Application of a three-electrode detection structure in preparation of an adenosine sensor

Abstract

This invention provides an application of a three-electrode detection structure in the fabrication of an adenosine sensor, specifically its application in acupuncture. The three-electrode detection structure can be used to fabricate adenosine, glucose, lactate, glutamate, uric acid, ascorbic acid, or dopamine sensors; the adenosine sensor is an implantable, continuously detectable adenosine sensor. The flexible, implantable adenosine sensor with a three-electrode structure described in this invention features good stability, high sensitivity, a wide linear detection range, short response time, and strong anti-interference capabilities. It can achieve continuous in vivo detection of changes in adenosine concentration in animals and the brain, and has been successfully applied to real-time in vivo monitoring of acupuncture-induced local adenosine release at acupoints, providing a novel and modern detection method for in-depth research on the mechanism of acupuncture efficacy.

Description

Technical Field

[0001] This invention belongs to the field of electrochemistry, and in particular relates to the application of a three-electrode detection structure in the preparation of an adenosine sensor. Background Technology

[0002] Acupuncture is effective, but its mechanism of action and biological basis remain to be elucidated. In recent years, numerous studies have shown that adenosine signaling plays a crucial role in the initiation of acupuncture. Adenosine, mainly generated from the hydrolysis of adenosine triphosphate (ATP), is an endogenous neuromodulator involved in various physiological and pharmacological processes, such as neurotransmission, inflammation, ischemic injury, and pain. As an extracellular messenger in most body fluids, adenosine interacts with four G protein-coupled receptor subtypes (A1, A2, ...). A A 2B These four receptors (A1, A2, and A3) interact to exert their functions, and their affinity for adenosine varies significantly. Importantly, different adenosine concentrations can mediate the activation of different adenosine receptor subtypes, thereby exerting different physiological regulatory functions. Therefore, the immediate detection of local adenosine release induced under different conditions or stimuli is a real need in the field of adenosine research. Currently, most methods for detecting adenosine in vivo require processing extracted blood or specific tissues under in vitro conditions before using traditional high-performance liquid chromatography (HPLC) or immunological analysis methods. This results in intermittent, delayed, highly variable, and unstable experimental results. Although microdialysis technology has been used for in vivo sampling and detection in recent years, each sampling requires continuous sampling for 5-20 minutes, which is a cumulative rather than a real-time quantity. Furthermore, after sampling, other techniques such as HPLC are still required for detection. Therefore, it is impossible to accurately reflect the concentration changes of adenosine throughout the entire acupuncture process and how it participates in real-time regulation. Summary of the Invention

[0003] In view of this, the present invention aims to overcome the defects in the prior art and proposes an application of a three-electrode detection structure in the preparation of an adenosine sensor.

[0004] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0005] This invention proposes an enzyme immobilization promoter comprising polyethyleneimine-functionalized graphene oxide and genipin in a mass ratio of 1-5:1.

[0006] The present invention also proposes an enzyme cross-linking fixative, wherein the enzyme cross-linking fixative comprises a polyethyleneimine-functionalized graphene oxide solution and a multi-enzyme cascade reaction solution in a volume ratio of 1-3:1.

[0007] Furthermore, the multi-enzyme cascade reaction solution comprises xanthine oxidase, purine nucleoside phosphorylase, adenosine deaminase, and genipin in a volume ratio of 1-2:1-2:1-2:1.

[0008] Furthermore, the concentration of the polyethyleneimine-functionalized graphene oxide solution is 1-10 mg / ml; the concentration of xanthine oxidase in the multi-enzyme cascade reaction solution is 0.5-5 U / ml; the concentration of purine nucleoside phosphorylase in the multi-enzyme cascade reaction solution is 0.5-5 U / ml; the concentration of adenosine deaminase in the multi-enzyme cascade reaction solution is 0.5-5 U / ml; and the concentration of genipin in the multi-enzyme cascade reaction solution is 1-10 mg / ml.

[0009] This invention also proposes a method for surface functionalization modification of the conductive reaction region of a working electrode, comprising the following steps:

[0010] (1) The conductive reaction region is immersed in the electrodeposition solution for electrodeposition to form an electron mediator layer on the conductive reaction region;

[0011] (2) A polyethyleneimine-functionalized graphene oxide solution is dropped onto the surface of the electron mediator layer, dried, and then the multi-enzyme cascade reaction solution is dropped onto its surface and dried to form an enzyme reaction layer.

