CN117647571A

CN117647571A - Electrochemical biosensor electrode suitable for analyte detection and preparation method thereof

Info

Publication number: CN117647571A
Application number: CN202311375842.0A
Authority: CN
Inventors: 赵天聪; 邹磊; 韩建美
Original assignee: Shenzhen Shanmei High Tech Research Institute Co ltd
Current assignee: Shenzhen Shanmei High Tech Research Institute Co ltd
Priority date: 2023-10-20
Filing date: 2023-10-20
Publication date: 2024-03-05

Abstract

The invention provides a preparation method of an electrochemical biosensor electrode suitable for analyte detection, which comprises the following steps: (1) forming an electrode layer on a substrate surface: forming electrode material on the surface of the substrate to form a patterned electrode system, conductive lines and contacts; (2) Coating Ag/AgCl slurry on the surface of the reference electrode, and drying to form an Ag/AgCl reference electrode; (3) Modifying an electron transfer mediator or a catalyst on the surface of the working electrode; (4) forming an enzyme layer on the surface of the working electrode; (5) Forming a mass transport limiting layer on the surface of the working electrode: (6) forming an anti-interference layer on the surface of the working electrode; (7) Forming a biocompatible coating on a sensor electrode end of the working electrode; (8) Immersing the whole modified electrode into 0.01M phosphate buffer solution with pH of 7.4 for 4-48h, so that each component and the coating are fully combined, the stability of the electrode is enhanced, and the prepared electrochemical biosensor electrode is obtained.

Description

Electrochemical biosensor electrode suitable for analyte detection and preparation method thereof

Technical Field

The present invention relates to the field of analyte detection devices, and in particular, to the field of analyte detection devices. To an electrochemical biosensor electrode.

Background

Prior art disclosures or literature: CN110044986B, CN111803086B, etc.

The similar technical characteristics are as follows: the design of electrode layering modification has certain similarity, and part of selected materials have certain similarity.

Background introduction:

the blood and body fluid components are directly related to physiological health level, and the detection and analysis of different components are of great medical significance for judging physiological condition of human body. Electrochemical biosensors are widely used in the field of analyte detection because they can convert biochemical signals into electrical signals for acquisition and amplification. As in continuous blood glucose monitoring devices for diabetes management, minimally invasive continuous monitoring of glucose levels to aid in insulin therapy; or various electrochemical detection chips and devices for in vitro analyte detection. Chinese patent No. CN201811640898.3 discloses a working electrode for a glucose monitoring probe, which comprises: a base layer; a glucose sensing layer formed on the substrate layer and having a glucose enzyme capable of chemically reacting with glucose; a semipermeable membrane formed on the glucose sensing layer for controlling the passage rate of glucose molecules; and a biocompatible film formed on the semipermeable film, wherein a nanoparticle layer that catalyzes a glucose reaction and is porous is provided between the base layer and the glucose sensing layer, and the glucose permeates the nanoparticle layer. According to the present disclosure, the working electrode working voltage can be reduced, interference can be reduced, the service life of the glucose monitoring probe can be prolonged, and the sensitivity of the reaction to glucose can be improved. According to the three-electrode subcutaneous implanted glucose sensor and the manufacturing method thereof disclosed by the invention, the sensor adopts a three-electrode form of a working electrode, a reference electrode and an auxiliary electrode, and the micro-current to be tested passes through the working electrode and the auxiliary electrode, and the reference electrode basically does not pass through the current, so that the service life of the reference electrode is prolonged; the manufacturing process of the sensor electrode is improved, so that the process is simple, the quality is controllable, the stability of the glucose oxidase of the working electrode is maintained, and the accuracy of sensor test data is ensured; in addition, carriers such as bovine serum albumin are omitted, and the activity and stability of glucose oxidase are ensured by adopting a glutaraldehyde and silane dual-function coupling mode, so that the potential safety hazard to a human body is reduced, and the rejection reaction of the human body is lightened; through the detachable block connected mode of transmitter and sensor for sensor and transmitter can conveniently separate, and the transmitter can also used repeatedly after realizing changing the sensor, has reduced user's use cost.

