A potential in-situ electrochemical sensor and a detection method thereof

Abstract

The application discloses a potential in-situ electrochemical sensor, which comprises a bipolar electrode and a first reference electrode, the bipolar electrode comprises an insulating tube, a conductive fiber, a second reference electrode and a probe solution arranged in the insulating tube; one end of the insulating tube is drawn into a tip, the conductive fiber extends to the outside of the tip of the insulating tube, and the surface of the conductive fiber extending to the outside of the tip of the insulating tube is combined with a phosphorus-sulfur substituted aptamer. The potential in-situ electrochemical sensor circuit of the application has almost no current, the influence of electrode implantation on the discharge frequency of adjacent neurons can be greatly avoided, the hydrophilic property of the surface of the bipolar electrode is increased after the bipolar electrode is modified by the aptamer, the adsorption of macromolecular proteins is reduced, and the sensitivity of the potential in-situ sensor is not influenced by the adsorption of proteins. In addition, the phosphorus-sulfur substituted aptamer is used, and the detection stability of the electrochemical sensor under various physiological conditions is further enhanced.

Description

Technical Field

[0001] This invention relates to the field of electrochemical analysis and detection technology. More specifically, it relates to a potentiometric in-situ electrochemical sensor and its detection method. Background Technology

[0002] Serotonin, ascorbic acid, uric acid, dopamine, and other reducing bioelectrically active substances play important roles in organisms. Therefore, real-time monitoring of the dynamic changes in the levels of these substances in living tissues provides an important tool for decoding the mechanisms by which tissues function in health and disease. Tissue-implanted microelectrode electrochemical methods offer high sensitivity and temporal and spatial resolution, making them a powerful technique for analyzing living tissues. However, the presence of precursors and metabolites with similar chemical properties, a large number of macromolecules, and complex signal transduction pathways, especially the high concentration of ascorbic acid (AA) in the brain, poses significant challenges to the monitoring of reducing bioelectrically active substances. Nucleic acid aptamers are sequence-specific nucleic acids selected through an evolutionary ligand system using the SELEX (Search Engine Exponential Enrichment) procedure. Chemically modifying electrode surfaces with target recognition ligands is an effective strategy to endow sensing systems with good selectivity. However, most current sensors require an external voltage to polarize the working electrode to generate an electrical signal, which in turn generates current in the system. This leads to biocompatibility issues. Potentiometric sensors have almost no current flowing through the circuit, which can alleviate biocompatibility problems. However, when applied to complex environments such as living tissues, they will face other challenges. For example, they may lose sensitivity due to protein adsorption in the brain, and the presence of nucleases in the extracellular fluid of the central nervous system (CNS) may affect the stability of DNA-based sensors.

[0003] Therefore, it is of great significance to provide a potentiometric in-situ electrochemical sensor with excellent stability and electrophysiological compatibility. Summary of the Invention

[0004] The first objective of this invention is to provide an electrophysiologically compatible potentiometric in-situ electrochemical sensor.

[0005] The second objective of this invention is to provide a detection method using the above-mentioned potentiometric in-situ electrochemical sensor.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a potential-type in-situ electrochemical sensor, comprising a bipolar electrode and a first reference electrode. The bipolar electrode includes an insulating tube, a conductive fiber placed inside the insulating tube, a second reference electrode, and a probe solution. The conductive fiber and the second reference electrode are partially immersed in the probe solution. One end of the insulating tube is drawn into a pointed tip, and the conductive fiber extends to the outside of the tip of the insulating tube.

[0008] Among them, the surface of the conductive fiber extending to the outside of the tip of the insulating tube is bound with phosphorus-sulfur aptamers.

[0009] Preferably, the conductive fiber extending to the outside of the tip of the insulating tube is bonded to the nucleic acid aptamer via covalent or non-covalent bonds.

[0010] More preferably, the non-covalent bond is formed by hydrophobic alkylamine chains modified on the surface of the conductive fiber and cholesterol modified on the nucleic acid aptamer.

[0011] Preferably, the nucleic acid aptamer is wherein the phosphorus oxygen bond in the nucleic acid aptamer is replaced by a phosphorus sulfur bond.

[0012] Preferably, the probe solution is used to create a chemical potential gradient between the conductive fibers in contact with the probe solution and the conductive fibers extending to the outside of the tip of the insulating tube.

[0013] More preferably, the 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.