[0012] (3) The conductive reaction region modified with the electron mediator layer and enzyme reaction layer is immersed in the anti-interference solution for electrodeposition, and the anti-interference layer is formed after drying.

[0013] Furthermore, the electrodeposition solution in step (1) comprises hydrochloric acid with a concentration of 2-5 mg / mL, potassium chloride solution with a concentration of 5-10 mg / mL, ferric chloride solution with a concentration of 2-5 mg / mL, potassium ferricyanide solution with a concentration of 2-10 mg / mL, and graphyne nanoparticles with a concentration of 0.1-1 mg / mL; the anti-interference solution in step (3) comprises o-phenylenediamine with a concentration of 10-50 mg / mL and bovine serum albumin with a concentration of 1-10 mg / mL.

[0014] The present invention also proposes a three-electrode detection structure, including a silver foil and two flexible film substrates. The flexible film substrates are respectively fixed to the upper and lower surfaces of the silver foil by adhesive layers. Each flexible film substrate is provided with a conductive layer, and each conductive layer is provided with an insulating layer.

[0015] The length of the insulating layer is less than the length of the conductive layer;

[0016] The portion of the conductive layer exposed relative to the insulating layer is a conductive reaction region. One of these conductive reaction regions is surface-functionalized using the method described in claim 5 or 6. The modified conductive reaction region serves as the working electrode, and the unmodified conductive reaction region serves as the counter electrode.

[0017] Furthermore, the length of the conductive layer is less than the length of the silver foil; the conductive layer is made of at least one of gold, platinum, titanium, palladium, copper, carbon black, graphite, or graphene; the flexible film substrate is made of at least one of polycarbonate, polytetrafluoroethylene, polyimide, polyethylene, polyethylene terephthalate, acrylonitrile-butadiene-styrene copolymer, or polymethyl methacrylate. The portion of the silver foil exposed relative to the conductive layer is electroplated with chlorine to form a dense and stable Ag / AgCl layer on its surface. It is then immersed in a 5% Nafion solution, left to stand for 1-10 seconds, and then removed and air-dried at room temperature in the dark to serve as a reference electrode.

[0018] The present invention also proposes an application of the aforementioned three-electrode detection structure in acupuncture.

[0019] Furthermore, the three-electrode detection structure is used in the preparation of adenosine sensors, glucose sensors, lactate sensors, glutamate sensors, uric acid sensors, ascorbic acid sensors, or dopamine sensors; the adenosine sensor is an implantable continuous adenosine detection sensor.

[0020] A flexible implantable adenosine sensor with a three-electrode structure, the sensor including the aforementioned three-electrode detection structure.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] Based on the advantages of electrochemical biosensing detection technology, such as low detection cost and no need for labeling or sampling, the flexible implantable adenosine sensor with a three-electrode structure described in this invention further adopts a number of high technologies, including a multi-enzyme cascade reaction system, composite nanomaterial enhancement, and MEMS microelectromechanical processing. This gives it the characteristics of good stability, high sensitivity, wide linear detection range, short response time, and strong anti-interference. It can realize in vivo, in situ, real-time, and continuous measurement of adenosine concentration in animal subcutaneous tissue and brain tissue. It has been successfully applied to the monitoring and research of the effect of acupuncture operations with different twisting rates on the local adenosine content changes in acupoints, providing a more effective high-tech analytical method for in-depth research on the mechanism of action of traditional acupuncture. Attached Figure Description

[0023] Figure 1 This is a perspective view of the three-electrode detection structure described in an embodiment of the present invention;

[0024] Figure 2 This is a side view of the three-electrode detection structure described in an embodiment of the present invention;

[0025] Figure 3 This is a linear fitting graph of the square root of the CV scan rate and the redox peak current value as described in the embodiments of the present invention;

[0026] Figure 4 This is a schematic diagram illustrating the working principle of the conductive reaction zone according to an embodiment of the present invention;

[0027] Figure 5 shows the optimal detection voltage selection diagram according to the embodiment of the present invention: 5-A is the current-time curve under different detection voltages, and 5-B is a comparison diagram of detection sensitivity under different detection voltages;

[0028] Figure 6 shows the continuous detection of adenosine in the in vitro environment according to the embodiment of the present invention: 6-A is the current-time curve, and 6-B is the linear fitting curve;

[0029] Figure 7 This is a test diagram illustrating the anti-interference capability as described in an embodiment of the present invention;

[0030] Figure 8 This is a continuous detection map of local adenosine in an animal body as described in an embodiment of the present invention;

[0031] Figure 9 This is a continuous detection map of local adenosine in the animal brain as described in an embodiment of the present invention;

[0032] Figure 10 This is a diagram illustrating the in vivo real-time monitoring of adenosine release induced by acupuncture in acupoints, as described in an embodiment of the present invention.