In general, electrochemical biosensors are used in devices for continuous blood glucose monitoring, and a current signal is measured and acquired in real time by using a chronoamperometry method, and the physiological glucose level is reflected by the current signal level; in the in vitro analyte detection process, various detection means including cyclic voltammetry, differential pulse voltammetry, electrochemical impedance spectroscopy, chronoamperometry and the like can be used to collect signals and establish the correspondence between electrical signals and analytes.

Various types of continuous testing devices and in vitro analytical devices are now successfully commercialized and put into medical testing applications. Especially in the fields of diabetes management and in-vitro analyte detection, the method has wide audience and high popularization rate. However, due to numerous problems that exist in practice, such as:

1. the reference electrode is continuously worn, and the continuous measurement time is long, so that the misalignment is easy to cause.

2. The noble metal electrode microfilaments have higher cost and serious resource waste.

3. Bovine serum albumin is used as an electrode anti-fouling agent, and when an electrode probe is used for minimally invasive implantable detection, the rejection reaction of the organism is easily induced.

4. Glutaraldehyde is used as a cross-linking agent, and biological toxicity is easy to occur to human bodies when inactivation is incomplete or residues exist.

5. The working electrode and the counter electrode in the electrode probe are far away, a stable loop is not easy to form, the electrode probe is easy to be interfered, and the measuring current fluctuates.

6. Each modification substance on the surface of the electrode is not firmly fixed, and the risk of falling off exists.

These problems lead to unstable performance, hidden safety hazards and high price in the application process of the electrochemical biosensor, and are questioned by partial patients and users. Therefore, how to build an electrochemical biosensor detection platform with stable performance, safety, low process cost and sustainable use is a key problem at present, and the above problems are needed to be solved and optimized.

Disclosure of Invention

In order to overcome the deficiencies of the prior art, the present invention provides an electrochemical biosensor electrode suitable for analyte detection,

the technical scheme of the invention is as follows: a method of making an electrochemical biosensor electrode suitable for analyte detection comprising the steps of:

(1) Forming an electrode layer on the surface of a substrate: forming electrode materials on the surface of a substrate to form patterned electrodes, conductive circuits and contacts; the electrode system comprises at least one counter electrode, one working electrode and one reference electrode;

(2) And coating Ag/AgCl slurry on the surface of the reference electrode, and drying to form the Ag/AgCl reference electrode.

(3) The surface of the working electrode is modified with an electron transfer mediator/catalyst: prussian blue is electrodeposited by chronoamperometry/cyclic voltammetry on the working electrode surface to form an electron transfer mediator.

(4) Forming an enzyme layer on the surface of the working electrode: glucose oxidase is dissolved in phosphate buffer solution, and 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide are added for treatment so as to activate carboxyl groups on the surface of the glucose oxidase.

(5) And dipping or coating or spraying an amino silane layer on the surface of the working electrode, and combining the activated glucose oxidase with the silane layer in a dipping or coating or spraying mode to complete the fixation of the enzyme layer.

(6) Forming a mass transport limiting layer on the surface of the working electrode: the polyethylene glycol-polyurethane blend liquid is coated on the surface of the working electrode to serve as a mass transportation limiting layer, and the polyethylene glycol-polyurethane film formed after drying can play a role in limiting the glucose transportation rate so as to achieve the purpose of ensuring sufficient oxygen in the enzyme layer.

(7) Forming an anti-interference layer on the surface of the working electrode: nafion solution is coated on the surface of the working electrode to be used as an anti-interference coating, and the coating is dried to form a film, so that foreign matters are prevented from being adsorbed on the surface of the electrode, and the working performance of the electrode is prevented from being rapidly reduced.

(8) Forming a biocompatible coating on the sensor electrode end: and (3) coating a polyvinylpyrrolidone solution on the surface of the working electrode as a biocompatible coating, drying the coating to form a film, and increasing the biocompatibility of the sensor electrode.