[0014] Preferably, the first reference electrode and the second reference electrode are made of the same material, and the first reference electrode is selected from any one of Ag / AgCl electrode, Hg / HgO electrode, calomel electrode and hydrogen electrode.

[0015] Preferably, the method for preparing the bipolar electrode includes the following steps:

[0016] 1) Pull one end of the insulating tube into a pointed tip, pass the conductive fiber through the inside of the insulating tube, and extend the conductive fiber to the outside of the tip of the insulating tube, and fix the conductive fiber.

[0017] 2) Seal one end of the insulating tube, insert the second reference electrode into the insulating tube, and then fill the insulating tube with probe solution so that the conductive fiber and the second reference electrode inside the insulating tube are at least partially immersed in the probe solution.

[0018] 3) Modify the surface of the conductive fiber extending to the outside of the insulating tube tip with phosphorus-sulfur substituted nucleic acid aptamers.

[0019] Preferably, the modified phosphorus-sulfur substituted nucleic acid aptamer specifically includes the following steps:

[0020] Hydrophobic alkylamine chains are modified on the surface of conductive fibers extending to the outside of the insulating tube tip;

[0021] Provides nucleic acid aptamers modified with cholesterol-containing phosphorus-sulfur substitutions;

[0022] Conductive fibers modified with hydrophobic alkyl chains and nucleic acid aptamers modified with cholesterol and phosphorus-sulfur substitutions in the presence of Mg 2+ Soak in the buffer solution for 10–15 hours.

[0023] Secondly, the present invention provides a method for detecting the content of reducing substances in a test component using the above-mentioned potentiometric in-situ electrochemical sensor, comprising the following steps:

[0024] The first reference electrode and the conductive fiber extending to the outside of the insulating tube tip are inserted into the same test component;

[0025] The open-circuit voltage between the first and second reference electrodes is tested, and the content of the reducing substance is determined by a standard curve.

[0026] Preferably, the reducing agent is ascorbic acid, uric acid, dopamine, phenol, bisphenol A, 5-hydroxytryptamine, etc.

[0027] Preferably, when the test component is a living biological tissue, the insulating tube is a capillary glass tube and the conductive fiber is carbon fiber.

[0028] 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.

[0029] The beneficial effects of this invention are as follows:

[0030] This invention constructs a potentiometric in-situ electrochemical sensor based on the redox potential method. By rationally selecting the probe solution, the electrochemical process in the entire circuit can proceed spontaneously. Almost no current flows through the entire circuit, and almost no reaction products are generated, which can exhibit superior stability and achieve stable detection over long periods of time.

[0031] The potentiometric in-situ electrochemical sensor provided by this invention has strong versatility. It can be used to detect different reducing bioelectrically active substances by modifying different nucleic acid aptamers. In addition, this invention can also be used for in vivo detection by adjusting the raw materials, size and other parameters of each electrode. Compared with the micro electrochemical sensor constructed by the current method, it can greatly avoid the influence of electrode implantation on the firing frequency of adjacent neurons.

[0032] The potentiometric in-situ electrochemical sensor of the present invention exhibits excellent selectivity, stability and neurocompatibility when used to detect dopamine (DA) in living tissue, and can record the dynamics and electrical signals of DA in important brain regions of live rats in real time and simultaneously. Attached Figure Description

[0033] 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.

[0034] Figure 1 The aptGRP shown in Example 1 DA A schematic diagram of the interface modification between the sensor structure and the bipolar carbon fiber electrode.

[0035] Figure 2 Show aptGRP DA Sensor feasibility test diagram; where a is aptGRP. DA Cyclic voltammetry of the sensor before and after adding 100 μM DA to aCSF, b is GRP DA Cyclic voltammetry of the sensor with and without 5mM K3Fe(CN)6.

[0036] Figure 3 Show aptGRP DA Sensors and GRP DA The sensor's potential response to DA.

[0037] Figure 4 Show aptGRP DA Sensors and GRP DA The sensor's potential response to DA and E OC Graph showing the relationship between DA concentration and logarithmic correlation.

[0038] Figure 5 The results show that aptGRP in aCSF containing 200 μM AA DA Sensors and GRP DA Graph showing the difference in sensitivity of the sensor to DA.

[0039] Figure 6 Show aptGRP DA Sensors, SCR GRP sensors and GRP DA Selectivity comparison chart of sensors.