[0033] Explanation of reference numerals in the attached figures:

[0034] 1. Silver foil; 2. Flexible film substrate; 3. Conductive layer; 4. Insulating layer; 5. Conductive reaction zone; 6. Adhesive layer. Detailed Implementation

[0035] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0036] The present invention will be described in detail below with reference to embodiments.

[0037] Example 1: Fabrication of Microelectrode Detection Structure

[0038] 1. A flexible polyethylene terephthalate film with a thickness of 0.1 mm is selected as the substrate. A nano-platinum conductive layer with a thickness of 20 μm is deposited on one side of the film using vapor deposition or magnetron sputtering. The film is then adhered to specific positions on both sides of a silver foil with a thickness of 0.05 mm (purity: 99.99%) using medical double-sided adhesive. The silver foil should not be completely covered on both sides, leaving conductive parts at both ends.

[0039] 2. Apply insulating medical single-sided adhesive to specific positions on both sides of the silver foil nano-platinum conductive layer, leaving conductive portions at both ends;

[0040] 3. The above sheet is cut into needle-shaped structures using an ultraviolet laser cutting machine. The exposed silver foil layer at the tip is 1mm × 0.4mm in size. The exposed nano-platinum layers on both sides serve as the working electrode and the counter electrode, respectively, and are both 1mm × 0.4mm in size. The other end of each layer can be exposed to expose a conductive part for connecting to an electrochemical workstation.

[0041] 4. Immerse the entire exposed silver foil layer at the tip in dilute hydrochloric acid. After electroplating and chlorination, an Ag / AgCl layer will form on the surface. Then immerse it in a 5% Nafion solution, let it stand for 3 seconds, and remove it. Air dry in a dark place at room temperature. Figure 1-2 As shown;

[0042] Example 2: Surface functionalization modification of the conductive reaction region of the working electrode

[0043] The surface of the conductive reaction region of the working electrode needs to be functionalized. The modification layers, from the inside out, are an electron mediator layer, an enzyme reaction layer, and an anti-interference layer.

[0044] Preparation of the electron mediator layer: 0.1M hydrochloric acid, 0.1M potassium chloride, 1.5mM ferric chloride, and 1.5mM potassium ferricyanide were mixed and dissolved. 0.1 mg / mL graphyne nanoparticles were slowly added under rapid stirring, along with an appropriate amount of co-solvent. The mixture was stirred rapidly for 1-2 hours. If the dispersion was insufficient, it could be sonicated for 0.5 hours until a uniformly dispersed electrodeposition solution was obtained. The cleaned conductive reaction zone was immersed in the electrodeposition solution, and cyclic voltammetry was used to scan 3 times in the range of 0.2-0.4V at a scan rate of 50 mV / s. After removal, the zone was rinsed with ultrapure water and activated in a deoxygenated electrolyte solution (containing 0.1M hydrochloric acid and 0.1M potassium chloride). Cyclic voltammetry was used to scan 50 times in the range of 0.2-0.4V at a scan rate of 100 mV / s. After removal, the zone was rinsed with ultrapure water and dried at 100℃ for 110 min. The electron mediator layer (Prussian blue and graphyne nanocomposite layer) was then obtained on the surface of the reaction zone.

[0045] Preparation of the enzyme reaction layer: 3 μL of polyethyleneimine-functionalized graphene oxide solution (5 mg / ml) was dropped onto the surface of the above electron mediator layer and dried at room temperature; then 3 μL of cross-linking enzyme solution (containing 2 U / ml xanthine oxidase, 2.5 U / ml purine nucleoside phosphorylase, 2.5 U / ml adenosine deaminase and 1 mg / ml genipin, in a volume ratio of 2:2:2:1) was added and dried at room temperature;

[0046] Preparation of the anti-interference layer: Immerse the conductive reaction area modified with the electron mediator layer and enzyme reaction layer into the o-phenylenediamine solution (containing 100mM o-phenylenediamine and 7mg / mL bovine serum albumin), apply a voltage of 0.75V for 15min using the constant voltage method, and then air dry at room temperature.

