阅读说明：本技术 一种差分阻抗电位型生物传感器及其制造方法 (Differential impedance potential type biosensor and manufacturing method thereof ) 是由 李远航 于 2018-06-14 设计创作，主要内容包括：本发明提供一种差分阻抗电位型生物传感器及其制造方法,通过加工一具备纵向厚度和具备信号输入区、信号输出区、反应检测区以及反应参考区的绝缘基板,再通过在信号输入区设置输入正极和输入负极,并在信号输出区设置第一输出电极,并在反应检测区设置间距尺寸相同且其反应端具备生物反应材料和惰性材料的第一检测电极和第二检测电极,并在反应参考区设置间距尺寸相同且其惰性端具备惰性材料的第一参考电极和第二参考电极,再通过将输入正极与第一检测电极电连接,将第二检测电极、第一参考电极以及第一输出电极共同电连接,将第二参考电极与所述输入负极电连接,从而制造出一种抗干扰能力强和灵敏度高的生物传感器,达到提升抗干扰能力和灵敏度的效果。(The invention provides a differential impedance potential type biosensor and a manufacturing method thereof, by processing an insulating substrate which is provided with a longitudinal thickness and is provided with a signal input area, a signal output area, a reaction detection area and a reaction reference area, arranging an input anode and an input cathode in the signal input area, arranging a first output electrode in the signal output area, arranging a first detection electrode and a second detection electrode which have the same space size and the same reaction end is provided with a biological reaction material and an inert material in the reaction detection area, arranging a first reference electrode and a second reference electrode which have the same space size and the same inert end is provided with an inert material in the reaction reference area, electrically connecting the input anode and the first detection electrode, electrically connecting the second detection electrode, the first reference electrode and the first output electrode together, and electrically connecting the second reference electrode and the input cathode, therefore, the biosensor with strong anti-interference capability and high sensitivity is manufactured, and the effects of improving the anti-interference capability and the sensitivity are achieved.)

1. A method for manufacturing a differential impedance potential type biosensor, comprising the steps of:

processing an insulating substrate which is provided with a longitudinal thickness and is provided with a signal input area, a signal output area, a reaction detection area and a reaction reference area by using an insulating material;

an input positive electrode and an input negative electrode are correspondingly arranged in the signal input area, and a first output electrode is arranged in the signal output area;

arranging a first detection electrode and a second detection electrode which have the same space size and reaction ends of which are provided with a biological reaction material and an inert material in the reaction detection area;

arranging a first reference electrode and a second reference electrode which have the same space size and inert ends of which are provided with inert materials in the reaction reference area;

and electrically connecting the input positive electrode with the first detection electrode, electrically connecting the second detection electrode, the first reference electrode and the first output electrode together, and electrically connecting the second reference electrode with the input negative electrode.

2. The method of manufacturing a differential impedance potentiometric biosensor as set forth in claim 1, further comprising the steps of:

a first reinforcing electrode with a reaction end provided with a biological reaction material and an inert material is arranged between the first detection electrode and the second detection electrode;

and a second reinforcing electrode with an inert end provided with an inert material is arranged between the first reference electrode and the second reference electrode.

3. The method of manufacturing a differential impedance potentiometric biosensor as set forth in claim 2, further comprising the steps of:

arranging a second output electrode in the signal output area;

arranging a third detection electrode and a fourth detection electrode which have the same space size and reaction ends of which are provided with a biological reaction material and an inert material in the reaction detection area;

arranging a third reference electrode and a fourth reference electrode which have the same space size and inert ends of which are provided with inert materials in the reaction reference area;

electrically connecting the third reference electrode to a connection point of the input positive electrode and the first detection electrode;

electrically connecting the fourth reference electrode, the second output electrode, and the third detection electrode in common;

and electrically connecting the fourth detection electrode with a connection point of the second reference electrode and the input cathode.

4. The method of manufacturing a differential impedance potentiometric biosensor as set forth in claim 3, further comprising the steps of:

a fourth enhanced electrode with a reaction end provided with a biological reaction material and an inert material is arranged between the third detection electrode and the fourth detection electrode;

and a third reinforcing electrode with an inert end provided with an inert material is arranged between the third reference electrode and the fourth reference electrode.