[0040] Figure 7 Show aptGRP DA The stability test results of the sensor; where a is aptGRP. DA Calibration graphs of the sensor before and after 2 hours of protein adsorption, b represents aptGRP. DA Calibration diagrams of the sensor before and after 4 hours of incubation in bovine fetal serum, c-d represent aptGRP. DA Calibration diagram of the sensor before and after 8 hours of in vivo implantation.

[0041] Figure 8 This diagram illustrates the DA sensing of in situ high potassium stimulation in the rat striatum and a typical E... OC Response diagram; where a is a schematic diagram of DA sensing under in situ high potassium stimulation of the rat striatum, and b is a typical E response diagram. OC Response diagram.

[0042] Figure 9 Show aptGRP DA Schematic diagram of DA sensing of the sensor in the medial forebrain tract-striatum and medial forebrain tract-nucleus accumbens and E OC Response diagram; where c is aptGRP DA Schematic diagram of DA sensing in the medial forebrain tract-striatum and medial forebrain tract-nucleus accumbens, where d represents aptGRP. DA The sensor is located in the medial forebrain tract-striatum and the medial forebrain tract-nucleus accumbens E. OC Response diagram.

[0043] Figure 10 This diagram illustrates the co-recording of chemical and electrical signals in vivo and E after high potassium stimulation. OC Response and neuronal firing diagram; where e is a schematic diagram of co-recording chemical and electrical signals in vivo, and f is E OC Response and neuronal firing diagram. Detailed Implementation

[0044] The following specific embodiments illustrate the potentiometric in-situ electrochemical sensor of the present invention and its usage method. This description is only intended to enable those skilled in the art to better understand the present invention, and does not limit the present invention in any way.

[0045] First, the present invention provides a potential-type in-situ electrochemical sensor, comprising a bipolar electrode and a first reference electrode. The bipolar electrode includes an insulating tube, a conductive fiber placed inside the insulating tube, a second reference electrode, and a probe solution. The conductive fiber and the second reference electrode are partially immersed in the probe solution. One end of the insulating tube is drawn into a tip, and the conductive fiber extends to the outside of the tip of the insulating tube.

[0046] Among them, the surface of the conductive fiber extending to the outside of the tip of the insulating tube is bound with phosphorus-sulfur aptamers.

[0047] It is understood that the bipolar electrode and the first reference electrode do not contact each other, and the conductive fiber and the second reference electrode do not contact each other.

[0048] In this invention, the potentiometric in-situ electrochemical sensor circuit constructed based on the redox potential method has almost no current. Compared with electrochemical sensors constructed using the current method, this greatly avoids the influence of electrode implantation on the firing frequency of adjacent neurons. Furthermore, traditional sensors suffer from sensitivity loss due to protein adsorption within tissues (such as the brain). The electrodes of this invention, after modification with nucleic acid aptamers, have increased surface hydrophilicity, reducing the adsorption of large protein molecules and preventing the sensitivity of the electrochemical sensor from being affected by protein adsorption. In addition, phosphorus-sulfur substituted nucleic acid aptamers effectively reduce nuclease degradation; therefore, the introduction of thiophosphate groups into the nucleic acid aptamers in this invention greatly enhances the testing stability of the electrochemical sensor.

[0049] In this type of potentiometric in-situ electrochemical sensor, the probe solution is filled inside the insulating tube during use, which can extend the lifespan of the sensor. Furthermore, it is understood that because the probe solution is to be filled into the insulating tube, we choose to seal the tip of the insulating tube, for example, using silicone, 502 glue, or AB glue.

[0050] In one specific embodiment of the present invention, the conductive fiber extending to the outside of the tip of the insulating tube is bonded to the nucleic acid aptamer by covalent or non-covalent bonds.

[0051] When the conductive fiber extending to the outside of the insulating tube tip binds to the nucleic acid aptamer via covalent bonds, group modification can be performed only on the nucleic acid aptamer to enable it to form covalent bonds with the conductive fiber, or group modification can be performed simultaneously on the surface of the conductive fiber to make the covalent bonds more stable. The covalent bonds can be NC covalent bonds, S-OH covalent bonds, CO-NH covalent bonds, etc., and the modifying groups can be -NH-, -NH2-, -OH, -SH, -CH, etc., respectively.

[0052] In another specific embodiment of the invention, the non-covalent bond is formed by a hydrophobic alkylamine chain modified on the surface of a conductive fiber and cholesterol modified on a nucleic acid aptamer. This binding method can further improve the testing stability of the bipolar electrode.

[0053] In one specific embodiment of the present invention, the alkylamine chain is C1 to C2. 12 Carboalkylamines, such as hexylamine and heptylamine.