(9) And immersing the whole electrode end part modified by each component and the coating into 0.01M phosphate buffer solution with the pH of 7.4 for 4-48 hours, so that each component and the coating are fully combined, the stability of the electrode is enhanced, and the prepared electrochemical biosensor electrode is obtained.

The electrochemical biosensor electrode suitable for analyte detection is characterized in that an electrode layer is formed on the surface of a substrate, and an electron transfer mediator/catalyst is modified on the surface of a working electrode to play a role in enhancing electron transfer efficiency, reducing the working potential of the sensor and catalyzing and decomposing the analyte under low potential.

Further, an enzyme layer is formed on the surface of the working electrode, and the enzyme layer screens, identifies and orients the analyte to be detected in the system to generate oxidation-reduction reaction, and conducts electrons to the electrode through the medium of an electron transfer mediator/catalyst so as to realize conversion from biochemical signals to electric signals.

Further, a mass transport limiting layer is formed on the surface of the working electrode, and the oxygen content fluctuation in the detection system can cause the sensor electrode to acquire signals to be influenced, for example, when the oxygen content is too high/too low, the sensitivity, the linear range and the response speed of the sensor for detecting certain biochemical reactions can be influenced, so that the mass transport limiting layer is required to control the ratio of oxygen to analyte in the enzyme layer so as to enable the biochemical reactions to stably proceed.

Further, an anti-interference layer is formed on the surface of the working electrode, and the anti-interference layer is required to be formed on the surface of the sensor electrode in order to avoid the rapid reduction of the working performance of the sensor caused by the adsorption and encapsulation of foreign matters in a long-time use environment based on the analysis of the implantation of the electrode end of the sensor into the skin layer of a human body.

Further, based on possible analysis of some application scenes of the skin layer implanted in the human body, in order to reduce rejection reaction related to the human body and occurrence of discomfort, inflammation, allergy and other phenomena of surrounding skin tissues after the sensor is implanted, a biocompatible coating is formed at the end part of the sensor electrode so as to ensure the safety of application.

The potential analytes which can be detected by the prepared electrochemical biosensor can be various substances such as lactic acid, alcohol, ketone, glucose, bilirubin, urea, uric acid, cholesterol, acetylcholine, ascorbic acid, amino acid, sodium ion, potassium ion, calcium ion, chloride ion, oxygen, carbon dioxide, pH, antibiotics, glutamine and the like.

The substrate can be a flexible or rigid substrate, and the flexible substrate is selected from polyimide, polyester, polyether, polydimethylsiloxane, polyvinylpyrrolidone, polyolefin, polytetrafluoroethylene and the like and copolymers formed by the above substances, or a flexible glass sheet, a flexible metal sheet, an inorganic compound oxide film and the like; the rigid substrate is selected from silicon dioxide, metal oxide, hard plastic, ceramic, polymethyl methacrylate, various resins and the like.

The electrode system specifically comprises at least one counter electrode, one working electrode and one reference electrode, wherein the electrode system can be a single counter electrode corresponding to a single working electrode or a single counter electrode corresponding to a plurality of working electrode systems, and the reference electrode only plays a role in providing reference potential. The electrode positional relationship may be coplanar, off-plane, or off-plane. The electrode material can be carbon-based material, metal material, inorganic compound material, organic biochemical material, etc., and the specific process method for forming the electrode coating can be screen printing, spraying, coating, soaking and drying, sputtering, electron beam evaporation, etc.

The electron transfer mediator may be a complex based on a transition metal element such as osmium, ruthenium, iron, cobalt, vanadium, or the like, ferricyanide, ferrocenyl derivative, or the like.

The catalyst can be platinum, silver, gold, lead, nickel, cobalt, titanium, iridium, ruthenium and other metal nano materials. The process used to form the electron transfer mediator/catalyst may be electroplating, electroless plating, sputtering, immersion drying, laser ablation, and the like.