5. A differential impedance potentiometric biosensor comprising:

an insulating substrate formed by processing an insulating material, having a longitudinal thickness, and having a signal input region, a signal output region, a reaction detection region, and a reaction reference region;

the input anode and the input cathode are correspondingly arranged in the signal input area;

a first output electrode disposed in the signal output region;

the first detection electrode and the second detection electrode are arranged in the reaction detection area at the same interval, have the same size and are provided with a biological reaction material and an inert material at the reaction end;

the inert ends of the first reference electrode and the second reference electrode are provided with inert materials and are arranged in the reaction reference area at the same size and interval;

the input positive electrode is electrically connected with the first detection electrode, the second detection electrode, the first reference electrode and the first output electrode are electrically connected together, and the second reference electrode is electrically connected with the input negative electrode.

6. The differential impedance potentiometric biosensor of claim 5, further comprising:

a first reinforcing electrode, the reaction end of which is provided with a biological reaction material and an inert material, and which is arranged between the first detection electrode and the second detection electrode;

and the inert end of the second reinforcing electrode is provided with an inert material and is arranged between the first reference electrode and the second reference electrode.

7. The differential impedance potentiometric biosensor of claim 6, further comprising:

a second output electrode disposed in the signal output region;

the third detection electrode and the fourth detection electrode are the same in size, reaction ends of the third detection electrode and the fourth detection electrode are provided with a biological reaction material and an inert material, and the third detection electrode and the fourth detection electrode are arranged in the reaction detection area at the same interval;

the inert ends of the third reference electrode and the fourth reference electrode are provided with inert materials and are arranged in the reaction reference area at the same interval;

wherein the third reference electrode is electrically connected to a connection point of the input positive electrode and the first detection electrode;

the fourth reference electrode, the second output electrode and the third detection electrode are electrically connected in common;

the fourth detection electrode is electrically connected to a connection point of the second reference electrode and the input cathode.

8. The differential impedance potentiometric biosensor of claim 7, further comprising:

a fourth reinforcing electrode, the reaction end of which is provided with a biological reaction material and an inert material, and which is arranged between the third detection electrode and the fourth detection electrode;

and the inert end of the third enhancement electrode is provided with an inert material and is arranged between the third reference electrode and the fourth reference electrode.

Technical Field

The invention belongs to the technical field of biosensor detection, and particularly relates to a differential impedance potential type biosensor and a manufacturing method thereof.

Background

Biosensors are typically a three-electrode system comprising a working electrode, a reference electrode, and a counter electrode. If the reference electrode and the counter electrode are combined into a whole, a two-electrode system can be formed by the reference electrode and the counter electrode. Biosensors are mainly classified into three types, a current type, a potential type, and an impedance spectrum type, according to a detected signal.

The mechanism of amperometric biosensors is: when the current response of certain electroactive substance under constant potential is analyzed, the different concentrations of the electroactive substance can cause different response currents. However, signal interference may be caused by the presence of other electroactive species in the test sample. Among them, the common amperometric biosensors include blood glucose and cholesterol test strips, etc.

The potential biosensor mechanism is as follows: the potential difference between two electrodes is measured by detecting the principle that the potential of balance electrode changes due to the concentration change of certain redox couple substance in the liquid or by using ion selective membrane to analyze the signal. However, the potentiometric biosensor has low sensitivity and slow response speed. Among them, the common potentiometric biosensing technologies include the pH detection by blood gas analysis and the detection of blood ions (such as K +, Ca +, etc.).

The impedance spectrum type biosensor has the following mechanism: an alternating voltage signal V is applied within a frequency band, a response current signal I is measured, and the impedance spectrum obtained by analyzing V/I is analyzed. However, the impedance spectrum type biosensor has low interference resistance and high requirements for a detection circuit, and therefore, is expensive and difficult to popularize.

In conclusion, the existing biosensor has the technical problems of weak interference resistance and low sensitivity.

Disclosure of Invention

The invention aims to provide a method for manufacturing a differential impedance potential type biosensor, so as to manufacture a biosensor with strong anti-interference capability and high sensitivity, and achieve the purpose of solving the technical problems of weak anti-interference capability and low sensitivity of the existing biosensor.