[0054] It is understood that the phosphorus-sulfur substituted nucleic acid aptamers of the present invention can be purchased directly or synthesized using existing technologies. The DNA sequence of the nucleic acid aptamers can be selected and adjusted according to the substance to be detected.

[0055] Furthermore, the probe solution is used to create a chemical potential gradient between the conductive fibers contacting the probe solution and the conductive fibers extending to the outside of the insulating tube tip. In this invention, by rationally selecting the electrochemical probe, 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. Moreover, the electrochemical probe and its concentration can be rationally designed and controlled, thereby regulating the sensitivity of the detection.

[0056] In one specific embodiment of the present invention, the 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.

[0057] In one specific embodiment of the present invention, the first reference electrode and the second electrode are made of the same material, and the first reference electrode is selected from any one of Ag / AgCl electrode, Hg / HgO electrode, calomel electrode and hydrogen electrode.

[0058] In one specific embodiment of the present invention, the method for preparing the bipolar electrode includes the following steps:

[0059] 1) Pull one end of the insulating tube into a pointed tip, pass the conductive fiber through the inside of the insulating tube, and extend the conductive fiber to the outside of the tip of the insulating tube, and fix the conductive fiber.

[0060] 2) Seal one end of the insulating tube, insert the second reference electrode into the insulating tube, and then fill the insulating tube with probe solution so that the conductive fiber and the second reference electrode inside the insulating tube are at least partially immersed in the probe solution.

[0061] 3) Modify the surface of the conductive fiber extending to the outside of the insulating tube tip with phosphorus-sulfur substituted nucleic acid aptamers.

[0062] In step 2), the order of sealing the tip of the insulating tube and inserting the reference electrode into the insulating tube can be interchanged, and the order of steps 2) and 3) can also be interchanged.

[0063] In one specific embodiment 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, used to add an electrochemical probe solution. At this point, 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.

[0064] In a specific embodiment of the present invention, the modified phosphorus-sulfur substituted nucleic acid aptamer specifically includes the following steps:

[0065] Hydrophobic alkylamine chains are modified on the surface of conductive fibers extending to the outside of the insulating tube tip;

[0066] Provides nucleic acid aptamers modified with cholesterol-containing phosphorus and sulfur substitutions;

[0067] Conductive fibers modified with hydrophobic alkyl chains and nucleic acid aptamers modified with cholesterol and phosphorus-sulfur substitutions in the presence of Mg 2+ Soak in the buffer solution for 10–15 hours.

[0068] In one specific embodiment of the present invention, the method for modifying hydrophobic alkylamine chains can be an electrochemical oxidation method.

[0069] Secondly, the present invention also provides a method for detecting the content of reducing substances in a test component using the above-mentioned potentiometric in-situ electrochemical sensor, comprising the following steps:

[0070] The first reference electrode and the conductive fiber extending to the outside of the insulating tube tip are inserted into the same test component;

[0071] The open-circuit voltage between the first and second reference electrodes is tested, and the content of the reducing substance is determined by a standard curve.

[0072] 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, living tissues, etc.

[0073] The standard curve can be plotted with a series of reducing substances of different concentrations on the x-axis and the corresponding open-circuit voltage values ​​on the y-axis. In a specific embodiment of the present invention, when the test component is a living biological tissue, the insulating tube is a capillary glass tube and the conductive fiber is carbon fiber.

[0074] 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.

[0075] Example 1

[0076] A potentiometric in-situ electrochemical sensor (aptGRP) DA The sensor includes a dual carbon fiber electrode and an Ag / AgCl reference electrode; the fabrication of the dual carbon fiber electrode includes the following steps:

[0077] (1) Fabrication of the bipolar carbon fiber electrode: First, carbon fibers were inserted into a glass capillary (7 / 10 μm), and then the glass capillary was drawn into two glass tubes with extremely fine tips and sealed with carbon fibers using a microelectrode drawing instrument. Next, the tubes were sealed and cured in prepared AB glue for 300 seconds to completely seal the tips of the glass tubes. Then, the electrode was placed at room temperature until the AB glue cured. Finally, the carbon fibers at the tips were cut under a microscope (approximately 300 μm in length), thus completing the fabrication of the unmodified bipolar carbon fiber electrode of this invention. The carbon fibers exposed outside the glass capillary serve as the anode of the sensor, while the carbon fibers remaining inside the glass capillary serve as the cathode.

