CN116898433A - Electrochemical sensor based on spiral electrode and preparation method thereof - Google Patents

Electrochemical sensor based on spiral electrode and preparation method thereof Download PDF

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Publication number
CN116898433A
CN116898433A CN202310678433.1A CN202310678433A CN116898433A CN 116898433 A CN116898433 A CN 116898433A CN 202310678433 A CN202310678433 A CN 202310678433A CN 116898433 A CN116898433 A CN 116898433A
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electrode
microwire
spiral
layer
electrochemical sensor
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谢曦
贺梦怡
陈惠琄
黄爽
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Sun Yat Sen University
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Sun Yat Sen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/1486—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using enzyme electrodes, e.g. with immobilised oxidase
    • A61B5/14865—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using enzyme electrodes, e.g. with immobilised oxidase invasive, e.g. introduced into the body by a catheter or needle or using implanted sensors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14507—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue specially adapted for measuring characteristics of body fluids other than blood
    • A61B5/1451—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue specially adapted for measuring characteristics of body fluids other than blood for interstitial fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14546—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue for measuring analytes not otherwise provided for, e.g. ions, cytochromes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/68—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient
    • A61B5/6846—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive
    • A61B5/6847—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive mounted on an invasive device
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/68—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient
    • A61B5/6846—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive
    • A61B5/6847—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive mounted on an invasive device
    • A61B5/6848—Needles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements

Abstract

The application discloses an electrochemical sensor based on a spiral electrode and a preparation method thereof, wherein the electrochemical sensor comprises a spiral microwire electrode, a microwire reference electrode, a microwire counter electrode and a circuit detection system; the first ends of the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode are connected and fixed with the circuit detection system; the microwire reference electrode and the microwire counter electrode penetrate through the spiral structure of the spiral microwire electrode; the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode all comprise a sensing area and a non-sensing area, and the non-sensing area is provided with an insulating layer; the sensing area of the spiral structure of the spiral microwire electrode is covered with multiple membrane layers. The embodiment of the application improves the living environment interference resistance of the electrochemical sensor, increases the sensing area of the microfilament electrode, enhances the sensing current signal intensity, improves the detection sensitivity of the sensor, realizes the real-time in-situ sensing of the concentration of biochemical molecules in subcutaneous tissue fluid, and can be widely applied to the field of sensors.

Description

Electrochemical sensor based on spiral electrode and preparation method thereof
Technical Field
The application relates to the field of sensors, in particular to an electrochemical sensor based on a spiral electrode and a preparation method thereof.
Background
Along with the micro-nano processing technology, artificial intelligence, the Internet of things technology, the appearance and development of flexible stretchable and biocompatible electronic products, the realization of monitoring health-related biochemical signals in real time and in situ and providing timely early warning and feedback targets has advanced to a certain extent, which is significant for developing new-generation diagnosis and treatment technologies. The semi-implanted device has wide application prospect in the aspect of real-time sensing of in-vivo physiological information, and the sensor is implanted under the skin, and a circuit of the heavy signal modulation and transmission module is externally arranged on the body surface, so that the change of the concentration of biochemical molecules in interstitial fluid can be monitored in real time and in situ, and the volume of the sensor implanted in the body is reduced. In particular, electrochemical sensors based on microneedles or microwires are capable of painless or minimally invasive sensing of the concentration of the analyte to be measured in interstitial fluid, thereby attracting increasing attention and research interest.
Currently, the semi-implantable electrode for commercial use has been used for monitoring physiological and biochemical clinical related values in living bodies, such as long-time in-vivo blood glucose monitoring of diabetics, pH monitoring of gastroesophageal reflux, blood pressure monitoring of heart failure and the like. However, the tiny size of the wire electrode limits the intensity and sensitivity of the amperometric signal of the electrode during in vivo electrochemical processes, and if the signal is too weak, the external physical signal or the environmental interference signal can easily affect the current signal of the microwire electrode. In addition, the "oxygen starved" environment within living tissue can also limit the detection linear range of small area wire electrodes.
Disclosure of Invention
Accordingly, an object of the embodiments of the present application is to provide an electrochemical sensor based on a spiral electrode and a method for manufacturing the same, which can improve the performance of resisting living environment interference and the test sensitivity.
