CN-115747200-B - Application of three-electrode detection structure in preparation of adenosine sensor

Abstract

The invention provides an application of a three-electrode detection structure in preparing an adenosine sensor, and an application of the three-electrode detection structure in acupuncture. The three-electrode detection structure is applied to the preparation of an adenosine sensor, a glucose sensor, a lactic acid sensor, a glutamic acid sensor, a uric acid sensor, an ascorbic acid sensor or a dopamine sensor, wherein the adenosine sensor is an implantable adenosine continuous detection sensor. The flexible implantable adenosine sensor with the three-electrode structure has the characteristics of good stability, high sensitivity, wide linear detection range, short response time, strong anti-interference performance and the like, can realize in-vivo continuous detection of the concentration change of the adenosine in the animal body and the brain, is successfully applied to in-vivo real-time monitoring of the local adenosine release in acupuncture-induced acupoints, and provides a brand-new modern detection means for the deep study of acupuncture action mechanisms.

Inventors

• ZHANG DI
• FANG YUXIN

Assignees

• 天津中医药大学

Dates

Publication Date

20260512

Application Date

20221122

Claims (4)

1. 1. A method for functionally modifying the surface of a conductive reaction zone of a working electrode is characterized by comprising the following steps: (1) Immersing a conductive reaction area into an electrodeposition solution for electrodeposition, and forming an electron mediator layer on the conductive reaction area; (2) Dripping a polyethylene imine functionalized graphene oxide solution onto the surface of the electron mediator layer, airing, dripping a multienzyme cascade reaction solution onto the surface of the graphene oxide solution, and airing to form an enzyme reaction layer; (3) Immersing the conductive reaction area modified with the electron mediator layer and the enzyme reaction layer into an anti-interference solution for electrodeposition, and airing to form an 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 0.5-5U/ml, the concentration of purine nucleoside phosphorylase in the multi-enzyme cascade reaction solution is 0.5-5U/ml, the concentration of adenosine deaminase in the multi-enzyme cascade reaction solution is 0.5-5U/ml, and the concentration of genipin in the multi-enzyme cascade reaction solution is 1-10 mg/ml; The electro-deposition solution in the step (1) comprises hydrochloric acid with the concentration of 2-5 mg/mL, potassium chloride solution with the concentration of 5-10 mg/mL, ferric chloride solution with the concentration of 2-5 mg/mL, potassium ferricyanide solution with the concentration of 2-10 mg/mL and graphite alkyne nano particles with the concentration of 0.1-1 mg/mL, and the anti-interference solution in the step (3) comprises o-phenylenediamine with the concentration of 10-50 mg/mL and bovine serum albumin with the concentration of 1-10mg/mL.
2. 2. The three-electrode detection structure is characterized by comprising a silver foil and 2 flexible film substrates, wherein the flexible film substrates are respectively fixed on the upper surface and the lower surface of the silver foil through adhesive layers, conductive layers are arranged on the flexible film substrates, and insulating layers are arranged on the conductive layers; the length of the insulating layer is smaller than that of the conducting layer; the exposed part of the conductive layer relative to the insulating layer is a conductive reaction zone, and one of the conductive reaction zones is subjected to surface functionalization modification by the method of claim 1.
3. 3. The three-electrode detection structure according to claim 2, wherein the length of the conductive layer is smaller than that of the silver foil, the conductive layer is made of at least one of gold, platinum, titanium, palladium, copper, carbon black, graphite or graphene, and the flexible film substrate is at least one of polycarbonate, polytetrafluoroethylene, polyimide, polyethylene terephthalate, acrylonitrile-butadiene-styrene copolymer or polymethyl methacrylate.
4. 4. The application of the three-electrode detection structure according to claim 2 or 3 is characterized in that the three-electrode detection structure is applied to the preparation of an adenosine sensor, and the adenosine sensor is an implantable adenosine continuous detection sensor.