[0047] Example 3: Verification of adenosine sensor performance using voltammetric scanning rate

[0048] The modified microelectrode detection structure was used as an adenosine sensor. To verify the effect of the voltammetric scan rate on the sensor's detection performance, the implanted end of the adenosine sensor was immersed in a 0.1M potassium deoxychloride solution. The other end of the sensor was connected to an electrochemical workstation for cyclic voltammetry detection. The voltage range was -0.1 to 1.5 V, and the scan rates were 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mV / s, with 10 scan cycles. The cyclic voltammetric scan results showed a pair of distinct redox peaks at 0.18 V and 0.9 V. The redox peak at 0.18 V was formed by the interconversion of Prussian blue and its reduced Prussian white deposited on the conductive layer surface; the redox peak at 0.9 V was formed by the interconversion of Prussian blue and its fully oxidized Prussian green, consistent with previous literature reports. The peak current value of the redox peak at 0.18 V was linearly fitted to the square root of the scan rate, as shown in the figure. Figure 3 As shown, there is a good linear relationship between the two, which indicates that the electrochemical signal generated by the prepared modified layer follows the diffusion-controlled surface reaction model.

[0049] Example 4: Optimal Detection Voltage Selection

[0050] The detection principle of the prepared adenosine sensor is based on a multi-enzyme cascade reaction system in the enzyme reaction layer. Specifically, the analyte adenosine is first deaminated by adenosine deaminase to form inosine. Inosine is then phosphorylated by purine nucleoside phosphorylase to produce hypoxanthine and ribose. Hypoxanthine is then catalyzed by xanthine oxidase to ultimately produce uric acid and hydrogen peroxide. Accompanying the rapid conversion from Prussian white to Prussian blue in the electron mediator layer on the conductive layer surface, hydrogen peroxide can be rapidly reduced to OH- via the transferred electrons. -Meanwhile, Prussian blue can be reduced back to Prussian white at very low catalytic potentials, typically below 0V (vs. Ag / AgCl). In this process, Prussian blue acts as an electron mediator, transferring electrons from the conductive layer surface to H₂O₂, thereby generating a detection current. Therefore, the prepared adenosine sensor can be used for the quantitative detection of the analyte adenosine using a chronoamperometry method.

[0051] The adenosine detection principle of the adenosine sensor is shown in the following formula:

[0052]

[0053]

[0054]

[0055] (Prussian white) K4Fe4 II [Fe II [CN)6]3+2H2O2→(Prussian blue)Fe4 III [Fe II (CN)6]3+4K + +4OH -

[0056] (Prussian blue)Fe4 III [Fe II (CN)6]3+4K + +4e - →(Prussian White) K4Fe4 II [Fe II (CN)6]3

[0057] The working principle of each functional modification layer is as follows: Figure 4 As shown.

[0058] To select the optimal detection voltage, the implanted end of the prepared adenosine sensor was immersed in a moderately stirred phosphate buffer solution (0.1 M, pH 7.4). The other end of the sensor was connected to an electrochemical workstation for chronocurrent detection. The applied detection voltages were -0.1, -0.05, 0, 0.1, and 0.2 V. After the baseline current stabilized, a certain volume of high-concentration adenosine solution was added dropwise to the buffer solution to create a 5 μM concentration gradient of the adenosine to be tested. This was repeated every 100 s, ultimately yielding multiple current-time detection curves, as shown below. Figure 5-A As shown. A linear fit was performed between adenosine concentration and detection current value to obtain and compare the detection sensitivity at different detection voltages. Figure 5-B It can be seen that the sensor has the highest sensitivity to adenosine when the detection voltage is -0.05V. Therefore, -0.05V is selected as the optimal adenosine detection voltage.