In a first aspect, an embodiment of the present application provides an electrochemical sensor based on a spiral electrode, including a spiral microwire electrode, a microwire reference electrode, a microwire counter electrode, and a circuit detection system; the first ends of the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode are connected and fixed with the circuit detection system; the microwire reference electrode and the microwire counter electrode penetrate through the spiral structure of the spiral microwire electrode; the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode all comprise a sensing area and a non-sensing area, and the non-sensing area is provided with an insulating layer; the sensing area of the spiral structure of the spiral microwire electrode is covered with multiple membrane layers.
Optionally, the spiral microwire electrode comprises a linear structure and a spiral structure, and the linear structure is connected with the circuit detection system; the area of the linear structure is a non-sensor area and is covered with an insulating layer; the area of the spiral structure is the sensing area.
Optionally, the spiral structure of the spiral microwire electrode sequentially comprises a substrate electrode, a first electron mediator layer, a second electron mediator layer, a conductive layer and a biological enzyme layer from inside to outside.
Optionally, the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode all comprise substrate electrodes, the material of the substrate electrodes comprises a high conductive material, and the high conductive material comprises any one of gold, carbon fiber, platinum iridium and nickel titanium.
Optionally, the area of the second end of the microwire reference electrode or microwire counter electrode comprises a sensing area of the electrode tip, the sensing area being located outside the area of the helical structure of the helical microwire electrode.
Optionally, the microfilament reference electrode comprises any of a silver/silver chloride/polyvinyl butyral electrode, a silver/silver chloride electrode, a calomel electrode, a hydrogen electrode, a mercury/mercury oxide electrode, or a mercury/mercurous sulfate electrode.
Optionally, the material of the insulating layer includes a material having insulation and biocompatibility, and the material of the insulating layer includes any one of parylene series material, polyimide, polydimethylsiloxane, or parylene.
In a second aspect, an embodiment of the present application provides a method for preparing an electrochemical sensor based on a spiral electrode, which is applied to the electrochemical sensor based on the spiral electrode, including:
insulating the substrate electrode on the non-sensing area corresponding to the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode;
preparing a microwire reference electrode and a microwire counter electrode;
preparing a spiral structure of a spiral microfilament electrode, and preparing a multi-film layer on the surface of the spiral structure of the spiral microfilament electrode;
and connecting the first ends of the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode with a circuit detection system for integration.
Optionally, preparing a spiral structure of the spiral microwire electrode, specifically comprising:
winding a microwire electrode of a sensing area on a columnar structure to construct a spiral structure;
separating the microwire electrode with the spiral structure from the columnar structure;
the helical structure is stretched such that there is a uniform gap between the helical rings of the helical structure.
Optionally, preparing a multi-film layer on the surface of the spiral structure of the spiral microwire electrode, specifically comprising:
the spiral structure is modified by sequentially using a first electron mediator, a second electron mediator, a conductive material and biological enzyme, so that a first electron mediator layer, a second electron mediator layer, a conductive layer and a biological enzyme layer are sequentially formed on the surface of the spiral structure.
The embodiment of the application has the following beneficial effects: the spiral microwire electrode is adopted in the embodiment, the spiral structure of the spiral microwire electrode improves the living body environment interference resistance of the electrochemical sensor, and the sensing area of the microwire electrode is increased while the vertical length of the electrode is ensured, so that the sensing current signal intensity is increased, and the detection sensitivity of the electrochemical sensor is improved; the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode which pass through the spiral microwire electrode form a three-electrode system, and are connected with the circuit detection system to form an electrochemical sensor based on the spiral electrode, so that the real-time in-situ sensing of the concentration of biochemical molecules in subcutaneous tissue fluid is realized, and the diagnosis and the subsequent treatment of a patient are facilitated; and the insulating layer on the surface of the microfilament electrode adopts a material with biocompatibility, so that the biosafety is improved.