Furthermore, the enzyme layer material formed is different according to different selected enzymes for the specific analytes, and the important marker glucose for diabetes blood glucose management is taken as an example, and enzymes such as glucose oxidase, glucose dehydrogenase and the like can be selected to be modified on the surface of the working electrode. The enzyme layer is not necessarily composed of a single enzyme, but may be composed of a plurality of enzymes, for example, a combination of a specific enzyme and a peroxidase. The enzyme layer may be formed by physical adsorption, crosslinking, layer-by-layer assembly, etc., and the crosslinking agent may be glutaraldehyde, a silane coupling agent, a succinimide substance, a carbodiimide substance, etc.

The material of the mass transport limiting film can be polyether, polyurea, polyurethane, polyvinyl pyridine, polyvinyl imidazole, polyvinylpyrrolidone, styryl polymer, polyethylene glycol, polytetrafluoroethylene, polyvinyl chloride, polymethyl methacrylate, nafion polymer, chitosan, sodium alginate, agar and the like, and copolymers formed among the above substances. The process method for forming the mass transport limiting layer can be spraying, coating, soaking, drying and the like.

The further formed anti-interference layer material can be polyether, polyurea, polyurethane, polyvinyl pyridine, polyvinyl imidazole, polyvinylpyrrolidone, styryl polymer, polyethylene glycol, polytetrafluoroethylene, polyvinyl chloride, polymethyl methacrylate, nafion polymer and the like and copolymers formed among the above substances. The process method for forming the mass transport limiting layer can be spraying, coating, soaking, drying and the like.

The further formed biocompatible coating material can be polylactic acid, polyurethane, polyurea, polyether, polyolefin, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, nafion polymer, chitosan, sodium alginate, agar substance and copolymer formed between the above substances.

The electron transfer mediator/catalyst and enzyme may be pre-attached and co-incubated on the surface of the working electrode.

The enzyme may be pretreated to allow the enzyme to unfold exposing the corresponding active site.

The enzyme and mass transport limiting material may be pre-mixed and co-incubated on the working electrode surface.

The mass transport limiting material, the anti-interference material and the biocompatible material in steps (5) (6) (7) may be pre-mixed and co-incubated on the same layer of the working electrode surface, or may be performed once, such that the mass transport limiting material, the anti-interference material and the biocompatible material are located on different layers of the working electrode surface.

The beneficial effects of the invention are as follows: the electrochemical biosensor has the advantages that the reference electrode area is larger, a sufficient action area and reserve quantity are provided for Ag/AgCl, the service life of the sensor electrode can be prolonged, the potential stability is enhanced, and the problem of sensor misalignment caused by rapid reference electrode loss due to chloride ion loss is avoided to a great extent.

According to the electrochemical biosensor, the carbon material and the polymer substrate are adopted as the electrode and the substrate, so that the production cost can be greatly reduced, and the waste of metal resources can be reduced.

The electrochemical biosensor adopts the anti-fouling layer formed by polyethylene glycol and polyurethane substances to block the foreign matter adsorption electrode, and the surface of the electrode treated by the silane coupling agent has a large number of hydrophilic groups to assist the anti-fouling coating to play a role together, so that the use of bovine serum albumin on the surface of the electrode is eliminated, and the rejection reaction possibly caused when the electrode is implanted into the skin layer of a human body is avoided.

The electrochemical biosensor adopts the interdigital electrode design, ensures the tight position relation between the working electrode and the counter electrode, forms a stable conductive loop in an electrolyte system, and can reduce signal fluctuation noise caused by instability of the electrode loop.

After the preparation and modification of each layer of the electrochemical biosensor electrode are finished, the electrochemical biosensor electrode is fully soaked in a wetting environment or a buffer solution environment, so that all layers are fully combined, and the connection is tight.

Description of the drawings:

FIG. 1 is a flow chart of a process for preparing an electrode for an electrochemical biosensor according to the present invention.