A method of manufacturing a differential impedance potential type biosensor, comprising the steps of:

processing an insulating substrate which is provided with a longitudinal thickness and is provided with a signal input area, a signal output area, a reaction detection area and a reaction reference area by using an insulating material;

an input positive electrode and an input negative electrode are correspondingly arranged in the signal input area, and a first output electrode is arranged in the signal output area;

arranging a first detection electrode and a second detection electrode which have the same space size and reaction ends of which are provided with a biological reaction material and an inert material in the reaction detection area;

arranging a first reference electrode and a second reference electrode which have the same space size and inert ends of which are provided with inert materials in the reaction reference area;

and electrically connecting the input positive electrode with the first detection electrode, electrically connecting the second detection electrode, the first reference electrode and the first output electrode together, and electrically connecting the second reference electrode with the input negative electrode.

Preferably, the method for manufacturing a differential impedance potential type biosensor further comprises the steps of:

a first reinforcing electrode with a reaction end provided with a biological reaction material and an inert material is arranged between the first detection electrode and the second detection electrode;

and a second reinforcing electrode with an inert end provided with an inert material is arranged between the first reference electrode and the second reference electrode.

Preferably, the method for manufacturing a differential impedance potential type biosensor further comprises the steps of:

arranging a second output electrode in the signal output area;

arranging a third detection electrode and a fourth detection electrode which have the same space size and reaction ends of which are provided with a biological reaction material and an inert material in the reaction detection area;

arranging a third reference electrode and a fourth reference electrode which have the same space size and inert ends of which are provided with inert materials in the reaction reference area;

electrically connecting the third reference electrode to a connection point of the input positive electrode and the first detection electrode;

electrically connecting the fourth reference electrode, the second output electrode, and the third detection electrode in common;

and electrically connecting the fourth detection electrode with a connection point of the second reference electrode and the input cathode.

Preferably, the method for manufacturing a differential impedance potential type biosensor further comprises the steps of:

a fourth enhanced electrode with a reaction end provided with a biological reaction material and an inert material is arranged between the third detection electrode and the fourth detection electrode;

and a third reinforcing electrode with an inert end provided with an inert material is arranged between the third reference electrode and the fourth reference electrode.

The invention also aims to provide a differential impedance potential type biosensor to solve the technical problems of weak interference resistance and low sensitivity of the existing biosensor.

A differential impedance potentiometric biosensor comprising:

an insulating substrate formed by processing an insulating material, having a longitudinal thickness, and having a signal input region, a signal output region, a reaction detection region, and a reaction reference region;

the input anode and the input cathode are correspondingly arranged in the signal input area;

a first output electrode disposed in the signal output region;

the first detection electrode and the second detection electrode are arranged in the reaction detection area at the same interval, have the same size and are provided with a biological reaction material and an inert material at the reaction end;

the inert ends of the first reference electrode and the second reference electrode are provided with inert materials and are arranged in the reaction reference area at the same size and interval;

the input positive electrode is electrically connected with the first detection electrode, the second detection electrode, the first reference electrode and the first output electrode are electrically connected together, and the second reference electrode is electrically connected with the input negative electrode.

Preferably, the differential impedance potentiometric biosensor further comprises:

a first reinforcing electrode, the reaction end of which is provided with a biological reaction material and an inert material, and which is arranged between the first detection electrode and the second detection electrode;

and the inert end of the second reinforcing electrode is provided with an inert material and is arranged between the first reference electrode and the second reference electrode.

Preferably, the differential impedance potentiometric biosensor further comprises:

a second output electrode disposed in the signal output region;

the third detection electrode and the fourth detection electrode are the same in size, reaction ends of the third detection electrode and the fourth detection electrode are provided with a biological reaction material and an inert material, and the third detection electrode and the fourth detection electrode are arranged in the reaction detection area at the same interval;

the inert ends of the third reference electrode and the fourth reference electrode are provided with inert materials and are arranged in the reaction reference area at the same interval;

wherein the third reference electrode is electrically connected to a connection point of the input positive electrode and the first detection electrode;

the fourth reference electrode, the second output electrode and the third detection electrode are electrically connected in common;

the fourth detection electrode is electrically connected to a connection point of the second reference electrode and the input cathode.