[0078] (2) Activation pretreatment of the bipolar carbon fiber electrode: First, the bipolar carbon fiber electrode was ultrasonically treated in acetone, 3.0M NO3, and 1.0M KOH solutions for 3–5 minutes to remove surface impurities. To electrochemically activate the carbon fiber, 3.0M KCl was injected into a glass capillary tube, and an Ag / AgCl wire was inserted into the glass tube. Subsequently, the bipolar carbon fiber electrode was 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.

[0079] (3) Nucleic acid aptamer modification of bipolar carbon fiber electrode: First, heptamine was modified on the surface of carbon fiber exposed outside the glass capillary: In 0.1M LiClO4 ethanol solution, heptamine (5mM) was electrochemically oxidized by applying a cyclic potential (-0.2~+1.6V vs. Ag / AgCl) at a scan rate of 10mV / s for 5 cycles to obtain heptamine modified carbon fiber electrode;

[0080] Prepare a solution containing 5mM Mg 2+ A 1 μM PBS buffer solution of phosphorus-sulfur-substituted nucleic acid aptamers was heated to 95°C and held for 3 min, then rapidly cooled to room temperature. A heptanyl-modified carbon fiber electrode was added and immersed at room temperature for 12 h. The electrode was then thoroughly rinsed with PBS and deionized water, and passivated in 0.5 mM cholesterol-TEG2000 for 0.5 h. The electrode was rinsed with deionized water and stored in phosphate buffer. The 3' end of the nucleotide chain of the phosphorus-sulfur-substituted nucleic acid aptamer was labeled with cholesterol (Chol). The phosphorus-sulfur-substituted nucleic acid aptamer sequence was: GGACGA CGC CAG TTT GAA GGT TCG TTC GCAGGT GTG GAG TGACGT CG TCC TTT TTT–Chol.

[0081] (4) After the modification is completed, the bipolar carbon fiber is thoroughly cleaned with deionized water and set aside for later use.

[0082] This example uses aptGRP. DA Sensor fabrication and interface modification of bipolar carbon fiber electrodes, such as Figure 1 As shown.

[0083] Comparative Example 1

[0084] A potentiometric in-situ electrochemical sensor (GRP) DA The sensor includes a dual carbon fiber electrode and an Ag / AgCl reference electrode; the fabrication of the dual carbon fiber electrode includes the following steps:

[0085] (1) Fabrication of the bipolar carbon fiber electrode: First, carbon fibers were inserted into a glass capillary (7 / 10 μm), and then the glass capillary was drawn into two glass tubes with extremely fine tips and sealed with carbon fibers using a microelectrode drawing instrument. Next, the tubes were sealed and cured in prepared AB glue for 300 seconds to completely seal the tips of the glass tubes. Then, the electrode was placed at room temperature until the AB glue cured. Finally, the carbon fibers at the tips were cut under a microscope (approximately 300 μm in length), thus completing the fabrication of the unmodified bipolar carbon fiber electrode of this invention. The carbon fibers exposed outside the glass capillary serve as the anode of the sensor, while the carbon fibers remaining inside the glass capillary serve as the cathode.

[0086] (2) Activation pretreatment of the bipolar carbon fiber electrode: First, the bipolar carbon fiber electrode was ultrasonically treated in acetone, 3.0M NO3, and 1.0M KOH solutions for 3–5 minutes to remove surface impurities. To electrochemically activate the carbon fiber, 3.0M KCl was injected into a glass capillary tube, and an Ag / AgCl wire was inserted into the glass tube. Subsequently, the bipolar carbon fiber electrode was 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.

[0087] Comparative Example 2

[0088] A potentiometric in-situ electrochemical sensor (ScrGRP sensor) includes a dual carbon fiber electrode and an Ag / AgCl reference electrode. The preparation of the dual carbon fiber electrode includes the following steps:

[0089] (1) Fabrication of the bipolar carbon fiber electrode: First, carbon fibers were inserted into a glass capillary (7 / 10 μm), and then the glass capillary was drawn into two glass tubes with extremely fine tips and sealed with carbon fibers using a microelectrode drawing instrument. Next, the tubes were sealed and cured in prepared AB glue for 300 seconds to completely seal the tips of the glass tubes. Then, the electrode was placed at room temperature until the AB glue cured. Finally, the carbon fibers at the tips were cut under a microscope (approximately 300 μm in length), thus completing the fabrication of the unmodified bipolar carbon fiber electrode of this invention. The carbon fibers exposed outside the glass capillary serve as the anode of the sensor, while the carbon fibers remaining inside the glass capillary serve as the cathode.

