A method for detecting the content of a target component in a test component based on redox potential

Abstract

This invention discloses a method for detecting the content of a target component in a test component based on redox potential, comprising the following steps: inserting a first electrode and a first reference electrode into a test component containing or not containing the target component; inserting a second electrode and a second reference electrode into an electrochemical probe solution, wherein the electrochemical probe solution is used to generate a chemical potential gradient between the first electrode and the second electrode; connecting the first electrode and the second electrode with a wire; measuring the open-circuit voltage between the first reference electrode and the second reference electrode; and determining the content of the target component according to a standard curve. This method, by rationally selecting the electrochemical probe to ensure that the reduction potential of the cathode is positive than the oxidation potential of the anolyte, allows the electrochemical process in the entire circuit to proceed spontaneously without the need for an external voltage, thereby avoiding the generation of oxidation products. It exhibits superior stability during long-term detection of the target component, especially for analytes such as 5-hydroxytryptamine that severely contaminate the electrode.

Description

Technical Field

[0001] This invention relates to the field of electrochemical analysis and detection technology. More specifically, it relates to a method for detecting the content of a target component in a test component based on redox potential method. Background Technology

[0002] Reducing bioelectroactive substances such as 5-hydroxytryptamine (5-HT), ascorbic acid, uric acid, and dopamine play important roles in organisms. Electrochemical methods, due to their minimal interference with the physiological environment, low background signal, and excellent spatiotemporal resolution, are well-suited for the direct detection of these reducing bioelectroactive molecules using implantable microsensors. Ideally, these reducing bioelectroactive molecules can be directly oxidized on the surface of a carbon electrode, generating a current as a signal output. However, in practice, the electrochemical oxidation process of these molecules is complex: for example, with the oxidation of 5-HT, the oxidation products rapidly polymerize into oligomers (mainly dimers), which are adsorbed onto the electrode surface through π-π stacking, forming an insulating film that affects the electrode's sensing performance. Even in short-term electrochemical detection, this electrode passivation significantly reduces current response and sensitivity, thus affecting the analytical detection of current-based sensors.

[0003] In recent years, researchers have shown promise in preventing the adsorption and passivation of electrodes by boron-doped diamond electrodes with fewer oxygen-containing functional groups on their surfaces. However, the large size and low electron density of these electrodes are detrimental to the biocompatibility and sensitivity of brain sensors. Furthermore, advancements in electrochemical techniques have offered different perspectives on addressing the contamination problem. Fast scanning cyclic voltammetry (FSCV) can achieve clean regeneration of electrode surfaces by applying ultra-high potentials or rapid scanning speeds, or shorten residence times to minimize the impact of product contamination. While these methods have practical application potential, the continuous accumulation of oxidation products remains the most pressing issue for long-term in vivo detection applications. Therefore, completely avoiding the generation of oxidation products is the best solution to address the fundamental challenge of electrochemical sensing of bioelectroactive molecules, particularly those heavily contaminated by oxidation products like 5-HT. Summary of the Invention

[0004] The first objective of this invention is to provide a method for detecting the content of a target component in a test component based on redox potential method.

[0005] A second objective of this invention is to provide an electrochemical sensor used in the above-described method.

[0006] The third objective of this invention is to provide a method for detecting the content of a target component in a test component using a bipolar electrode based on redox potential.

[0007] The fourth objective of this invention is to provide an electrochemical sensor for detecting reducing substances in the living environment, particularly for analytical substances such as 5-hydroxytryptamine whose oxidation products severely contaminate the electrode.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for detecting the content of a target component in a test component based on the redox potential method, comprising the following steps:

[0010] The first electrode is used as the anode and is inserted together with the first reference electrode into the test component, which may or may not contain the target component.

[0011] The second electrode is used as the cathode and is inserted into the electrochemical probe solution together with the second reference electrode. The electrochemical probe solution is used to generate a chemical potential gradient between the first electrode and the second electrode.

[0012] Connect the first electrode and the second electrode with a wire;

[0013] The open-circuit voltage between the first and second reference electrodes is tested, and the content of the target component is determined according to the standard curve.

[0014] The first reference electrode and the second reference electrode are made of the same material.

[0015] Preferably, the plotting of the standard curve includes the following steps:

[0016] The first electrode is used as the anode and is inserted together with the first reference electrode into a solution containing different concentrations of the target component;

[0017] The second electrode is used as the cathode and is inserted into the electrochemical probe solution together with the second reference electrode. The electrochemical probe solution is used to generate a chemical potential gradient between the first electrode and the second electrode.

[0018] The first electrode and the second electrode are connected by a wire;

[0019] Test the open-circuit voltage between the first reference electrode and the second reference electrode;

[0020] A standard curve is plotted with the logarithm of the concentration of the target component on the x-axis and the corresponding open-circuit voltage value on the y-axis.

[0021] The first reference electrode and the second reference electrode are made of the same material.

