analysis of the intracellular H2O2 concentration. Figure 8 , 9 As shown in Figures 10, in cells not treated with drugs, the ionization sensor showed a lower frequency, while cells treated with PMA, MPTP, and L-DOPA, respectively, showed a concentration-dependent increase in frequency, which indicates that the ionization sensor can effectively sense changes in intracellular H2O2.

[0128] In the real-time monitoring of intracellular H2O2 changes stimulated by drugs, as shown in the attached Figure 11 As shown in the figure, after the ionization sensor obtained a stable current response in SH-SY5Y cells, the addition of 1mM L-DOPA induced further current changes and higher frequencies, indicating that the intracellular H2O2 level increased. This result shows that the ionization sensor can effectively detect the real-time dynamic changes of H2O2 at the single-cell level, providing valuable information for cell physiology and pathology research.

[0129] The present invention has been described in detail above. For those skilled in the art, within the scope not departing from the gist and scope of the present invention, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application is intended to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made with conventional techniques known in the art.

Claims

1. A hydrogen peroxide sensing electrode, characterized in that, It includes a tube body, the tube body has a first end and a second end, the first end has a first opening, the second end is a tip, the tip has a second opening, the first opening and the second opening are through, and the diameter of the second opening is smaller than that of the first opening; The inside of the tip is filled with a solid hydrogel embedding catalase, and the pore diameter of the solid hydrogel is 20-50 nm; The tube body is filled with an electrolyte solution, and the electrolyte solution is a solution with ionic conductivity; The electrolyte solution contacts the solid hydrogel.

2. The hydrogen peroxide sensing electrode according to claim 1, characterized in that: The hydrogel is 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium] propane-1-sulfonate inner salt hydrogel.

3. The hydrogen peroxide sensing electrode according to any one of claims 1-2, characterized in that: The mass ratio of the catalase to the monomer of the solid hydrogel is (1-5):

50.

4. The hydrogen peroxide sensing electrode according to any one of claims 1 to 3, characterized in that: The inner diameter of the tube body between the first end and the second end of the tube body is 1.1 mm; The length of the tip is 5-10 mm; The gel filling length inside the tip is 5-10 μm; The semi-cone angle of the tip is 5°; The diameter of the second opening is 200-300 nm.

5. The hydrogen peroxide sensing electrode according to any one of claims 1-4, characterized in that: The electrolyte solution is phosphate buffer solution, Tris-HCl buffer solution or HEPES buffer solution.

6. The preparation method of the hydrogen peroxide sensing electrode according to any one of claims 1-5, comprising the following steps: S1. Provide the tube body; S2. Fill the solid hydrogel embedding catalase in the tip; S3. Inject the electrolyte solution into the tube body from the first opening to obtain the hydrogen peroxide sensing electrode.

7. The preparation method of the hydrogen peroxide sensing electrode according to claim 6, characterized in that: The monomer for preparing the hydrogel is 3-[N,N-dimethyl-[2-(2-methylprop-2-enoyloxy)ethyl]ammonium] propane-1-sulfonate inner salt, and step S2 includes: 1) adding a catalyst to a precursor solution composed of the monomer, the catalase, a crosslinking agent, an initiator and a buffer solution to obtain a mixed solution; 2) inserting the tip of the tube body into the mixed solution, enabling the mixed solution to enter the tip through capillary action and performing free radical polymerization in the tip to form the hydrogel embedding catalase.

8. The preparation method of the hydrogen peroxide sensing electrode according to claim 7, characterized in that: The crosslinking agent is N,N'-methylenebisacrylamide; The initiator is ammonium persulfate; The catalyst is tetramethylethylenediamine; The buffer solution is phosphate buffer solution; The ratio of the monomer, the crosslinking agent, the initiator, the catalyst and the buffer solution is (200-300) mg:(5-8) mg:(5-6) mg:(5-10) μL:(500-1000) μL; The temperature of the crosslinking reaction is 20-40 °C, and the time is 1-3 minutes.

9. An electrochemical hydrogen peroxide sensor, characterized in that, It includes: An electrochemical workstation; A hydrogen peroxide sensing electrode, which is the hydrogen peroxide sensing electrode according to any one of claims 1-5 or the hydrogen peroxide sensing electrode prepared by the method according to any one of claims 6-8; A working electrode, one end of the working electrode contacts the electrolyte solution inside the tube body of the hydrogen peroxide sensing electrode; A counter electrode.

10. The electrochemical hydrogen peroxide sensor according to claim 9, wherein: Both the working electrode and the counter electrode are Ag / AgCl electrodes.

11. Use of the hydrogen peroxide sensing electrode according to any one of claims 1-5, the hydrogen peroxide sensing electrode prepared by the method according to any one of claims 6-8, or the electrochemical hydrogen peroxide sensor according to any one of claims 9-10 in any one of the following or for preparing a product having any one of the following functions: A1) Monitoring the concentration of hydrogen peroxide in a solution; A2) Monitoring the concentration of hydrogen peroxide in cells; A3) Evaluating the oxidative stress effect of drugs related to Parkinson's disease.

12. A method for monitoring the concentration of hydrogen peroxide in cells or in vitro, characterized in that, Using the electrochemical hydrogen peroxide sensor according to any one of claims 9-10, comprising the following steps: (1) Insert the hydrogen peroxide sensing electrode into the electrolyte solution containing the sample to be tested, the electrolyte solutions inside and outside the tube are the same, and the other end of the working electrode is connected to the electrochemical workstation; Insert one end of the counter electrode into the electrolyte solution containing the sample to be tested, and the other end is connected to the electrochemical workstation; (2) Apply a voltage between the working electrode and the counter electrode, test the ionic current of the sample to be tested, and monitor the concentration of hydrogen peroxide in the sample to be tested according to the ionic current.

13. The method for monitoring the intracellular hydrogen peroxide or extracellular hydrogen peroxide concentration according to claim 12, wherein: Before monitoring the sample to be tested, the method includes the following steps: 1) Insert the hydrogen peroxide sensing electrode into the external electrolyte solution of the tube, the electrolyte solutions inside and outside the tube are the same, the other end of the working electrode is connected to the electrochemical workstation, and insert one end of the counter electrode into the external electrolyte solution of the tube, and the other end is connected to the electrochemical workstation; 2) Apply a voltage between the working electrode and the counter electrode and keep it for a period of time to pre-concentrate the catalase at the tip of the tube; preferably, the applied voltage is -100 to -300 mV and the duration is 100 to 300 s.

14. The method for monitoring the intracellular hydrogen peroxide or extracellular hydrogen peroxide concentration according to any one of claims 12-13, characterized in that: In step (2), the applied voltage of the amperometric method is -100 to -300 mV.

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