Fig. 2 is a schematic diagram of the patterned electrode, conductive line and contact prepared in step 1.

FIG. 3a is a schematic view of a coplanar single electrode

Fig. 3b is a schematic illustration of a coplanar multi-electrode.

Fig. 4a is a schematic front view of an out-of-plane electrode.

FIG. 4b is a schematic reverse side view of an electrode with different sides.

Fig. 5 is a schematic view of a dissimilar electrode.

FIG. 6 is a schematic diagram of the working electrode, counter electrode and reference electrode supported on three substrates, respectively

Detailed Description

In order to make the technical scheme and technical effects of the invention more clear, the invention is further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Example 1

As shown in fig. 1, a method for preparing an electrochemical biosensor electrode suitable for analyte detection, comprising the steps of:

(1) And (3) carrying out screen printing on the surface of the polyimide substrate with a carbon paste electrode, wherein the proportion of the carbon nano tubes in the carbon paste is 1% -20%, and drying to form the patterned carbon electrode, the conductive circuit and the contact after printing is completed. The explanation about the electrode, the conductive line, the contact, etc. is shown in fig. 2, wherein the two interdigital electrodes, namely, the electrode 1 and the electrode 2 are respectively a working electrode and a counter electrode; the square electrode, electrode 3, is the reference electrode. It should be noted that the preferred results are described in this embodiment, and the electrodes may be exchanged with each other, for example, electrode 1 may be a counter electrode or a reference electrode, electrode 2 may be a working electrode or a reference electrode, and electrode 3 may be a working electrode or a counter electrode. Each electrode is connected with one contact, and at least one conductive line is arranged between the electrode and the corresponding contact. The region formed by the whole of each electrode is a detection region, that is, a region that plays a role in detection in practical use. The region formed by the whole of each contact is a contact region, namely, a region electrically connected with external electric equipment in practical application. The working electrode may be one or more, and is shown in fig. 3a as a schematic diagram of a coplanar single electrode, and in fig. 3b as a schematic diagram of a coplanar multiple electrode, where at least one counter electrode and one reference electrode are respectively present in addition to the working electrode. As shown in fig. 4a and 4b, the electrode may be located on different sides, for example, the working electrode, the counter electrode and the reference electrode are located on opposite sides of the substrate. As shown in fig. 5, the electrode may be in a different type, and the working electrode, the counter electrode and the reference electrode are supported on three substrates, respectively.

(2) And (3) coating Ag/AgCl slurry on the surface of the reference electrode, and drying to form the Ag/AgCl reference electrode.

(3) Prussian blue is electrodeposited by chronoamperometry/cyclic voltammetry on the working electrode surface to form an electron transfer mediator.

(4) Glucose oxidase is dissolved in phosphate buffer solution, and 1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide are added for treatment so as to activate carboxyl groups on the surface of the glucose oxidase.

(6) The polyethylene glycol-polyurethane blend liquid is coated on the surface of the working electrode to serve as a mass transportation limiting layer, and the polyethylene glycol-polyurethane film formed after drying can play a role in limiting the glucose transportation rate so as to achieve the purpose of ensuring sufficient oxygen in the enzyme layer.

(7) Nafion solution is coated on the surface of the working electrode to be used as an anti-interference coating, and the coating is dried to form a film, so that foreign matters are prevented from being adsorbed on the surface of the electrode, and the working performance of the electrode is prevented from being rapidly reduced.

(8) And (3) coating a polyvinylpyrrolidone solution on the surface of the working electrode as a biocompatible coating, drying the coating to form a film, and increasing the biocompatibility of the sensor electrode.

(9) The electrode end after the modification of each component and coating is immersed in 0.01M phosphate buffer solution with pH of 7.4 for 4-48h to enable each component and coating to be fully combined, so that the stability of the electrode is enhanced, and the prepared electrochemical biosensor electrode is obtained, which is schematically shown in figure 6, wherein a substance 1 represents a substrate, a substance 2 represents an electron transfer mediator/catalyst, a substance 3 represents an enzyme layer, a substance 4 represents a mass transport limiting layer, a substance 5 represents an anti-interference layer, and a substance 6 represents a biocompatible coating.