[0022] Preferably, the first reference electrode and the second reference electrode are selected from any one of Ag / AgCl electrode, Hg / HgO electrode, calomel electrode or hydrogen electrode.

[0023] Preferably, the first electrode or the second electrode is selected from any one of carbon fiber electrode, glassy carbon electrode or gold electrode.

[0024] Preferably, the electrochemical probe solution is a K3IrCl6 / K2IrCl6 solution with an equal concentration ratio or a K3[Fe(CN6)] / K4[Fe(CN6)] solution with an equal concentration ratio.

[0025] Preferably, the target component is a reducing substance.

[0026] More preferably, the reducing substance is ascorbic acid, uric acid, dopamine, phenol, bisphenol A, 5-hydroxytryptamine, etc.

[0027] Secondly, the present invention provides an electrochemical sensor for the above-described method, comprising an anode cell and a cathode cell; the anode cell contains a first electrode and a first reference electrode, wherein the first electrode and the first reference electrode are not in contact with each other; the cathode cell contains a second electrode and a second reference electrode, wherein the second electrode and the second reference electrode are not in contact with each other; the anode cell is used to add a test component; and the cathode cell is used to add an electrochemical probe solution.

[0028] Preferably, the first reference electrode and the second reference electrode are made of the same material and are selected from any one of Ag / AgCl electrode, Hg / HgO electrode, calomel electrode or hydrogen electrode.

[0029] Preferably, the first electrode or the second electrode is selected from any one of carbon fiber electrode, glassy carbon electrode and gold electrode.

[0030] Thirdly, the present invention provides a method for detecting the content of a target component in a test component using a bipolar electrode based on the redox potential method, comprising the following steps:

[0031] A conductive fiber is passed through the inside of an insulating tube, with one end of the conductive fiber extending to the outside of the insulating tube. The conductive fiber is fixed, and one end of the insulating tube is sealed. Then, an electrochemical probe solution is placed inside the insulating tube, so that the conductive fiber inside the insulating tube comes into contact with the electrochemical probe solution.

[0032] The first reference electrode and the conductive fiber extending to the outside of the insulating tube are inserted into the same test component, which may or may not contain the target component.

[0033] The second reference electrode is inserted into the electrochemical probe solution inside the insulating tube;

[0034] The open-circuit voltage between the first and second reference electrodes is tested, and the content of the target component is determined by a standard curve.

[0035] The first and second reference electrodes are made of the same material, and the electrochemical probe solution is used to generate a chemical potential gradient between the conductive fibers in contact with the electrochemical probe solution and the conductive fibers extending to the outside of the insulating tube.

[0036] Preferably, the plotting of the standard curve includes the following steps:

[0037] A conductive fiber is passed through the inside of an insulating tube, with one end of the conductive fiber extending to the outside of the insulating tube. The conductive fiber is fixed, and one end of the insulating tube is sealed. Then, an electrochemical probe solution is placed inside the insulating tube, so that the conductive fiber inside the insulating tube comes into contact with the electrochemical probe solution.

[0038] The first reference electrode and the conductive fiber extending to the outside of the insulating tube are inserted into the same solution containing different concentrations of the target component;

[0039] The second reference electrode is inserted into the electrochemical probe solution inside the insulating tube;

[0040] Test the open-circuit voltage between the first reference electrode and the second reference electrode;

[0041] A standard curve is plotted with the logarithm of the concentration of the target component on the x-axis and the open-circuit voltage value on the y-axis.

[0042] The first and second reference electrodes are made of the same material, and the electrochemical probe solution is used to generate a chemical potential gradient between the conductive fibers in contact with the electrochemical probe solution and the conductive fibers extending to the outside of the insulating tube.

[0043] Preferably, the first reference electrode and the second reference electrode are selected from any one of Ag / AgCl electrode, Hg / HgO electrode, calomel electrode and hydrogen electrode.

[0044] Preferably, the electrochemical probe solution is a K3IrCl6 / K2IrCl6 solution with an equal concentration ratio or a K3[Fe(CN6)] / K4[Fe(CN6)] solution with an equal concentration ratio.

[0045] Preferably, the target component is a reducing substance.

[0046] Preferably, the reducing substance is ascorbic acid, uric acid, dopamine, phenol, bisphenol A, 5-hydroxytryptamine, etc.

[0047] Preferably, when the test component is living tissue, the insulating tube is a capillary glass tube and the conductive fiber is carbon fiber.

[0048] Fourthly, the present invention provides an electrochemical sensor for detecting reducing substances in living tissue.

[0049] The electrochemical sensor includes a bipolar carbon fiber electrode; the bipolar carbon fiber electrode includes a fixative, carbon fiber, an electrochemical probe solution, and a conical capillary glass tube, wherein the carbon fiber passes through the conical capillary glass tube and its end portion extends beyond the tip of the capillary glass tube, and the fixative is used to fix the carbon fiber and close the tip opening of the capillary glass tube.