Drawings
FIG. 1 is a schematic view of an electrochemical sensor based on a spiral electrode according to an embodiment of the present application;
fig. 2 is a schematic structural view of an electrochemical sensor implanted in a human body based on a spiral electrode according to an embodiment of the present application;
FIG. 3 is a schematic structural view of a spiral microwire electrode according to an embodiment of the present application;
FIG. 4 is a schematic flow chart of a method for manufacturing an electrochemical sensor based on a spiral electrode according to an embodiment of the present application;
FIG. 5 is a schematic diagram of a process for preparing a spiral microwire electrode according to an embodiment of the present application;
FIG. 6 is a schematic SEM diagram of a spiral microfilament glucose sensing system after integration of three electrodes and three electrodes; wherein, figure (a) is an SEM schematic diagram of the surface structure of the gold substrate; FIG. b is a SEM schematic view of the surface structure of the CNT layer; FIG. C is a SEM schematic diagram of the surface structure of PEDOT: PSS layer; FIG. d is a SEM schematic view of the surface structure of the Pt layer; FIG. (e) is a schematic view of a microwire electrode insulation structure; FIG. (f) is an SEM schematic of the surface of an Ag/AgCl/PVB microfilament reference electrode; FIG. (g) is an SEM schematic of the surface of a platinum microfilament counter electrode; FIG. (h) is an SEM schematic of an integrated three-electrode system;
FIG. 7 is a graph of glucose concentration response test results for a spiral electrode-based electrochemical sensor according to an embodiment of the present application; wherein, the graph (a) is a graph of the glucose concentration response test result of the electrochemical sensor with 1 spiral turn; FIG. (b) is a graph showing the glucose concentration response test result of an electrochemical sensor having 4 spiral turns; FIG. (c) is a graph showing the glucose concentration response test result of an electrochemical sensor having 7 spiral turns; FIG. (d) is a graph showing the glucose concentration response test result of an electrochemical sensor having 10 spiral turns;
FIG. 8 is a graph of the results of testing the anti-interference performance of an electrochemical sensor based on a spiral electrode according to an embodiment of the present application;
FIG. 9 is a graph showing the results of a test of subcutaneous glucose concentration in rats within 3 hours for an electrochemical sensor based on a spiral electrode according to an embodiment of the present application;
reference numerals illustrate: 1. a spiral microwire electrode; 2. a microfilament reference electrode; 3. a microwire counter electrode; 4. an insulating layer; 5. a circuit detection system; 22. a skin layer; 23. a dermis layer; 24. a glucose molecule; 11. an electrode substrate; 12. a first electron mediator layer; 13. a second electron mediator layer; 14. a conductive layer; 15. a biological enzyme layer.
Detailed Description
The application will now be described in further detail with reference to the drawings and to specific examples. The step numbers in the following embodiments are set for convenience of illustration only, and the order between the steps is not limited in any way, and the execution order of the steps in the embodiments may be adaptively adjusted according to the understanding of those skilled in the art.
In the following description, reference is made to "some embodiments" which describe a subset of all possible embodiments, but it is to be understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with one another without conflict.
In the following description, the terms "first", "second", "third" and the like are merely used to distinguish similar objects and do not represent a specific ordering of the objects, it being understood that the "first", "second", "third" may be interchanged with a specific order or sequence, as permitted, to enable embodiments of the application described herein to be practiced otherwise than as illustrated or described herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the embodiments of the application is for the purpose of describing embodiments of the application only and is not intended to be limiting of the application.
Before describing embodiments of the present application in further detail, the terms and terminology involved in the embodiments of the present application will be described, and the terms and terminology involved in the embodiments of the present application will be used in the following explanation.
As shown in fig. 1, a first embodiment of the present application provides an electrochemical sensor based on a spiral electrode, which comprises a spiral microwire electrode 1, a microwire reference electrode 2, a microwire counter electrode 3 and a circuit detection system 5; the first ends of the spiral microwire electrode 1, the microwire reference electrode 2 and the microwire counter electrode 3 are connected and fixed with the circuit detection system 5; the microwire reference electrode 2 and the microwire counter electrode 3 penetrate through the spiral structure of the spiral microwire electrode 1; the spiral microwire electrode 1, the microwire reference electrode 2 and the microwire counter electrode 3 all comprise a sensing area and a non-sensing area, and the non-sensing area is provided with an insulating layer 4; the sensing area of the spiral structure of the spiral microwire electrode 1 is covered with multiple membrane layers.