The electrochemical biosensor electrode is matched with a hollow stainless steel microneedle to assist in puncturing skin, can be implanted into a skin layer, is further connected with electrical equipment through a contact, applies working voltage to a sensor, collects measurement data, performs post-treatment on the data, and can realize continuous glucose measurement of a human body.

Example 2

The difference from example 1 is that:

in the step (1), a carbon paste electrode is screen-printed on the surface of the polyethylene terephthalate substrate.

In the step (3), silver nano particles are deposited on the surface of the working electrode by a chronoamperometric method to serve as a catalyst.

In the step (5), lactate oxidase is immobilized as an enzyme layer on the surface of the working electrode.

In the step (7), polyethylene glycol-polyurethane-Nafion blend liquid is coated on the surface of the working electrode, and the mixture is dried to form a film which is used as a mass transportation limiting layer and an anti-interference coating.

In the step (8), polylactic acid is coated on the surface of the working electrode as a biocompatible coating.

The electrochemical biosensor electrode prepared by the embodiment can be used for lactic acid detection in human blood/body fluid.

Example 3

The difference from example 1 is that:

in the step (1), a screen printing carbon paste electrode is carried out on the surface of a polydimethylsiloxane substrate, and 1% -20% of carbon nanotubes containing active groups such as amino groups, carboxyl groups, hydroxyl groups, succinimide groups and the like are doped.

In step (4), glucose oxidase is pre-blended with an osmium complex, and the osmium complex is supported on the surface of the glucose oxidase.

In the step (5), glucose oxidase loaded with osmium complex is coupled to the surface of the working electrode as an electron transfer mediator-enzyme layer.

In the step (7), the polyurethane-polyurea segmented copolymer is coated on the surface of the working electrode to serve as a mass transportation limiting layer and an anti-interference layer.

In the step (8), a polyvinyl pyridine-styrene copolymer is coated on the surface of the working electrode as a biocompatible coating.

Example 4

The difference from example 1 is that:

in the step (3), the ferrocene solution is infiltrated on the surface of the working electrode, and the ferrocene electron transfer mediator is formed by drying.

In step (8), a biocompatible coating is not required.

The electrochemical biosensor electrode prepared by the embodiment can be used for detecting lactic acid components in a liquid phase system in vitro.

The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. For those skilled in the art, the architecture of the invention can be flexible and changeable without departing from the concept of the invention, and serial products can be derived. But a few simple derivatives or substitutions should be construed as falling within the scope of the invention as defined by the appended claims.

Claims

1. A method of making an electrochemical biosensor electrode suitable for analyte detection comprising the steps of:

(1) Forming an electrode layer on the surface of a substrate: forming electrode material on the surface of the substrate to form a patterned electrode system, conductive lines and contacts; the electrode system comprises at least one counter electrode, one working electrode and one reference electrode; each electrode is connected with one contact, and at least one conductive circuit is arranged between the electrode and the corresponding contact; the region formed by the whole electrodes is a detection region; the area formed by the whole contacts is a contact area, namely an area electrically connected with external electrical equipment;

(2) Coating Ag/AgCl slurry on the surface of the reference electrode, and drying to form an Ag/AgCl reference electrode;

(3) Modifying an electron transfer mediator or a catalyst on the surface of the working electrode;

(4) Forming an enzyme layer on the surface of the working electrode;

(5) Forming a mass transport limiting layer on the surface of the working electrode:

(6) Forming an anti-interference layer on the surface of the working electrode;

(7) Forming a biocompatible coating on a sensor electrode end of the working electrode;

(8) Immersing the whole modified electrode into 0.01M phosphate buffer solution with pH of 7.4 for 4-48h, so that each component and the coating are fully combined, the stability of the electrode is enhanced, and the prepared electrochemical biosensor electrode is obtained.