[0050] Preferably, it also includes an electrochemical probe solution filled inside the capillary glass tube.

[0051] Preferably, the reducing substance is ascorbic acid, uric acid, dopamine, phenol, bisphenol A, 5-hydroxytryptamine, etc.

[0052] Preferably, the electrochemical probe solution is a K3IrCl6 / K2IrCl6 solution with an equal concentration ratio or a K3[Fe(CN6)] / K4[Fe(CN6)] solution with an equal concentration ratio.

[0053] In addition, unless otherwise specified, any range described in this invention includes the endpoint, any value between the endpoints, and any subrange consisting of the endpoint or any value between the endpoints.

[0054] The beneficial effects of this invention are as follows:

[0055] The present invention provides a method for detecting the content of a target component in a test component based on redox potential. By rationally selecting the cathode electrochemical probe, the electrochemical process in the entire circuit can proceed spontaneously. Furthermore, the species and concentration of the cathode electrochemical probe can be rationally designed and controlled to achieve the construction of a spontaneous reaction. Almost no current flows through the entire circuit, thus almost no reaction products are generated, resulting in superior stability and enabling stable detection over long periods.

[0056] The present invention further provides a method for detecting the content of a target component in a test component using a bipolar electrode based on the redox potential method. This method not only does not require an external voltage, but the bipolar electrode structure can effectively avoid cross-interference of electrochemical reactions between the anode and cathode, thus exhibiting superior stability and enabling real-time detection over a longer period of time in vivo.

[0057] This invention also provides an electrochemical sensor for detecting reducing substances in a living environment. When this electrochemical sensor is used to detect the content of serotonin in a living brain, almost no current flows, minimizing the generation of oxidation products and avoiding electrode passivation by serotonin oxidation products, thus achieving long-term stable monitoring in a living brain. This can be extended to the study of other molecules with similar serious pollution problems. Attached Figure Description

[0058] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0059] Figure 1 A schematic diagram of the operation of the electrochemical sensor in Example 1 is shown.

[0060] Figure 2 A schematic diagram of the electrochemical sensor of Example 2 is shown.

[0061] Figure 3 The electrochemical sensor of Example 1 is shown to respond to a 10 μM 5-HT injection potential.

[0062] Figure 4 The results show that at a potential of +0.30 V, bare carbon fiber electrodes recorded the current response to the addition of 10 μM 5-HT to artificial cerebrospinal fluid.

[0063] Figure 5 The changes in the potential response of the electrochemical sensor and the current response of the current-type sensor in Example 1 are shown after the addition of 10 μM 5-HT to artificial cerebrospinal fluid.

[0064] Figure 6 The in-situ Raman spectra recorded on the surfaces of the current-type sensor electrode and the electrochemical sensor electrode of Example 2 in artificial cerebrospinal fluid containing 10 mM 5-HT are shown; where A is the in-situ Raman spectrum recorded on the surface of the current-type sensor electrode and B is the in-situ Raman spectrum recorded on the surface of the electrochemical sensor electrode of Example 2.

[0065] Figure 7 The electrochemical sensor of Example 1 is shown to respond to different concentrations of 5-HT and E0. OC The graph shows the logarithmic relationship between E and 5-HT concentration; where A represents the potential response of the electrochemical sensor in Example 1 with continuous addition of 5-HT to artificial cerebrospinal fluid containing a baseline concentration of 10 nM 5-HT, and B represents the potential response of E. OC Graph showing the logarithmic relationship between 5-HT concentration and concentration.

[0066] Figure 8The time response and calibration curves of the electrochemical sensor of Example 1 before and after 4 hours of adsorption in bovine serum albumin solution and before and after 6 hours of implantation in the dorsal raphe nucleus of a guinea pig brain are shown. Among them, A is the time response curve of the electrochemical sensor before and after 4 hours of adsorption in bovine serum albumin solution, B is the calibration curve of the electrochemical sensor before and after 4 hours of adsorption in bovine serum albumin solution, C is the time response curve of the electrochemical sensor before and after 6 hours of implantation in the dorsal raphe nucleus of a guinea pig brain, and D is the calibration curve of the electrochemical sensor before and after 6 hours of implantation in the dorsal raphe nucleus of a guinea pig brain.

[0067] Figure 9 The Ei of the electrochemical sensor in Example 1 is shown when 50 μM 5-HT is microinjected locally into the guinea pig cortex. OC response.

[0068] Figure 10 It shows 70mM K + Typical E was recorded under local microinjection. OC response.