Specifically, the microwire reference electrode 2 and microwire counter electrode 3 are longer than the helical microwire electrode 1 in length, passing vertically through the helical structure of the helical microwire electrode 1.
Specifically, the spiral microwire electrode 1, the microwire reference electrode 2 and the microwire counter electrode 3 form a three-electrode system. The spiral microwire electrode 1 is used as a sensing working electrode for detecting the concentration of biochemical molecules in subcutaneous tissue interstitial fluid (ISF). The microwire reference electrode 2 is used for precisely controlling the electrode point position of the spiral microwire electrode 1. The microwire counter electrode 3 is used as an auxiliary electrode for conducting current and forms a loop with the spiral microwire electrode 1.
Specifically, the diameter of the three microwire electrodes as described above may range from 5 to 200 μm.
Specifically, the multilayer film on the spiral structure of the spiral-shaped microwire electrode 1 is formed by modifying with a set material.
In particular, the electrochemical sensor based on the spiral electrode of the embodiment is used for detecting the concentration of glucose in a human body, and can also detect the concentration of other biochemical molecules, including but not limited to Na + ，K + ，Ca 2+ pH, uric acid, lactic acid,cholesterol, monoamine, ethanol, phospholipids, L-amino acids, tyrosine, pyruvic acid, etc.
Specifically, detection of other biochemical molecules in the human body is achieved by changing the material of the multilayer film on the spiral microwire electrode.
In particular, electrochemical sensors based on spiral electrodes may also be applied to analyze biological fluids, including cerebral spinal fluid, lymph fluid, blood or urine.
Optionally, the spiral microwire electrode 1 comprises a linear structure and a spiral structure, and the linear structure is connected with the circuit detection system 5; the area of the linear structure is a non-sensor area and is covered with an insulating layer 4; the area of the spiral structure is the sensing area.
Specifically, as shown in fig. 2, the sensing area is fully implanted subcutaneously at the time of measurement; the non-sensing area is mainly arranged outside the body, and part of the non-sensing area is contacted with human tissues.
Specifically, as shown in fig. 2, the spiral microwire electrode 1, microwire reference electrode 2 and microwire counter electrode 3 are arranged in the non-sensing area outside the human body, and the proper length can be selected according to actual requirements.
Optionally, as shown in fig. 3, the spiral structure of the spiral microwire electrode 1 sequentially comprises a substrate electrode 11, a first electron mediator layer 12, a second electron mediator layer 13, a conductive layer 14 and a biological enzyme layer 15 from inside to outside.
Specifically, the substrate electrode is used to transmit an electrical signal to the circuit detection system 5.
Specifically, the electron mediator layer is used to improve the electrochemical performance of the sensing electrode. In this embodiment, the material of the first electron mediator layer adopts Carbon Nanotubes (CNTs) to form an electron mediator CNT layer; the second electron mediator layer is made of poly 3, 4-ethylene-dioxythiophene: polystyrene sulfonate (PEDOT: PSS) to form an electron mediator PEDOT: PSS layer.
Specifically, the material of the electron mediator layer also comprises ferrocene, tetrathiafulvalene and the like.
Specifically, the conductive layer is used for catalyzing oxidation-reduction reaction and conducting electric signals emitted by the biological enzyme layer. In this embodiment, the material of the conductive layer includes, but is not limited to, a metal material, such as platinum, to form a platinum layer.
Specifically, the bio-enzyme layer is used to convert the biochemical molecular concentration signal in subcutaneous ISF into an electrical signal. In this embodiment, the material of the bio-enzyme layer includes, but is not limited to, glucose oxidase, forming a glucose oxidase layer.
In particular, the glucose oxidase layer acts as a glucose catalytic element with high specificity, such that electrochemical glucose sensors based on helical electrodes can convert glucose concentration into amperometric signals. The detection of other biochemical molecule concentrations can be achieved by using different biological enzymes to form different layers of biological enzymes.
Optionally, the spiral microwire electrode 1, the microwire reference electrode 2 and the microwire counter electrode 3 all comprise substrate electrodes, the material of the substrate electrodes comprises a high conductive material, and the high conductive material comprises any one of gold, carbon fiber, platinum iridium and nickel titanium.