Preferably, the differential impedance potentiometric biosensor further comprises:

a fourth reinforcing electrode, the reaction end of which is provided with a biological reaction material and an inert material, and which is arranged between the third detection electrode and the fourth detection electrode;

and the inert end of the third enhancement electrode is provided with an inert material and is arranged between the third reference electrode and the fourth reference electrode.

The invention provides a differential impedance potentiometric biosensor and a manufacturing method thereof, wherein an insulating substrate which is provided with a longitudinal thickness and is provided with a signal input area, a signal output area, a reaction detection area and a reaction reference area is formed by processing an insulating material, an input anode and an input cathode are correspondingly arranged in the signal input area, a first output electrode is arranged in the signal output area, a first detection electrode and a second detection electrode which have the same space size and the same reaction end are provided with a biological reaction material and an inert material are arranged in the reaction detection area, a first reference electrode and a second reference electrode which have the same space size and the same inert end are provided with an inert material are arranged in the reaction reference area, the input anode is electrically connected with the first detection electrode, and the second detection electrode, the first reference electrode and the first output electrode are electrically connected together, and the second reference electrode is electrically connected with the input cathode, so that the biosensor with strong anti-interference capability and high sensitivity is manufactured, and the effects of improving the anti-interference capability and the sensitivity are achieved.

Drawings

FIG. 1 is a flowchart showing the steps of a method for producing a differential impedance potential type biosensor as provided in example 1;

FIG. 2 is a flowchart showing the steps of a method for producing a differential impedance potential type biosensor as provided in example 2;

FIG. 3 is a flowchart showing the steps of a method for producing a differential impedance potential type biosensor provided in example 3;

FIG. 4 is a flowchart showing the steps of a method for producing a differential impedance potential type biosensor provided in example 4;

FIG. 5 is a view showing a differential impedance potential type biosensor manufactured and formed by the method for manufacturing a differential impedance potential type biosensor provided in example 1;

FIG. 6 is a view showing a differential impedance potential type biosensor manufactured and formed by the method for manufacturing a differential impedance potential type biosensor provided in example 2;

FIG. 7 is a view showing a differential impedance potential type biosensor manufactured and formed by the method for manufacturing a differential impedance potential type biosensor provided in example 3;

FIG. 8 is a view showing a differential impedance potential type biosensor manufactured and formed by the method for manufacturing a differential impedance potential type biosensor according to example 4;

FIG. 9 is a schematic diagram of a partial structure (part B) and a corresponding schematic diagram of an equivalent circuit (part A) of the differential impedance potentiometric biosensor of FIG. 6;

fig. 10 is an equivalent circuit schematic diagram of the differential impedance potential type biosensor in fig. 8.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Referring to fig. 1 to 10, an object of the embodiments of the present disclosure is to provide a method for manufacturing a differential impedance potentiometric biosensor, so as to manufacture a biosensor with strong interference rejection and high sensitivity, so as to solve the technical problems of weak interference rejection and low sensitivity of the existing biosensor, and achieve the effect of improving the interference rejection and the sensitivity.

Referring to fig. 1 to 4, the method for manufacturing the differential impedance potential type biosensor includes the steps of: s1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, and S15. Next, the disclosed embodiments will be presented in a plurality of disclosed embodiments for a method of manufacturing the differential impedance potential type biosensor.

Referring to fig. 1, embodiment 1 provides a method for manufacturing a differential impedance potential type biosensor, comprising the steps of: s1, S2, S3, S4 and S5.

On the one hand, step S1: an insulating substrate having a longitudinal thickness and having a signal input region, a signal output region, a reaction detection region and a reaction reference region is formed by processing an insulating material.

It should be noted that the insulating substrate may be a rectangular flat plate with a longitudinal thickness, and may also be a flat plate with other shapes according to specific application, such as a triangular flat plate, a pentagonal flat plate, and other flat plates with other shapes.

The insulating substrate may be made of a polymer such as polyethylene, polypropylene, polyacrylamide, polystyrene, polyvinyl chloride, polyetherimide, or polyethylene terephthalate.

The insulating substrate can also be made of epoxy resin, epoxy glass cloth laminated board, polyimide resin and other resins or composites.

The insulating substrate can also be made of cellulose, glass, ceramics, paper and other insulating materials. In addition, the thickness and size of the insulating substrate may be set according to specific needs.