[0069] Figure 11 The electrochemical sensor of Example 1 is shown in the sensory-related change graph for measuring 5-HT signal in vivo, where A represents the change in 5-HT signal at a concentration of 2 μL / min. -1 When 70 mM KCl or artificial cerebrospinal fluid is injected locally into the dorsal raphe nucleus (DRN) at a rapid rate, the real-time E of 5-HT... OC Response B is the E recorded when 5-HT is released after local injection of 70 mM KCl into the DRN before or after escitalopram injection. OC Response, C represents the time before and after escitalopram injection, 70 mM K + The 5-HT overflow time induced by local stimulation, where t 1 / 2 For E OC The duration of the full width at half maximum (FWHM), D is 70 mm K. + E caused by local stimulation OC A statistical chart showing the changes. Detailed Implementation

[0070] The following specific embodiments illustrate the gel electrolyte membrane of the present invention and its preparation method. This description is only intended to enable those skilled in the art to better understand the present invention, but does not limit the present invention in any way.

[0071] In a first aspect, the present invention provides a method for detecting the content of a target component in a test component based on the redox potential method, comprising the following steps:

[0072] The first electrode is used as the anode and is inserted together with the first reference electrode into the test component, which may or may not contain the target component.

[0073] The second electrode is used as the cathode and is inserted into the electrochemical probe solution together with the second reference electrode. The electrochemical probe solution is used to generate a chemical potential gradient between the first electrode and the second electrode.

[0074] Connect the first electrode and the second electrode with a wire;

[0075] The open-circuit voltage between the first and second reference electrodes is tested, and the content of the target component is determined according to the standard curve.

[0076] The first reference electrode and the second reference electrode are made of the same material.

[0077] It is understood that the test component is a liquid or other component that allows electrons to move freely, including but not limited to buffer solutions, organic solutions, gels, etc.

[0078] In the above scheme, by rationally selecting the electrochemical probe to ensure that the reduction potential of the cathode is positive than the oxidation potential of the anolyte, the electrochemical process in the entire circuit can proceed spontaneously without the need for an external voltage, thereby avoiding the generation of oxidation products to a certain extent. Furthermore, the species and concentration of the electrochemical probe at the cathode can be rationally designed and controlled, thereby regulating the sensitivity of the detection.

[0079] In some embodiments of the present invention, the first electrode and the second electrode may be made of the same or different materials.

[0080] The device produced by the above method is also within the scope of protection of this invention. In some embodiments of this invention, an electrochemical sensor is provided, including an anode cell and a cathode cell; the anode cell contains a first electrode and a first reference electrode, the first electrode and the first reference electrode being non-contacting; the cathode cell contains a second electrode and a second reference electrode, the second electrode and the second reference electrode being non-contacting; the first electrode and the second electrode are connected by a wire; the first reference electrode and the second reference electrode are made of the same material; the anode cell is used to add a test component; the cathode cell is used to add an electrochemical probe solution; the electrochemical probe solution is used to generate a chemical potential gradient between the first electrode and the second electrode.

[0081] Secondly, the present invention provides a method for detecting the content of a target component in a test component using a bipolar electrode based on the redox potential method, comprising the following steps:

[0082] A conductive fiber is passed through the inside of an insulating tube, with one end of the conductive fiber extending to the outside of the insulating tube. The conductive fiber is fixed, and one end of the insulating tube is sealed. Then, an electrochemical probe solution is placed inside the insulating tube, so that the conductive fiber inside the insulating tube comes into contact with the electrochemical probe solution.

[0083] The first reference electrode and the conductive fiber extending to the outside of the insulating tube are inserted into the same test component, which may or may not contain the target component.

[0084] The second reference electrode is inserted into the electrochemical probe solution inside the insulating tube;

[0085] The open-circuit voltage between the first and second reference electrodes is tested, and the content of the target component is determined by a standard curve.

[0086] The first reference electrode and the second reference electrode are made of the same material.

[0087] It is understood that the test component is a liquid or other state that allows electrons to move freely, including but not limited to buffer solutions, organic solutions, gels, etc.

[0088] This method is based on the first method. This method not only does not require external pressure, but the entire circuit can spontaneously carry out electrochemical reactions. Moreover, by using bipolar electrodes, it effectively avoids cross-interference between the electrochemical reactions of the anode and cathode. Specifically, the conductive fiber exposed to the outside serves as the anode, while the conductive fiber remaining in the insulating tube and in contact with the electrochemical probe solution that can undergo reduction serves as the cathode.

[0089] In some embodiments of the present invention, the conductive fibers are fixed with an adhesive colloid, such as silicone, 502 glue, or AB glue, and one end of the insulating tube is sealed. The sealed end of the insulating tube forms a closed space capable of containing liquid for adding the electrochemical probe solution. In this case, the conductive fibers can be considered as two parts: the part inside the insulating tube and the part exposed outside the tube. The conductive fibers inside the insulating tube, due to contact with the electrochemical probe solution, are considered the cathode, while the conductive fibers outside the insulating tube are considered the anode.

[0090] The electrochemical probe solution is used to create a chemical potential gradient between the conductive fibers in contact with the electrochemical probe solution and the conductive fibers extending to the outside of the insulating tube.