2. The method of making an electrochemical biosensor electrode suitable for analyte detection according to claim 1, wherein the substrate can be a flexible or rigid substrate; the flexible substrate is selected from polyimide, polyester, polyether, polydimethylsiloxane, polyvinylpyrrolidone, polyolefin, polytetrafluoroethylene or any copolymer thereof, or flexible glass sheet, flexible metal sheet, inorganic compound oxide film and the like; the rigid substrate is selected from silicon dioxide, metal oxide, hard plastic, ceramic, polymethyl methacrylate, resin or any copolymer thereof;

the electrode material is selected from one of a carbon-based material, a metal material, an inorganic compound material and an organic biochemical material, and the method for forming the electrode coating is screen printing, spraying, coating, infiltration drying, sputtering or electron beam evaporation;

the electrode systems are coplanar, different-surface and different-body.

3. The method of making an electrochemical biosensor electrode suitable for analyte detection according to claim 1, wherein the electron transfer mediator is selected from complexes of the transition metal elements osmium, ruthenium, iron, cobalt, vanadium, or ferricyanide, ferrocenyl derivatives; the catalyst is selected from platinum, silver, gold, lead, nickel, cobalt, titanium, iridium and ruthenium metal nano materials; the process for forming the electron transfer mediator/catalyst is electroplating, electroless plating, sputtering, immersion drying or laser ablation.

4. The method for preparing an electrochemical biosensor electrode for analyte detection according to claim 1, wherein the enzyme layer is formed on the surface of the working electrode by physical adsorption, crosslinking, layer-by-layer assembly, wherein the crosslinking agent is selected from glutaraldehyde, silane coupling agents, succinimide substances, or carbodiimides.

5. The method for preparing an electrochemical biosensor electrode for analyte detection according to claim 1, wherein the material forming the mass transport limiting layer on the surface of the working electrode is selected from the group consisting of polyether, polyurea, polyurethane, polyvinylpyridine, polyvinylimidazole, polyvinylpyrrolidone, styrene-based polymer, polyethylene glycol, polytetrafluoroethylene, polyvinylchloride, polymethyl methacrylate, nafion polymer, chitosan, sodium alginate, agar, and any copolymer thereof; the method for forming the mass transport limiting layer on the surface of the working electrode is spraying, coating or soaking and drying.

6. The method for preparing an electrochemical biosensor electrode for analyte detection according to claim 1, wherein the material forming the anti-interference layer on the surface of the working electrode is selected from the group consisting of polyether, polyurea, polyurethane, polyvinylpyridine, polyvinylimidazole, polyvinylpyrrolidone, styryl polymer, polyethylene glycol, polytetrafluoroethylene, polyvinylchloride, polymethyl methacrylate, nafion polymer and any copolymer thereof; the method for forming the anti-interference layer on the surface of the working electrode is spraying, coating or soaking and drying.

7. The method for preparing an electrochemical biosensor electrode for analyte detection according to claim 1, wherein the material forming the biocompatible coating on the surface of the working electrode is selected from the group consisting of polylactic acid, polyurethane, polyurea, polyether, polyolefin, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, nafion polymer, chitosan, sodium alginate, agar, and any copolymer thereof.

8. The method of preparing an electrochemical biosensor electrode for analyte detection according to claim 1, wherein after step (2), the electron transfer mediator/catalyst and enzyme are pre-attached and co-incubated on the surface of the working electrode, followed by step (5).

9. The method of preparing an electrochemical biosensor electrode suitable for analyte detection according to claim 1, wherein after step (3), the enzyme and mass transport limiting material are pre-mixed and co-incubated on the working electrode surface; then, step (6) is carried out.

10. The method of preparing an electrochemical biosensor electrode suitable for analyte detection according to claim 1, wherein the mass transport limiting material, the anti-interference material and the biocompatible material in steps (5) (6) (7) are pre-mixed and co-incubated on the same layer of the working electrode surface.