Specifically, the substrate electrode is a flexible microwire electrode. The material of the substrate electrode in this embodiment includes, but is not limited to, a metal material, such as gold.
Optionally, the area of the second end of the microwire reference electrode 2 or microwire counter electrode 3 comprises a sensing area of the electrode tip, the sensing area being located outside the area of the helical structure of the helical microwire electrode 1.
Specifically, both the microwire reference electrode 2 and the microwire counter electrode 3 are longer than the helical microwire electrode 1.
Optionally, the microfilament reference electrode 2 comprises a silver/silver chloride/polyvinyl butyral (Ag/AgCl/PVB) electrode, a silver/silver chloride (Ag/AgCl) electrode, a calomel electrode, a hydrogen electrode, a mercury/mercury oxide (Hg/HgO) electrode, or mercury/mercurous sulfate (Hg/Hg) 2 SO 4 ) Any one of the electrodes.
Specifically, the microwire reference electrode 2 in the present embodiment includes, but is not limited to, the above materials, for example, ag/AgCl/PVB microwire reference electrode is used.
Specifically, the microwire counter electrode 3 in the present embodiment includes, but is not limited to, a metal counter electrode, such as a microwire platinum counter electrode.
Optionally, the material of the insulating layer 4 includes a material having insulation and biocompatibility, and the material of the insulating layer 4 includes any one of parylene series materials (such as parylene C/D/F/N), polyimide (PI), polydimethylsiloxane (PDMS), or parylene.
Specifically, the material of the insulating layer 4 in this embodiment adopts parylene C, and the thickness of the insulating layer 4 may be in the range of 1-50 μm.
In particular, the insulating layer 4 is used to eliminate additional signal interference and prevent short circuit accidents.
In a second aspect, as shown in fig. 4, an embodiment of the present application provides a method for preparing an electrochemical sensor based on a spiral electrode, which is applied to the electrochemical sensor based on a spiral electrode, and includes:
s100, insulating the substrate electrode on a non-sensing area corresponding to the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode;
s200, preparing a microwire reference electrode and a microwire counter electrode;
s300, preparing a spiral structure of a spiral microfilament electrode, and preparing a multi-film layer on the surface of the spiral structure of the spiral microfilament electrode;
and S400, connecting the first ends of the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode with a circuit detection system for integration.
Specifically, the insulating treatment is performed on the non-sensing area of the electrode, and the insulating treatment comprises covering an insulating layer on the non-sensing area of the electrode. Specifically, a layer of parylene C is deposited on the non-sensing area corresponding to the substrate electrode in vacuum by a chemical vapor deposition method.
Specifically, a microwire reference electrode and a microwire counter electrode are prepared, which comprise corresponding film layers respectively covered on substrate electrodes thereof.
Specifically, a spiral structure of a spiral microwire electrode is prepared, and a multi-film layer is prepared on the surface of the spiral structure of the spiral microwire electrode, including a substrate electrode is wound on a columnar structure to form a spiral structure, and a multi-film layer is sequentially prepared on the spiral structure by using corresponding materials.
Optionally, preparing a spiral structure of the spiral microwire electrode 1, specifically comprising:
s310, winding the microwire electrode of the sensing area on the columnar structure to construct a spiral structure;
s320, separating the microwire electrode with the spiral structure from the columnar structure;
s330, stretching the spiral structure to enable uniform gaps to exist among spiral rings of the spiral structure.
Specifically, the columnar structure includes a needle, and the outer diameter is 0.7mm.
In particular, the size of the columnar structure may be less than 0.7mm, thereby obtaining a spiral-like structure of smaller size.
Specifically, stretching is performed on the spiral structure, and stretching can be performed through the electric displacement platform, so that uniform gaps are formed between spiral rings of the spiral structure.
Optionally, preparing a multi-film layer on the surface of the spiral structure of the spiral microwire electrode, specifically comprising:
and S340, sequentially using a first electron mediator, a second electron mediator, a conductive material and biological enzyme to modify the spiral structure, so that a first electron mediator layer, a second electron mediator layer, a conductive layer and a biological enzyme layer are sequentially formed on the surface of the spiral structure.