[0091] Thirdly, the present invention provides an electrochemical sensor for detecting reducing substances in living tissue.

[0092] The electrochemical sensor includes a bipolar carbon fiber electrode; the bipolar carbon fiber electrode includes a fixative, carbon fiber, an electrochemical probe solution, and a conical capillary glass tube, wherein the carbon fiber passes through the conical capillary glass tube and its end portion extends beyond the tip of the capillary glass tube, and the fixative is used to fix the carbon fiber and close the tip opening of the capillary glass tube.

[0093] The electrochemical sensor, proposed based on the second method, is used to detect the content of target components in living tissue.

[0094] In some embodiments of the invention, an electrochemical probe solution is also included, which is filled inside the capillary glass tube.

[0095] In some embodiments of the present invention, when the electrochemical sensor is used, the electrochemical probe solution is then filled inside the capillary glass tube.

[0096] In some embodiments of the present invention, the electrochemical probe solution is used to generate a chemical potential gradient between the carbon fibers in contact with the electrochemical probe solution and the carbon fibers extending to the outside of the capillary glass tube.

[0097] In some embodiments of the present invention, the selectivity of the above-mentioned electrochemical sensor in in vivo detection mainly relies on brain region specificity. For example, when detecting serotonin, the dorsal raphe nucleus, which is rich in serotonergic neurons, is selected as the research object. In non-serotonin-rich brain regions, the surface of the positively charged carbon fiber can be modified with Nafion, which can weaken the potential response of ascorbic acid through the electrostatic repulsion between Nafion and ascorbic acid.

[0098] To make the present invention, its technical solutions, and advantages clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0099] Example 1

[0100] An electrochemical sensor includes a bipolar carbon fiber electrode and an Ag / AgCl reference electrode; the preparation of the bipolar carbon fiber electrode includes the following steps:

[0101] (1) Preparation of bipolar carbon fiber electrodes: First, carbon fibers were threaded into glass capillaries and then drawn into two glass tubes with extremely fine tips and sealed with carbon fibers using a microelectrode drawing instrument. Next, a small amount of liquid silica gel was injected into the tips of the glass tubes from the open end, and then the tubes were centrifuged to ensure that the tips were completely sealed. Then, the electrode was placed at room temperature until the silica gel solidified. The carbon fibers exposed on the outside of the glass capillaries were used as the working electrodes. Finally, the carbon fibers at the tips (100-300 μm) were cut under a microscope, thus completing the preparation of the bipolar carbon fiber electrodes.

[0102] (2) Pretreatment of the bipolar carbon fiber electrode: First, the bipolar carbon fiber electrode was ultrasonically treated sequentially in acetone, 3.0M HNO3, and 1.0M KOH solutions for 3-5 minutes to remove surface impurities. 3.0M KCl was injected into the glass capillary of the bipolar carbon fiber electrode, and an Ag / AgCl wire was inserted into the glass tube. The bipolar carbon fiber electrode was then immersed in 1.0M NaOH solution and treated with a constant voltage (+1.5V) for 80 seconds to achieve preliminary electrochemical activation of the microelectrode. Finally, cyclic voltammetry was performed at a scan rate of 0.05V / s within a voltage range of 0.0-+1.0V until a stable cyclic voltammogram was obtained, thus completing the activation pretreatment process of the bipolar carbon fiber electrode.

[0103] (3) After the pretreatment is completed, the bipolar carbon fiber is thoroughly cleaned with deionized water and set aside for later use.

[0104] Example 2

[0105] An electrochemical sensor includes an anolyte and a cathode. The anolyte contains a glassy carbon electrode 1 and an Ag / AgCl reference electrode 1, which are not in contact with each other. The anolyte also contains a glassy carbon electrode 2 and an Ag / AgCl reference electrode 2, which are also not in contact with each other. The glassy carbon electrode 2 and the glassy carbon electrode 1 are connected by a wire. The anolyte is used to add a test component, and the cathode is used to add an electrochemical probe solution.

[0106] The method for detecting the content of a target component in a test component using the above-mentioned electrochemical sensor includes the following steps:

[0107] 1) Add artificial cerebrospinal fluid, with or without 5-hydroxytryptamine, to the anode tank;

[0108] 2) Add K3IrCl6 / K2IrCl6 solution of equal concentration ratio to the cathode cell;

[0109] 3) Test the open-circuit voltage between the Ag / AgCl reference electrode 1 and the Ag / AgCl reference electrode 2; determine the content of the target component 5-hydroxytryptamine according to the standard curve; wherein, the reduction potential of K2IrCl6 on the glassy carbon electrode 2 is positive to the oxidation potential of 5-hydroxytryptamine on the glassy carbon electrode 1.