[0059] Example 5: Continuous detection of adenosine in the in vitro environment

[0060] To verify the continuous detection performance of the prepared adenosine sensor in vitro, the implanted end of the adenosine sensor was immersed in a moderately stirred phosphate buffer solution (0.1 M, pH 7.4). The other end of the sensor was connected to an electrochemical workstation for chronocurrent detection at a detection voltage of -0.05 V. After the baseline current stabilized, a certain volume of high-concentration adenosine solution was added dropwise to the buffer solution to create concentration gradients of 0.5 and 5 μM adenosine, with additions every 100 s. After multiple additions, the final adenosine concentration reached 50 μM, and the current-time detection curve was obtained, as shown below. Figure 6-A As shown. A linear fit was performed between adenosine concentration and detection current value (…). Figure 6-B The linear regression equation y = -1.1x - 45.09 was obtained, and the linear correlation coefficient (R²) was [missing value]. 2 The adenosine concentration was 0.997, and the detection sensitivity was 1.1 nA / μM, indicating that the adenosine concentration in the range of 0-50 μM showed a good linear relationship with the detection current, further verifying that the prepared adenosine sensor has good continuous detection capability in the in vitro environment.

[0061] Example 6 Anti-interference capability test

[0062] To verify the detection specificity of the prepared adenosine sensor, its anti-interference capability was tested. The implanted end of the adenosine sensor was immersed in a moderately stirred phosphate buffer solution (0.1M, pH 7.4). The other end of the sensor was connected to an electrochemical workstation for chronoamperometry detection at a voltage of -0.05V. After the baseline current stabilized, a high-concentration adenosine solution was added dropwise twice to the buffer solution to increase the concentration of the adenosine to be measured by 5 μM. Then, based on the physiological concentrations of common electrochemical interfering substances in the body environment, 5 μM dopamine, 400 μM ascorbic acid, and 100 μM uric acid were added to the buffer solution sequentially. Finally, adenosine was added twice more. Figure 7 It can be seen that, compared with the detection current generated by adenosine, the interference current generated by dopamine, ascorbic acid and uric acid, which are common in vivo interfering substances at the three physiological concentrations, is negligible. This indicates that the adenosine sensor has good anti-interference ability. This is due to the Prussian blue nanocomposite material significantly reducing the detection voltage, and the combined use of the poly(o-phenylenediamine) anti-interference layer.

[0063] Example 7: Serial Detection of Local Adenosine in Animals

[0064] SPF-grade healthy male Wistar rats, weighing 230±20g, were used in the experiment. After anesthetizing the rats with sodium phenobarbital via intraperitoneal injection, the skin and hair on the right thigh were shaved, and the rats were then fixed to a surgical board. The prepared adenosine sensor implantation end was inserted into the local muscle tissue of the thigh using a disposable sterile syringe needle or a notched needle, and externally secured with medical plaster to prevent it from being pulled out upon awakening. The other end of the sensor was connected to an electrochemical workstation for timing current detection at a voltage of -0.05V. Once the baseline current reached stability (20 min), 50 μL of adenosine solution (4 mM) and 50 μL of physiological saline (9%) were slowly injected sequentially at a distance of 3-5 mm from the sensor implantation site using a syringe needle, and the changes in the detection current were recorded in real time. Figure 8 As shown, after injecting a higher concentration of adenosine, the local adenosine concentration rapidly increases, and the detection current intensity also increases rapidly, reaching a peak after about 30 seconds. Subsequently, the detection current intensity gradually decreases. This is likely due to the slow decrease in local adenosine concentration caused by diffusion and metabolism. Furthermore, the peak detection current values ​​after two adenosine injections are essentially consistent, further indicating good in vivo repeatability. In contrast, injecting saline solution shows a significant difference; it only causes a momentary fluctuation in the detection current, which quickly returns to baseline levels. This may be because the mechanical damage caused by the injection itself induces the rapid release and breakdown of local adenosine in vivo, a neuroprotective mechanism. These results demonstrate that the prepared adenosine sensor possesses excellent continuous in vivo adenosine detection capability.