It should be noted that the signal input region, the signal output region, the reaction detection region, and the reaction reference region are functional region divisions performed on the plate surface of the insulating substrate, and may be used to set corresponding functional structures.

On the other hand, step S2: an input positive electrode and an input negative electrode are correspondingly arranged in the signal input area, and a first output electrode is arranged in the signal output area.

The input positive electrode, the input negative electrode and the first output electrode can be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the input positive electrode, the input negative electrode and the first output electrode can be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

In addition, the input anode, the input cathode and the first output electrode can be made of the same or different materials. For example, the input positive electrode is made of a carbon material, and the first output electrode is made of an alloy material.

In addition, the input anode, the input cathode and the first output electrode can be adjusted according to different application conditions in terms of the size, the distance and other parameters.

The positive input electrode, the negative input electrode, and the first output electrode each function as a potential signal input, and a potential signal output.

On the other hand, step S3: in the reaction detection area, a first detection electrode and a second detection electrode which have the same space size and the reaction ends of which are provided with a biological reaction material and an inert material are arranged.

The biological reaction material includes, but is not limited to, enzymes, antibodies, DNA, RNA, and the like.

The reaction end having a biological reaction material means that a biological reaction material for performing a biological reaction with a solution to be measured is provided at the reaction end.

It should be noted that the inert materials include, but are not limited to: protein blocking agents such as Bovine Serum Albumin (BSA) and albumin (OVA); organic blocking agents such as 6-mercaptohex-1-ol (MCH), and 1, 6-Hexanedithiol (HDT); stabilizers such as sucrose, glycerin, gelatin, amino acids, and polyvinylpyrrolidone; enzyme, antibody, DNA, etc. for inactivation treatment.

The inert end is provided with an inert material, which means that the inert end of the electrode is provided with an inert material that does not undergo biochemical reactions.

It should be noted that the first detection electrode and the second detection electrode may be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the first detection electrode and the second detection electrode can also be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

In addition, the first detection electrode and the second detection electrode can be made of the same or different materials. For example, the first detection electrode is made of a carbon material, and the second detection electrode is made of a gold material.

In addition, the size, the spacing and other parameters of the first detection electrode and the second detection electrode can be adjusted according to different application situations.

It should be noted that, the first detection electrode and the second detection electrode respectively perform specific biochemical reactions with the solution to be detected through the biological reaction material at the reaction end thereof.

It should be noted that the first detection electrode and the second detection electrode may be disposed in a parallel rectangular structure to form a plate capacitor structure. An interdigitated structure or other deformed structure may be provided, but it is required that these deformed structures satisfy that the sizes of the first detection electrode and the second detection electrode are the same and the pitch between the first detection electrode and the second detection electrode is the same.

On the other hand, step S4: in the reaction reference area, a first reference electrode and a second reference electrode which have the same space size and inert ends of which are provided with inert materials are arranged.

It should be noted that the inert materials include, but are not limited to: protein blocking agents such as Bovine Serum Albumin (BSA) and albumin (OVA); organic blocking agents such as 6-mercaptohex-1-ol (MCH), and 1, 6-Hexanedithiol (HDT); stabilizers such as sucrose, glycerin, gelatin, amino acids, and polyvinylpyrrolidone; enzyme, antibody, DNA, etc. for inactivation treatment.

The inert end is provided with an inert material, which means that the inert end of the electrode is provided with an inert material that does not undergo biochemical reactions.

It should be noted that the first reference electrode and the second reference electrode may be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the first reference electrode and the second reference electrode can also be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

In addition, the first reference electrode and the second reference electrode can be made of the same or different materials. For example, the first reference electrode is made of a carbon material, and the second reference electrode is made of a gold material.

In addition, the size, the spacing and other parameters of the first reference electrode and the second reference electrode can be adjusted according to different application situations.

It is also noted that the first reference electrode and the second reference electrode function to have the same change as the first detection electrode and the second detection electrode when facing the interfering object by forming the same inert material layer as the first detection electrode and the second detection electrode. Further, the influence of the interfering object can be removed by the difference.

It should be noted that the first reference electrode and the second reference electrode may be disposed in a parallel rectangular structure to form a plate capacitor structure. Interdigitated or other shape-changing structures may be provided, but these shape-changing structures are required to satisfy the condition that the first reference electrode and the second reference electrode are the same in size and the spacing between the first reference electrode and the second reference electrode is the same.