[0110] Test Example 1

[0111] Testing the stability of the electrochemical sensor of this invention:

[0112] 5-HT was detected using the electrochemical sensor described in Example 1 on an electrochemical workstation or multimeter. The bipolar carbon fiber electrode and Ag / AgCl reference electrode from the electrochemical sensor were inserted into artificial cerebrospinal fluid. During detection, a K3IrCl6 / K2IrCl6 (5 mM, 1:1 concentration ratio) solution was filled into a glass capillary, and 10 μM 5-HT was added to the artificial cerebrospinal fluid. This resulted in a steady-state potential change of approximately 20 mV. The open-circuit potential remained essentially unchanged over the 2-hour measurement period (see [link to example 1]). Figure 3 ).

[0113] To verify that the electrochemical sensor in Example 1 provides a stable signal recording for 5-hydroxytryptamine (5-HT), the response of 5-HT was compared with that of a conventional current-type electrode. At a potential of +0.30 V, a bare carbon fiber electrode recorded the current response upon addition of 10 μM 5-HT to artificial cerebrospinal fluid. The results are shown below. Figure 4 In traditional current-type sensors, the current drops significantly starting from 5-HT oxidation, and within 30 minutes, the current signal falls below the detection baseline. Figure 4 The passivation effect of 5-HT in current-based sensors was confirmed.

[0114] right Figure 3 and Figure 4 The real-time records are statistically analyzed, and the results are shown in [link to statistics]. Figure 5 ,in, and E OC (I) represents the potential (current) values ​​at the start time and the given time, respectively. Figure 5 The results show that the potential response of the electrochemical sensor based on the present invention changes by only 3% within 30 minutes, while the conventional current-type sensor loses all signal within 30 minutes. Therefore, the electrochemical sensor of the present invention exhibits superior stability in the detection of 5-HT compared to conventional sensors.

[0115] To further verify that the detection method of the present invention has exceptionally high stability due to the almost absence of oxidation products on the electrode surface, this example uses in-situ Raman spectroscopy to analyze the changes in the chemical composition of the electrode surface before and after 5-HT detection. Figure 6 The results show that in artificial cerebrospinal fluid containing 10 mM 5-HT, the current-type sensor ( Figure 6 A) and the electrochemical sensor of Example 2 of the present invention ( Figure 6 B) In-situ Raman spectra recorded on the electrode surface. Specifically, 5-HT was detected using a conventional current-type sensor, and as oxidation began, the spectrum was observed at 1450 cm⁻¹. -1A new Raman peak appeared, caused by the characteristic HCH signal on the indole ring. This indicates that the formation and adsorption of oligomers at the electrochemical interface are strongly correlated with the decrease in oxidation current. As the oxidation time increased to 30 min, the intensity ratio of the D-band and G-band defect parameters on the electrode surface (I0) decreased. D / I G The value decreased from 1.40 to 0.71, indicating that the oxidation reaction caused a significant change in the chemical composition of the electrode surface (see...). Figure 6 A). And based on Figure 2 The detection device in the experiment showed no significant change in the Raman signal after 30 minutes (see...). Figure 6 B), Electrode surface I D / I G No significant changes were observed. These results demonstrate that using a sensor based on redox potential ensures high stability for long-term 5-HT analysis because almost no redox reaction occurs on the electrode surface.

[0116] Test Example 2

[0117] 5-HT was detected using the electrochemical sensor described in Example 1. The detection was performed on an electrochemical workstation or multimeter. The bipolar carbon fiber electrode and Ag / AgCl reference electrode of the electrochemical sensor were inserted into artificial cerebrospinal fluid. During the detection process, a K3IrCl6 / K2IrCl6 (5 mM, concentration ratio 1:1) solution was filled into a glass capillary. Simultaneously, the potential response caused by different concentrations of 5-HT was detected in artificial cerebrospinal fluid (composition: NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM), CaCl2 (1.1 mM)). The relationship between the potential response and the concentration of 5-HT was plotted. When different concentrations of 5-HT (0.05 μM, 0.2 μM, 0.5 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM) were added to the system, the peak current response showed an increasing trend. Furthermore, within the concentration range of 50 nM to 50 μM, the potential response signal of the bipolar carbon fiber electrode in this invention showed a linear correlation with the logarithm of the 5-HT concentration (see...). Figure 7 (As shown).

[0118] Figure 7 The results show that the bipolar electrochemical sensor based on the redox potential method of this invention has good detection sensitivity under physiological conditions, and the range of 5-HT concentration change in the brain is within its linear response range. Therefore, this electrode can meet the requirements of in vivo electrochemical detection.

[0119] Test Example 3

[0120] Test 1: The electrochemical sensor of Example 1 was tested in bovine serum albumin (BSA) solution (40 mg / mL). -1 Adsorption was performed for 4 hours, and then the time response and calibration curve before and after adsorption were measured respectively (during the detection process, a K3IrCl6 / K2IrCl6 (5mM, concentration ratio 1:1) solution was filled into the glass capillary). The results are shown in […]. Figure 8 As shown in AB.