[0065] Example 8: Serial Detection of Local Adenosine in Animal Brain

[0066] SPF-grade healthy male Wistar rats, weighing 230±20g, were used in the experiment. After anesthetizing the rats with intraperitoneal injection of sodium phenobarbital, the top of the head was shaved and the skin prepared. The rats were then fixed back-up to a stereotaxic apparatus. The skin of the skull and face was cut with surgical scissors, and the four corners of the skin were secured with vascular clamps to fully expose the cranial wound. The periosteum was bluntly dissected and removed. The anterior fontanelle was located based on the cranial sutures and used as the center point of the three-dimensional coordinate system. The three-dimensional manipulator was manipulated to position itself at the anterior fontanelle, and marked as the zero point. The manipulator was then moved to the position (XYZ) = (3.0.0), marked with a marker, and the manipulator was removed. A cranial drill was used to create a hole. The prepared adenosine sensor implantation end was fixed to the holder and moved to the cranial foramen, then lowered to a Z-value of 5 (striatum). The other end of the sensor was connected to an electrochemical workstation for timing current detection at a voltage of -0.05V. Once the baseline current stabilized (10 min), 4 mM and 10 mM adenosine solutions were sequentially injected into the brain using a microsyringe, with each injection volume being 20 μL. The changes in the detected current were recorded in real time. Figure 9As shown, both adenosine injections increased the detection current intensity, and the increase in current caused by higher concentrations of adenosine was more significant. These results demonstrate that the prepared adenosine sensor can achieve in vivo real-time detection of changes in adenosine concentration in the animal brain.

[0067] Example 9: Real-time in vivo monitoring of acupuncture-induced local adenosine release at acupoints.

[0068] SPF-grade healthy male Wistar rats, weighing 230±20g, were used in the experiment. The "Zusanli" acupoint was located according to the animal acupoint location method in *Experimental Acupuncture*. Zusanli is located in the muscle groove approximately 5mm below the fibular head on the lateral aspect of the rat's hind leg, below the knee joint. First, the rats were anesthetized by intraperitoneal injection of sodium phenobarbital. After complete anesthesia, the hair at the right "Zusanli" acupoint was shaved, and the rat was then fixed to a surgical board. Using a disposable sterile syringe needle or a notched needle, the implantable end of the prepared adenosine sensor was implanted into the muscle tissue of the rat's lower leg at the Zusanli acupoint. Medical plaster was applied externally for fixation to prevent the sensor from being pulled out upon awakening. The other end of the sensor was connected to an electrochemical workstation for timing current detection at a voltage of -0.05V. Once the baseline current stabilized (20-40 min), disposable sterile acupuncture needles (0.35×25 mm) were inserted into the Zusanli acupoint of the rats. A rotating manipulation technique was applied, using a balanced tonifying and reducing method, at a frequency of 180 times / min for 2 minutes. This was followed by manipulation every 8 minutes for a total of 3 times, with a total acupuncture time of 30 minutes. The changes in current were recorded in real time. The rotation frequency was controlled by a metronome, and all acupuncture procedures were performed by a single person. To ensure the stability of the acupuncture technique, the operator repeatedly practiced the technique on an acupuncture technique parameter analyzer before and during the experiment. Figure 10 As shown, twisting caused rapid fluctuations and increases in the detection current. After twisting stopped, the detection current slowly decreased. The changes in the detection current were consistent across the three twisting processes, and the intensity of the detection current remained higher than the initial value throughout the entire acupuncture process. This fully demonstrates that acupuncture induces a sustained release of adenosine in the acupoint area, and the degree and duration of adenosine concentration changes are correlated with the acupuncture technique and intensity. The above experimental results preliminarily verify that adenosine, as a neurotransmitter, plays a certain mediating role in the mechanism of acupuncture efficacy, and further prove that the prepared adenosine sensor can be effectively applied to the study of acupuncture mechanisms.

[0069] In contrast, the conventional method of microdialysis-high performance liquid chromatography-mass spectrometry (HPLC-MS) was used to detect the effect of acupuncture at the Zusanli acupoint on local adenosine concentration in rats. The anesthesia and fixation procedures for rats were as described above. Before using the microdialysis equipment, the tubing system was checked for integrity. Then, all tubing was connected, and the skin at the Zusanli acupoint was cut open with surgical scissors. A syringe needle with a tear-tube attached was inserted into the local muscle tissue at the acupoint, leaving the tear-tube in place. The syringe needle was then slowly withdrawn. A probe was then inserted into the tear-tube, and the probe was advanced while the tear-tube was withdrawn until the probe was fully implanted into the local muscle tissue at the acupoint. The probe could be fixed with tape or surgical sutures. Ringer's solution was infused at a uniform rate (2 μL / min). After equilibration for 2 hours, tissue fluid collection began. Because the amount of local tissue fluid produced was extremely small, one sample tube was collected every 30 minutes. Tissue fluid samples were collected during the first 30 minutes of acupuncture and during the first 30 minutes of acupuncture, using the acupuncture method described above. During dialysate collection, the collection tubes should be placed on an ice pack. After collection, all samples should be immediately stored at -20°C and transferred to a -80°C freezer for analysis after dialysis. High-performance liquid chromatography-mass spectrometry (HPLC-MS) was used for detection. The collected samples were first thawed at room temperature, then centrifuged at 13000 rpm for 10 minutes. The supernatant was then removed and analyzed. The final experimental results showed that, compared to before acupuncture, 30 minutes of acupuncture resulted in an approximately four-fold increase in local adenosine concentration at the acupoint.