In a specific embodiment, an Ag/AgCl/PVB microfilament reference electrode is prepared comprising the steps of:
PVB solution was prepared by dissolving 791mg PVB powder and 500ml sodium chloride (NaCl) powder in 10ml methanol;
cleaning the surface of the substrate electrode with 75% alcohol for 2 minutes;
uniformly covering the sensing area corresponding to the insulated substrate electrode with Ag/AgCl slurry, and drying in an oven at 80 ℃;
a layer of PVB film is adsorbed on the sensing area covered with Ag/AgCl by a pulling method, and the PVB film is dried at room temperature.
Specifically, each time a PVB solution is used, it is sufficiently dissolved by ultrasonic heating at 65 ℃ for several hours.
Specifically, the preparation of the Ag/AgCl layer can also be achieved by an electroplating process.
Specifically, the preparation method of the microfilament platinum counter electrode comprises the following steps:
adding a proper amount of platinum sodium sulfite electroplating platinum solution into a beaker, and carrying out electrochemical deposition on a sensing area corresponding to an insulated substrate electrode by using a multi-step constant current method, wherein the specific parameters are as follows:
the first stage: 6e -5 A，10s；
And a second stage: -6e -5 A，10s；
And a third stage: -5e -4 A，600s。
The microfilament platinum counter electrode was taken out and dried at room temperature for more than 2 hours.
As shown in fig. 5, a spiral structure of a spiral microwire electrode is prepared, and a multi-film layer is prepared on the surface of the spiral structure of the spiral microwire electrode, comprising the following steps:
a 50mg/ml glucose oxidase (GOx) solution, a 80mg/ml Bovine Serum Albumin (BSA) solution, and a 2.5wt% glutaraldehyde solution (in PBS with ph=7.2) were prepared and stored in an environment at 4 ℃;
BSA solution, glutaraldehyde solution and GOx solution were mixed at a ratio of 5:2:1 to obtain GOx/BSA/glutaraldehyde solution;
winding the electrode of the corresponding sensing area of the insulated substrate electrode on the columnar structure, thereby forming a spiral structure;
separating the substrate electrode with the spiral structure from the columnar structure;
stretching the two ends of the spiral structure to ensure that uniform gaps are reserved between spiral rings;
adsorbing a layer of CNT on the surface of the microfilament spiral structure by a pulling method, and placing the CNT in a fume hood for drying at room temperature to form an electron mediator CNT layer;
modifying PEDOT to PSS outside the CNT layer by a constant current electrochemical polymerization method to form an electron mediator PEDOT to PSS layer;
plating a platinum layer outside the PEDOT PSS layer by an electroplating method to form a platinum layer;
the glucose oxidase is modified outside the platinum layer by adsorption to form a glucose oxidase layer.
Specifically, PEDOT PSS is modified outside the CNT layer by a constant current electrochemical polymerization method, and the method comprises the following steps:
10ml of water, 11ml of 3, 4-Ethylenedioxythiophene (EDOT) and 223mg of sodium poly (4-styrenesulfonate) (NaPSS) were prepared as a mixed solution;
the spiral microwire electrode 1 was placed in the mixed solution, a constant current of 10 μa was applied to the spiral microwire electrode 1, and electrochemical polymerization was performed for 400 seconds.
Specifically, a layer of platinum is plated outside the PEDOT/PSS layer by an electroplating method, and the method comprises the following steps:
adding a proper amount of platinum sodium sulfite electroplating platinum solution into a beaker;
electrochemical deposition of the helical structure was performed using a multi-step constant current method with the following parameters:
the first stage: 6e -5 A，10s；
And a second stage: -6e -5 A，10s；
And a third stage: -5e -4 A，600s。
As shown in fig. 6, SME characterization is performed on the surface layered morphology of each structure in the process of the preparation method of the electrochemical sensor based on the spiral electrode in the embodiment; as shown in fig. 6 (a) and (b), after the CNT layer is modified on the surface of the gold-substrate electrode, the electrode surface shows a rough porous structure, which means that the CNT layer has a large specific surface area; as shown in fig. 6 (c), a layer of PEDOT: PSS layer is modified outside the CNT layer, the PEDOT: PSS layer shows the morphology of the polymer, and the surface area is further increased; as shown in FIG. 6 (d), a platinum layer is modified outside the PEDOT: PSS layer, and the surface area is increased continuously; as shown in fig. 6 (e), the parylene C insulating layer 4 is coated on the gold substrate electrode to form a transparent polymer plastic film coating layer with a thickness of about 15 μm.