Description

Application of three-electrode detection structure in preparation of adenosine sensor Technical Field The invention belongs to the field of electrochemistry, and particularly relates to application of a three-electrode detection structure in preparation of an adenosine sensor. Background The therapeutic effect of acupuncture is definite, but the mechanism of action and biological basis thereof need to be explained. In recent years, a great deal of research has shown that adenosine signaling plays a key role in the initiation of acupuncture. Adenosine is produced by the hydrolysis of Adenosine Triphosphate (ATP), an endogenous neuromodulator, involved in a variety of physiological and pharmacological processes, such as neurotransmission, inflammation, ischemic injury and pain. As an extracellular messenger in most body fluids, adenosine functions by interacting with four G-protein coupled receptor subtypes (a 1、A2A、A2B and a 3), which differ significantly in affinity for adenosine. Importantly, different adenosine concentrations can mediate activation of different adenosine receptor subtypes, thereby exerting different physiological regulatory functions. Thus, the immediate detection of induced local adenosine release under different conditions or stimuli is a real need in the art of adenosine research. At present, most of the detection methods for the in-vivo adenosine are to treat the extracted blood or specific tissues under the in-vitro condition and then detect the extracted blood or specific tissues by adopting the traditional high performance liquid chromatography technology or the immunological analysis method, and the like, which can cause the defects of intermittent, delayed, large difference, unstable and the like of experimental results. In-vivo sampling detection is performed by using a microdialysis technology in recent years, but sampling is performed continuously for 5-20 minutes each time, so that the accumulated amount is rather than the real-time amount, and detection is performed by using other technologies such as a high performance liquid chromatography technology after sampling, so that the concentration change rule of adenosine in the whole needling effect process cannot be truly and accurately reflected in the first time, and how the concentration change participates in real-time regulation. Disclosure of Invention In view of the above, the present invention aims to overcome the defects in the prior art, and proposes an application of a three-electrode detection structure in preparing an adenosine sensor. In order to achieve the above purpose, the technical scheme of the invention is realized as follows: The invention provides an enzyme immobilization promoter, which comprises polyethylene imine functionalized graphene oxide and genipin in a mass ratio of 1-5:1. The invention also provides an enzyme crosslinking fixing solution, which comprises a polyethyleneimine functionalized graphene oxide solution and a multienzyme cascade reaction solution in a volume ratio of 1-3:1. Further, the multienzyme 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. Further, the concentration of the polyethyleneimine functionalized graphene oxide solution is 1-10mg/ml, the concentration of xanthine oxidase in the multi-enzyme cascade reaction solution is 0.5-5U/ml, the concentration of purine nucleoside phosphorylase in the multi-enzyme cascade reaction solution is 0.5-5U/ml, the concentration of adenosine deaminase in the multi-enzyme cascade reaction solution is 0.5-5U/ml, and the concentration of genipin in the multi-enzyme cascade reaction solution is 1-10mg/ml. The invention also provides a method for functionally modifying the surface of the conductive reaction zone of the working electrode, which comprises the following steps: (1) Immersing a conductive reaction area into an electrodeposition solution for electrodeposition, and forming an electron mediator layer on the conductive reaction area; (2) Dripping a polyethylene imine functionalized graphene oxide solution onto the surface of the electron mediator layer, airing, dripping the multienzyme cascade reaction solution onto the surface of the graphene oxide solution, and airing to form an enzyme reaction layer; (3) Immersing the conductive reaction area modified with the electron mediator layer and the enzyme reaction layer into an anti-interference solution for electrodeposition, and airing to form an anti-interference layer. Further, the electrodeposition solution in the step (1) comprises hydrochloric acid with the concentration of 2-5mg/mL, potassium chloride solution with the concentration of 5-10mg/mL, ferric chloride solution with the concentration of 2-5mg/mL, potassium ferricyanide solution with the concentration of 2-10mg/mL and graphite alkyne nano particles with the concentration of 0.1-1mg/mL, and the anti-interference solution