[0070] Compared with conventional detection methods, although both sets of experimental results demonstrate that acupuncture can induce an increase in local adenosine concentration in the acupoint area, the conventional method of microdialysis-high performance liquid chromatography-mass spectrometry requires strict and accurate calibration of the extracted samples, which is cumbersome in terms of probe recovery rate determination. Furthermore, the sampling time interval is long, and other techniques are still needed for detection after sampling, thus failing to accurately reflect the changes in adenosine during acupuncture in real time. The prepared adenosine sensor is a flexible, miniature composite sensing needle structure integrating a working electrode, counter electrode, and reference electrode. It features high detection sensitivity, fast response speed, simple operation, and portability. It can achieve in vivo, in-situ, real-time, and dynamic detection of local adenosine concentration changes in the acupoint area throughout the entire acupuncture process, providing a more intuitive and effective modern biological detection method for the study of the acupuncture effect principle, and further providing strong scientific evidence for guiding clinical practice and improving clinical efficacy.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for surface functionalization modification of the conductive reaction region of a working electrode, characterized in that: Includes the following steps: (1) The conductive reaction region is immersed in the electrodeposition solution for electrodeposition to form an electron mediator layer on the conductive reaction region; (2) A polyethyleneimine-functionalized graphene oxide solution is dropped onto the surface of the electron mediator layer, dried, and then a multi-enzyme cascade reaction solution is dropped onto its surface and dried to form an enzyme reaction layer. (3) The conductive reaction region modified with the electron mediator layer and enzyme reaction layer is immersed in the anti-interference solution for electrodeposition, and then dried to form the anti-interference layer. The concentration of the polyethyleneimine-functionalized graphene oxide solution is 1-10 mg / ml; the concentration of xanthine oxidase in the multi-enzyme cascade reaction solution is 0.5-5 U / ml; the concentration of purine nucleoside phosphorylase in the multi-enzyme cascade reaction solution is 0.5-5 U / ml; the concentration of adenosine deaminase in the multi-enzyme cascade reaction solution is 0.5-5 U / ml; and the concentration of genipin in the multi-enzyme cascade reaction solution is 1-10 mg / ml. The electrodeposition solution in step (1) comprises hydrochloric acid at a concentration of 2-5 mg / mL, potassium chloride solution at a concentration of 5-10 mg / mL, ferric chloride solution at a concentration of 2-5 mg / mL, potassium ferricyanide solution at a concentration of 2-10 mg / mL, and graphyne nanoparticles at a concentration of 0.1-1 mg / mL; the anti-interference solution in step (3) comprises o-phenylenediamine at a concentration of 10-50 mg / mL and bovine serum albumin at a concentration of 1-10 mg / mL.

2. A three-electrode detection structure, characterized in that: The device includes a silver foil and two flexible film substrates. The flexible film substrates are fixed to the upper and lower surfaces of the silver foil by adhesive layers. Each flexible film substrate has a conductive layer, and each conductive layer has an insulating layer. The length of the insulating layer is less than the length of the conductive layer; The portion of the conductive layer exposed relative to the insulating layer is a conductive reaction region, and one of the conductive reaction regions is surface functionalized using the method described in claim 1.

3. The three-electrode detection structure according to claim 2, characterized in that: The length of the conductive layer is less than the length of the silver foil; the material of the conductive layer is at least one of gold, platinum, titanium, palladium, copper, carbon black, graphite or graphene; the flexible film substrate is at least one of polycarbonate, polytetrafluoroethylene, polyimide, polyethylene, polyethylene terephthalate, acrylonitrile-butadiene-styrene copolymer or polymethyl methacrylate.

4. An application of the three-electrode detection structure according to claim 2 or 3, characterized in that: The application of the described three-electrode detection structure in the fabrication of an adenosine sensor; the described adenosine sensor is an implantable continuous adenosine detection sensor.

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