[0121] Test 2: The electrochemical sensor from Example 1 was implanted into the dorsal raphe nucleus of a guinea pig brain for 6 hours. Its time response and calibration curve were measured before and after adsorption (during the detection process, a K3IrCl6 / K2IrCl6 (5mM, concentration ratio 1:1) solution was filled into the glass capillary). The results are shown in […]. Figure 8 CD shown.

[0122] Figure 8 The results showed that the sensitivity of the electrochemical sensor in Example 1 was independent of the electroactive surface area, even in high concentrations of bovine serum albumin solution (BSA, 40 mg / mL). -1 After incubation for 4 hours in a solution or implantation in the brain of a guinea pig for 6 hours, the sensor maintained its excellent and consistent potential response and sensitivity. This result demonstrates that the electrochemical sensor of this invention can still stably and effectively sense 5-HT in protein-rich biological environments.

[0123] Test Example 4

[0124] In vivo application of the electrochemical sensor in Example 1

[0125] To verify the sensor of this invention's ability to achieve long-term stable monitoring of 5-HT during in vivo detection, we used the electrochemical sensor of Example 1 to detect the release of 5-HT in the brain of guinea pigs. During the detection process, a K3IrCl6 / K2IrCl6 (5 mM, concentration ratio 1:1) solution was filled into a glass capillary. First, the tip of a bipolar carbon fiber electrode was implanted near the guinea pig cortex, and 1.0 μL of 5-HT solution (50 μM) was micro-injected into this area. A micro-reference electrode (Ag / AgCl) was placed on the surface of the rat brain or in the skull to record changes in open-circuit potential. Following in-situ microinjection of 5-HT, the open-circuit potential significantly increased within 30 seconds (see results). Figure 9 This result demonstrates that the electrochemical sensor of the present invention can detect 5-HT in vivo.

[0126] Test Example 5

[0127] Test 1: We filled the glass capillary of the electrochemical sensor from Example 1 with K3IrCl6 / K2IrCl6 (5mM, concentration ratio 1:1), and then implanted it into the dorsal raphe nucleus of the mouse brain. Endogenous 5-HT release was induced using a high-potassium (70mM) or artificial cerebrospinal fluid stimulation model (results are shown in...). Figure 11 A). K + Following stimulation, a dramatic increase in potential of more than 2.7-fold (concentration change of 1.4-fold) was observed within 50 seconds. Subsequently, the potential gradually decreased to basal levels within 90 seconds. This observation is very similar to previously reported results and may be a consequence of the body's intrinsic systems maintaining extracellular 5-HT homeostasis. Furthermore, after three consecutive high-K... + Following stimulation, the continuously recorded potential responses showed a similar release pattern (see results). Figure 10 Among them, the small rectangular bars represent local injections of K. + The duration of the effect further demonstrates the high stability of this electrochemical sensor. Importantly, the use of artificial cerebrospinal fluid instead of high-potassium solution for in-situ microinjection did not significantly alter the potential change, further highlighting the sensor's ability to reliably detect endogenous 5-HT changes with good spatial-temporal resolution.

[0128] Test 2: To explore whether the electrochemical sensing sensor of Example 1 can be used to study changes in 5-HT in the living brain induced by drug treatment (wherein, during the detection process, a K3IrCl6 / K2IrCl6 (5mM, concentration ratio 1:1) solution was filled into a glass capillary). For this purpose, escitalopram (10 mg / kg) was injected intraperitoneally into guinea pigs 15 minutes before high potassium stimulation. -1 Escitalopram, a commonly used selective serotonin reuptake inhibitor (SSRI), was used to compare the potential responses before and after escitalopram injection (see...). Figure 11 B), Figure 11 CD showed a 1.7-fold increase in spillover time and a 1.2-fold increase in potential after drug treatment (concentration change of 1.1-fold). This indicates that SSRIs are more effective at slowing the reuptake process than at regulating SSRI-related extracellular 5-HT concentrations, which may have important implications for the treatment of several mental illnesses. These results demonstrate that the bipolar carbon fiber electrode potential-type sensor based on redox potential in this invention can not only be used for real-time sensitive detection of in vivo 5-HT, but also provides a detection tool for studying drug-regulated 5-HT kinetic changes.

[0129] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for detecting the content of a target component in a test component based on redox potential method, characterized in that, The method is based on an electrochemical sensor comprising an anolyte and a cathode. The anolyte contains a first electrode and a first reference electrode, which are not in contact with each other. The cathode contains a second electrode and a second reference electrode, which are not in contact with each other. The anolyte is used to add the test component, and the cathode is used to add an electrochemical probe solution. The method includes the following steps: The first electrode is used as the anode and is inserted together with the first reference electrode into the test component, which may or may not contain the target component. The second electrode is used as the cathode and is inserted into the electrochemical probe solution together with the second reference electrode. The electrochemical probe solution is used to generate a chemical potential gradient between the first electrode and the second electrode. Connect the first electrode and the second electrode with a wire; The open-circuit voltage between the first and second reference electrodes is tested, and the content of the target component is determined according to the standard curve. The first reference electrode and the second reference electrode are made of the same material. The first and second reference electrodes are selected from Ag / AgCl electrodes; The first electrode or the second electrode is selected from carbon fiber electrodes; The electrochemical probe solution is a K3IrCl6 / K2IrCl6 solution with an equal concentration ratio; The target component is 5-hydroxytryptamine, and the test component is a biological non-living tissue.