As shown in fig. 7, electrochemical sensors with different spiral turns in a spiral structure are respectively sensed in glucose solutions with different concentrations, and the concentration response is tested; wherein, fig. 7 (a), 7 (b), 7 (c) and 7 (d) are concentration response tests of electrochemical sensors with spiral turns of 1, 4, 7 and 10 turns, respectively; the surface current response signal of the test structure is influenced by the change of the glucose concentration, the intensity of the current signal is increased along with the increase of the spiral turns, and the fact that the area of the sensing area is increased can effectively improve the sensitivity of the electrochemical sensor is indicated.
Specifically, the concentration of the solution was increased in a gradient of 2mM in the range of 0mM-30mM.
As shown in FIG. 8, the electrochemical sensor of the present example was subjected to an anti-interference performance test, and physiological-related interferents including lactic acid (2 mM), potassium chloride (10 mM), uric acid (50. Mu.M) and ascorbic acid (10. Mu.M) were sequentially added to a PBS solution. As can be seen from fig. 8, the effect of these interfering substances is not more than 15% of the glucose signal, and the electrochemical sensor of this embodiment has good selectivity for detecting glucose, indicating its potential for monitoring glucose in complex liquid environments.
As shown in fig. 9, the electrochemical sensor of this example was fixed subcutaneously in a semi-implanted rat, and the glucose concentration of the rat was measured in vivo for 3 hours, and data was recorded every 15 minutes, to obtain a graph of the glucose monitoring results in vivo. The star point is the reference blood glucose concentration measured by the blood glucose meter, the curve is the calibrated data sensing result, and the electrochemical sensor can be found to continuously measure the subcutaneous ISF glucose level of the rat at different time points, and the fluctuation of the glucose concentration of the rat is relatively small in a short time.
The embodiment of the application has the following beneficial effects: the spiral microwire electrode and microwire reference electrode form a three-electrode system, so that the damage to the skin when the multi-electrode is implanted is reduced; the modification of the surface of the spiral structure with CNT and PEDOT: PSS can increase the specific surface area of the electrode, thereby increasing the adsorption of the electrode to enzyme and promoting the electron transfer between the substrate electrode and the enzyme active site; the spiral structure of the spiral microwire electrode improves the living body environment interference resistance of the electrochemical sensor, and increases the sensing area of the microwire electrode while guaranteeing the vertical length of the electrode, thereby increasing the sensing current signal intensity and improving the detection sensitivity of the electrochemical sensor; the insulating layer is made of a material with good biocompatibility, so that the possibility of thrombus initiation is reduced, and the biosafety is improved; by implanting the helical microwire electrode into the subcutaneous ISF environment, rapid, continuous, in-situ and environmental interference resistant glucose signal acquisition is achieved without obtaining a large sample size.
The present application also provides a second embodiment, wherein the spiral-structured bio-enzyme layer of the spiral-shaped microfilament electrode is prepared using urate oxidase to form a urate oxidase layer; the rest of the electrochemical sensor based on the spiral electrode is the same as the above embodiment, so that the electrochemical sensor based on the spiral electrode with uric acid sensing function is obtained and can be used for measuring the uric acid concentration in subcutaneous ISF.
It can be seen that the contents of the second embodiment are all applicable to the first embodiment, and the functions specifically implemented by the second embodiment are the same as those of the first embodiment, and the advantages achieved by the second embodiment are the same as those achieved by the first embodiment.
The application also provides a third embodiment, the electrochemical sensor based on the spiral electrode provided by the embodiment is the same as the electrochemical sensor based on the spiral electrode provided by the first embodiment, the electrochemical sensor based on the spiral electrode is fixed under the cerebral cortex of the skull, the three-electrode system is fixed on the skull by using dental cement, and the circuit detection system is arranged outside a human body and fixed on the body surface, so that the real-time in-situ sensing of the glucose concentration in the body fluid environment of the brain is realized.