[0090] (2) Activation pretreatment of the bipolar carbon fiber electrode: First, the bipolar carbon fiber electrode was ultrasonically treated in acetone, 3.0M NO3, and 1.0M KOH solutions for 3–5 minutes to remove surface impurities. To electrochemically activate the carbon fiber, 3.0M KCl was injected into a glass capillary tube, and an Ag / AgCl wire was inserted into the glass tube. Subsequently, the bipolar carbon fiber electrode was 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.

[0091] (3) Nucleic acid aptamer modification of bipolar carbon fiber electrode: First, heptamine was modified on the surface of carbon fiber exposed outside the glass capillary: in 0.1M LiClO4 ethanol solution, heptamine (5mM) was electrochemically oxidized by applying a cyclic potential (-0.2~+1.6V vs. Ag / AgCl) at a scan rate of 10mV / s for 5 cycles to obtain heptamine modified carbon fiber electrode.

[0092] Prepare a solution containing 5mM Mg 2+ A 1 μM PBS buffer solution of phosphorus-sulfur-substituted nucleic acid aptamers was heated to 95°C and held for 3 min, then rapidly cooled to room temperature. A heptanyl-modified carbon fiber electrode was added and immersed at room temperature for 12 h. The electrode was then thoroughly rinsed with PBS and deionized water, and passivated in 0.5 mM cholesterol-TEG2000 for 0.5 h. The electrode was rinsed with deionized water and stored in phosphate buffer. The 3' end of the nucleotide chain of the phosphorus-sulfur-substituted nucleic acid aptamer was labeled with cholesterol (Chol). The sequence of the phosphorus-sulfur-substituted nucleic acid aptamer is: NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN TTT TTT–Chol.

[0093] (4) After the modification is completed, the bipolar carbon fiber is thoroughly cleaned with deionized water and set aside for later use.

[0094] Test case

[0095] Test Example 1

[0096] Testing the sensing performance of potentiometric in-situ electrochemical sensors

[0097] The aptGRP of Example 1 was tested on an electrochemical workstation. DA Sensor and Comparative Example 1 GRP DA The electrochemical sensing performance of the sensor was investigated.

[0098] Test 1: Using a bipolar electrode as the working electrode, an Ag / AgCl electrode as the reference electrode, a Pt wire as the counter electrode, and aCSF as the electrolyte, 100 μM DA was first added to aCSF, and the electrochemical test of the system was performed using cyclic voltammetry. Then, 5 mM potassium ferricyanide was added to aCSF, and the electrochemical test of the system was performed using cyclic voltammetry.

[0099] Test 2: A traditional three-electrode system was used, with an Ag / AgCl wire inserted into artificial cerebrospinal fluid (aCSF) as the working electrode and an Ag / AgCl wire inserted into a probe solution that can undergo a reduction reaction as the reference electrode. 1-50 μM DA was added to aCSF successively, and the open-circuit voltage of the system was recorded.

[0100] Test 3: Using an Ag / AgCl wire inserted into aCSF as the working electrode and an Ag / AgCl wire inserted into a probe solution that can undergo a reduction reaction as the reference electrode, 200 μM AA was added to aCSF, followed by 1–50 μM DA, and the open-circuit voltage of the system was recorded.

[0101] Result: (1) Figure 2 The results show that when 100 μM DA is added to aCSF, DA peaks at approximately 0 V. When 5 mM potassium ferricyanide is added to aCSF, a reduction peak appears at approximately 0.38 V. Therefore, the potential difference between the reduction reaction potential generated by potassium ferricyanide at the cathode and the potential difference between the dopamine at the anode is sufficient to constitute a spontaneous reaction.

[0102] (2) When 5 μM DA is added to artificial cerebrospinal fluid, aptGRP DA The sensor generated a rapid potential change of approximately 48mV and reached equilibrium in about 2 seconds, while the GRP DA The sensor generated a potential change of approximately 28 mV and reached equilibrium in approximately 254 seconds. Figure 3 As shown.

[0103] (3) In the concentration range of 1–50 μM, GRP DASensors and aptGRP DA The sensor produced a stepwise potential response during the continuous addition of DA (the detection concentrations of DA were 1, 2, 5, 10, 20, and 50 μM). OC It has a linear relationship with the logarithm of DA concentration, such as Figure 4 As shown.