2. The method according to claim 1, characterized in that, The plotting of the standard curve includes the following steps: The first electrode is used as the anode and is inserted together with the first reference electrode into a solution containing different concentrations of the target component; The second electrode is used as the cathode and is inserted into the electrochemical probe solution together with the second reference electrode. The electrochemical probe solution is used to generate a chemical potential gradient between the first electrode and the second electrode. The first electrode and the second electrode are connected by a wire; Test the open-circuit voltage between the first reference electrode and the second reference electrode; A standard curve is plotted with the logarithm of the concentration of the target component on the x-axis and the corresponding open-circuit voltage value on the y-axis. The first reference electrode and the second reference electrode are made of the same material.

3. An electrochemical sensor for use in the method according to any one of claims 1-2, characterized in that, It includes an anolyte and a cathode; the anolyte contains a first electrode and a first reference electrode, the first electrode and the first reference electrode being non-contacting; the cathode contains a second electrode and a second reference electrode, the second electrode and the second reference electrode being non-contacting; the anolyte is used to add the test component; the cathode is used to add an electrochemical probe solution. The first and second reference electrodes are selected from Ag / AgCl electrodes; The first electrode or the second electrode is selected from carbon fiber electrodes; The electrochemical probe solution is a K3IrCl6 / K2IrCl6 solution with an equal concentration ratio.

4. A method for detecting the content of a target component in a test component using a bipolar electrode based on redox potential, characterized in that, Includes the following steps: A conductive fiber is passed through the inside of an insulating tube, with one end of the conductive fiber extending to the outside of the insulating tube. The conductive fiber is fixed, and one end of the insulating tube is sealed. Then, an electrochemical probe solution is placed inside the insulating tube, so that the conductive fiber inside the insulating tube comes into contact with the electrochemical probe solution. The first reference electrode and the conductive fiber extending to the outside of the insulating tube are inserted into the same test component, which may or may not contain the target component. The second reference electrode is inserted into the electrochemical probe solution inside the insulating tube; The open-circuit voltage between the first and second reference electrodes is tested, and the content of the target component is determined by a standard curve. The first reference electrode and the second reference electrode are made of the same material, and the electrochemical probe solution is used to generate a chemical potential gradient between the conductive fibers in contact with the electrochemical probe solution and the conductive fibers extending to the outside of the insulating tube. The first and second reference electrodes are selected from Ag / AgCl electrodes; The electrochemical probe solution is a K3IrCl6 / K2IrCl6 solution with an equal concentration ratio; The target component is 5-hydroxytryptamine; The conductive fiber is carbon fiber, and the test component is a biological non-living tissue.

5. The method as described in claim 4, characterized in that, The plotting of the standard curve includes the following steps: A conductive fiber is passed through the inside of an insulating tube, with one end of the conductive fiber extending to the outside of the insulating tube. The conductive fiber is fixed, and one end of the insulating tube is sealed. Then, an electrochemical probe solution is placed inside the insulating tube, so that the conductive fiber inside the insulating tube comes into contact with the electrochemical probe solution. The first reference electrode and the conductive fiber extending to the outside of the insulating tube are inserted into the same solution containing different concentrations of the target component; The second reference electrode is inserted into the electrochemical probe solution inside the insulating tube; Test the open-circuit voltage between the first reference electrode and the second reference electrode; A standard curve is plotted with the logarithm of the concentration of the target component on the x-axis and the open-circuit voltage value on the y-axis. The first and second reference electrodes are made of the same material, and the electrochemical probe solution is used to generate a chemical potential gradient between the conductive fibers in contact with the electrochemical probe solution and the conductive fibers extending to the outside of the insulating tube.

6. An electrochemical sensor for detecting reducing substances in a living environment, characterized in that, It includes a bipolar carbon fiber electrode; the bipolar carbon fiber electrode includes a fixative, carbon fiber and a conical capillary glass tube, wherein the carbon fiber passes through the conical capillary glass tube and the end portion extends beyond the tip of the capillary glass tube, and the fixative is used to fix the carbon fiber and close the tip opening of the capillary glass tube. The electrochemical sensor also includes an electrochemical probe solution filled inside the capillary glass tube; The reducing substance is 5-hydroxytryptamine; The electrochemical probe solution was a K3IrCl6 / K2IrCl6 solution with an equal concentration ratio.

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