It can be seen that the contents of the third embodiment are all applicable to the first embodiment, and the functions specifically implemented by the third embodiment are the same as those of the first embodiment, and the advantages achieved by the third embodiment are the same as those achieved by the first embodiment.
While the preferred embodiment of the present application has been described in detail, the application is not limited to the embodiment, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the application, and these equivalent modifications and substitutions are intended to be included in the scope of the present application as defined in the appended claims.

Claims (10)

1. An electrochemical sensor based on a spiral electrode is characterized by comprising a spiral microwire electrode, a microwire reference electrode, a microwire counter electrode and a circuit detection system; the first ends of the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode are connected and fixed with the circuit detection system; the microwire reference electrode and the microwire counter electrode pass through the spiral structure of the spiral microwire electrode; the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode all comprise a sensing area and a non-sensing area, and the non-sensing area is provided with an insulating layer; the sensing area of the spiral structure of the spiral microwire electrode is covered with a multi-film layer.
2. The spiral electrode-based electrochemical sensor of claim 1, wherein the spiral microwire electrode comprises a linear structure and a spiral structure, the linear structure being connected to the circuit detection system; the area of the linear structure is a non-sensor area and is covered with the insulating layer; the area of the spiral structure is a sensing area.
3. The spiral electrode-based electrochemical sensor according to claim 1, wherein the spiral structure of the spiral microwire electrode comprises a substrate electrode, a first electron mediator layer, a second electron mediator layer, a conductive layer, and a bio-enzyme layer in order from inside to outside.
4. The spiral electrode-based electrochemical sensor of claim 1, wherein the spiral microwire electrode, the microwire reference electrode, and the microwire counter electrode each comprise a substrate electrode, the material of the substrate electrode comprising a highly conductive material comprising any one of gold, carbon fiber, platinum iridium, and nickel titanium.
5. The spiral electrode-based electrochemical sensor of claim 1, wherein the area of the second end of the microwire reference electrode or microwire counter electrode comprises a sensing area of an electrode tip, the sensing area being located outside of the area of the spiral structure of the spiral microwire electrode.
6. The spiral electrode-based electrochemical sensor of claim 1, wherein the microfilament reference electrode comprises any one of a silver/silver chloride/polyvinyl butyral electrode, a silver/silver chloride electrode, a calomel electrode, a hydrogen electrode, a mercury/mercury oxide electrode, or a mercury/mercurous sulfate electrode.
7. The spiral electrode-based electrochemical sensor according to claim 1, wherein the material of the insulating layer comprises a material having insulation and biocompatibility, and the material of the insulating layer comprises any one of parylene series materials, polyimide, polydimethylsiloxane, or parylene.
8. A method of manufacturing a spiral electrode-based electrochemical sensor according to any one of claims 1 to 7, comprising:
insulating the substrate electrode on the non-sensing area corresponding to the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode;
preparing the microwire reference electrode and the microwire counter electrode;
preparing a spiral structure of the spiral microfilament electrode, and preparing a multi-film layer on the surface of the spiral structure of the spiral microfilament electrode;
and connecting the first ends of the spiral microwire electrode, the microwire reference electrode and the microwire counter electrode with the circuit detection system for integration.
9. The method for manufacturing a spiral electrode-based electrochemical sensor according to claim 8, wherein the manufacturing the spiral structure of the spiral microwire electrode specifically comprises:
winding a microwire electrode of a sensing area on a columnar structure to construct the spiral structure;
separating the microwire electrode with the spiral structure from the columnar structure;
stretching the helical structure so that there is a uniform gap between the helical rings of the helical structure.
10. The method for manufacturing an electrochemical sensor based on a spiral electrode according to claim 8, wherein the preparation of the multi-film layer on the surface of the spiral structure of the spiral microwire electrode specifically comprises:
the spiral structure is modified by sequentially using a first electron mediator, a second electron mediator, a conductive material and biological enzyme, so that a first electron mediator layer, a second electron mediator layer, a conductive layer and a biological enzyme layer are sequentially formed on the surface of the spiral structure.
CN202310678433.1A 2023-06-08 2023-06-08 Electrochemical sensor based on spiral electrode and preparation method thereof Pending CN116898433A (en)

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