[0104] (4) The presence of AA greatly reduces GRP DA The sensor's response to DA, and aptGRP DA The sensor's response is GRP DA The sensor's performance is 10 times higher, specifically, it can still detect DA at concentrations as low as 100 nM. Furthermore, aptGRP... DA The sensor's sensitivity is higher than that of GRP. DA The sensor performance has improved significantly by 1.7 times (e.g.) Figure 5 (As shown).

[0105] The electrochemical measurements in Test Examples 2-4 below were performed on an electrochemical workstation using a conventional three-electrode system. An Ag / AgCl wire inserted into a supporting electrolyte or the surface of a mouse brain served as the working electrode, while an Ag / AgCl wire inserted into a probe solution capable of reduction served as the reference electrode. When the target molecule DA was present, the bipolar system enabled spontaneous electrochemical detection, and the DA molecule content was determined by recording the system's open-circuit voltage.

[0106] Test Example 2

[0107] Selectivity of potentiometric in-situ electrochemical sensors

[0108] In the central nervous system, numerous electroactive molecules, such as 3,4-dihydroxyphenylacetic acid (DOPAC), levodopa (L-DOPA), norepinephrine (NE), ascorbic acid (AA), and uric acid (UA), overlap within the oxidation potential window of dopamine (DA). The presence of high baseline AA levels, in particular, necessitates higher selectivity from conventional neuronal sensors to meet the requirements. Therefore, this example focuses on the aptGRP of Example 1. DA Sensor and Comparative Example 1 GRP DA The selectivity of the scrGRP sensor, compared to scale 2, was tested.

[0109] Test method: Interfering substances of the same concentration (10 μM) were sequentially added to aCSF containing 200 μM AA. These interfering substances included DOPAC, L-DOPA, NE, AA, and UA. OC The reaction was negligible, and then the aptGRP of Example 1 was tested. DA Sensor and Comparative Example 1 GRP DA open circuit voltage of the sensor

[0110] Result: As Figure 6 As shown, the SCR GRP sensor and GRP DA The sensor produced a small response to all species, including DA, demonstrating the crucial role of nucleic acid aptamers in sensor selectivity.

[0111] Test Example 3

[0112] Stability of potentiometric in-situ electrochemical sensors in complex systems

[0113] Test aptGRP separately DA The sensor was tested in a high concentration of bovine serum albumin solution (BSA, 40 mg / mL). -1 The open-circuit voltages before and after 2 hours of incubation in 10% bovine fetal serum (FBS), before and after 4 hours of incubation in 10% bovine fetal serum (FBS), and before and after 8 hours of implantation in the rat brain were measured. Results are shown in […]. Figure 7 As shown.

[0114] Results: Typically, the presence of abundant proteins in biological systems passivates the sensor surface, reducing its sensitivity in vivo. Compared to traditional current-type sensors, the aptGRP of this invention... DA The sensitivity of the sensor is independent of the electroactive surface area, even in high concentrations of bovine serum albumin solution (40 mg / mL). -1 After incubation for 2 hours in 10% FBS, 4 hours in 10% FBS, or 8 hours after implantation in the rat brain, the sensor maintained its excellent and consistent potential response and sensitivity. This demonstrates the superior performance of the aptGRP of the present invention. DA The sensor is still able to stably and effectively sense DA even in protein-rich biological environments.

[0115] Test Example 4

[0116] In vivo applications of potentiometric in-situ electrochemical sensors

[0117] 1.1 In order to evaluate aptGRP DA The applicability of the sensor to detect DA dynamics in the brain of a living animal in real time. Testing method: We first stimulated the striatum (Str) of rats (300-350 g adult male Sprague-Dawley rats) with 70 mM KCl. Then, aptGRP... DA After injecting 70 mM KCl (2 μL / min, for 30 s) into the nearby striatum, we observed a sharp change in E. OC The response was related to an immediate increase in the local concentration of DA, while the control experiment, which injected the same volume of aCSF at the same flow rate, showed no significant potential fluctuation (e.g. Figure 8(As shown).

[0118] 1.2 aptGRP DA Sensors are applied to more complex neural circuits to form functional units of the brain. We use aptGRP. DA The sensor monitored the release of DA in rat str and NAc via electrical stimulation (60 Hz, ±350 μA, 2 ms / phase) of rat MFB. Results are as follows: Figure 9 As shown, under electrical stimulation, a rapid increase in potential signals attributable to DA was observed in both the MFB-Str and MFB-NAc neural circuits, followed by a rapid decay to baseline. This confirms the efficacy of aptGRP. DA The sensor is effective and reliable in selectively monitoring the dynamics of dopamine (DA) in different brain regions.

[0119] 1.3 Research on aptGRP DA Can sensors be combined with electrophysiological techniques to achieve simultaneous recording of chemical signals and neuronal activity? We first used aptGRP... DA The sensor was implanted in combination with a commercial microelectrode array (MEA) into the Str of rats, and aptGRP was recorded. DA Changes in the electrical activity of neurons near the sensor, as shown in the following figures. Figure 10 As shown. We combine aptGRP DA Sensors and MEA were used to investigate the real-time correlation between Str DA dynamics and neuronal firing frequency induced by 70 mM KCl stimulation. Under in situ KCl-induced neuronal depolarization, aptGRP... DA The sensor recorded rapid potential fluctuations, reflecting an increase in DA concentration in Str. Simultaneously, when the potential reached its maximum value, the electrophysiological recording showed a sharp increase in neuronal firing.

[0120] Conclusion: In summary, the results in sections 1.1 to 1.3 all prove the validity of aptGRP. DA The sensor's ability to jointly detect dopamine and neuronal activity in key brain regions of live animals.

[0121] 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 potentiometric in-situ electrochemical sensor, characterized in that, The device includes a bipolar electrode and a first reference electrode. The bipolar electrode includes an insulating tube, a conductive fiber placed inside the insulating tube, a second reference electrode, and a probe solution. The conductive fiber and the second reference electrode are partially immersed in the probe solution. One end of the insulating tube is drawn into a pointed tip, and the conductive fiber extends to the outside of the tip of the insulating tube. Among them, the surface of the conductive fiber extending to the outside of the tip of the insulating tube is bound with phosphorus-sulfur aptamers; The first reference electrode and the second reference electrode are made of the same material, and the first reference electrode is selected from an Ag / AgCl electrode; The conductive fiber extending to the outside of the insulating tube tip is bonded to the nucleic acid aptamer via covalent or non-covalent bonds; the non-covalent bonds are formed by hydrophobic alkylamine chains modified on the surface of the conductive fiber and cholesterol modified on the nucleic acid aptamer. Specifically, the nucleic acid aptamer is a nucleic acid aptamer in which the phosphorus oxygen bond is replaced by a phosphorus sulfur bond. The probe solution is used to create a chemical potential gradient between the conductive fibers in contact with the probe solution and the conductive fibers extending to the outside of the tip of the insulating tube. The probe solution is either a K3IrCl6 / K2IrCl6 solution or a K3[Fe(CN6)] / K4[Fe(CN6)] solution with an equal concentration ratio. The method for preparing the bipolar electrode includes the following steps: 1) Pull one end of the insulating tube into a pointed tip, pass the conductive fiber through the inside of the insulating tube, and extend the conductive fiber to the outside of the tip of the insulating tube, and fix the conductive fiber. 2) Seal the tip of the insulating tube, insert the second reference electrode into the insulating tube, and then fill the insulating tube with probe solution so that the conductive fiber and the second reference electrode inside the insulating tube are at least partially immersed in the probe solution. 3) Modify the surface of conductive fibers extending to the outside of the insulating tube tip with phosphorus-sulfur substituted nucleic acid aptamers; The modified phosphorus-sulfur substituted nucleic acid aptamer specifically includes the following steps: Hydrophobic alkylamine chains are modified on the surface of conductive fibers extending to the outside of the insulating tube tip; Provides nucleic acid aptamers modified with cholesterol-containing phosphorus and sulfur substitutions; Conductive fibers modified with hydrophobic alkyl chains and nucleic acid aptamers modified with cholesterol and phosphosulfur substitutions in the presence of Mg 2+ Soak in the buffer solution for 10-15 hours.

2. A method for detecting the content of reducing substances in a test component using the potentiometric in-situ electrochemical sensor as described in claim 1, characterized in that, Includes the following steps: The first reference electrode and the conductive fiber extending to the outside of the insulating tube tip are inserted into the same test component; The open-circuit voltage between the first reference electrode and the second reference electrode is tested, and the content of the reducing substance is determined by a standard curve.

3. The method according to claim 2, characterized in that, The reducing agent is dopamine.

4. The method according to claim 2, characterized in that, When the test component is a living biological tissue, the insulating tube is a capillary glass tube and the conductive fiber is carbon fiber.

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