It is noted that the methods of disposing the bio-reactive material at the reaction end and disposing the inert material at the inert end in steps S3 and S4, respectively, include, but are not limited to, the following methods:

first, the protein-based bioreaction material is set.

First, covalent crosslinking method.

For example, the surface of the electrode is modified with a monomolecular layer with carboxyl at the end, and then activated by 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to be combined with protein so as to fix the protein.

In another example, the monolayer with amino groups at the ends is modified on the surface of the electrode to be combined with the carboxyl of the protein to realize protein immobilization.

For another example, the surface of the electrode is modified with a monomolecular layer with an amino group at the end, and then glutaraldehyde is used for crosslinking the monomolecular layer and the amino group on the protein to realize protein immobilization.

For another example, the electrode surface is modified with a monomolecular layer with a disulfide bond or maleimide group at the end to be further combined with a thiol group of a protein to realize immobilization and the like.

Second, adsorption immobilization method.

For example, proteins can be immobilized by interaction of polar, hydrogen, and hydrophobic bonds of the adsorption carrier and the protein molecules. Wherein, the adsorption carrier can be selected from graphite powder, active carbon, graphite-polytetrafluoroethylene and the like.

For another example, the protein is adsorbed and immobilized by immobilizing negatively charged gold nanoparticles.

Third, an electropolymerization method.

For example, pyrrole monomers are doped with a protein a solution, and then protein a is immobilized by cyclic voltammetric electropolymerization.

Fourthly, embedding and fixing method.

For example, a protein or the like is embedded in an electrode using a specific membrane material. Wherein the membrane material includes, but is not limited to, synthetic polymer (such as Nafion), carbon paste (prepared by mixing antigen or antibody with solvent such as paraffin oil, and adding graphite powder to obtain paste), hydrogel and sol-gel.

Next, nucleic acids such as DNA and RNA are set.

A monomolecular layer with carboxyl at the tail end can be modified on the surface of the electrode, and the carboxyl is activated by 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) and combined with DNA with amino at the tail end.

The surface of the electrode is irreversibly adsorbed and fixed with avidin, and biotin-labeled DNA is further fixed through specific binding of biotin-avidin.

And directly fixing the DNA modified by sulfydryl on the surface of the electrode through the action of gold sulfydryl bonds.

On the other hand, step S5: the input positive electrode is electrically connected with the first detection electrode, the second detection electrode, the first reference electrode and the first output electrode are electrically connected together, and the second reference electrode is electrically connected with the input negative electrode.

It should be noted that the electrical connection may be made by a wire.

In addition, the conducting wire can be made into a single-layer structure by adopting a metal material or a conductive non-metal material. The conductive non-metallic material can be carbon or graphene.

In addition, the conducting wire can be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material.

In addition, the parameters such as the size, the spacing and the like of the conducting wires can be adjusted according to different application situations.

In addition, the relative positions of the signal input area, the signal output area, the reaction detection area and the reaction reference area can be adjusted according to different application situations. The shape of the wires for electrical connection can also be adjusted accordingly.

Referring to fig. 2, embodiment 2 is proposed as an improvement on embodiment 1, and embodiment 2 provides a method for manufacturing a differential impedance potential type biosensor, comprising the steps of: s1, S2, S3, S4, S5, S6, and S7. To avoid the repeated description, embodiment 2 will explain only the modification steps S6 and S7.

On the one hand, step S6: a first reinforcing electrode having a biological reaction material and an inert material at its reaction end is provided between the first detection electrode and the second detection electrode.

It should be noted that the first enhanced electrode may be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the first enhanced electrode can also be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

It should be further noted that the first reinforcing electrode functions as: the variation range of the dielectric constant of the capacitor formed by the first detection electrode and the second detection electrode is increased, and the variation range of the capacitor value is further increased.

On the other hand, step S7: between the first reference electrode and the second reference electrode, a second reinforcing electrode having an inert end provided with an inert material is disposed.

It should be noted that the second enhanced electrode can be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the second enhanced electrode can also be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

It should be noted that the second reinforcing electrode functions as: and increasing the variation amplitude of the dielectric constant of the capacitor formed by the first reference electrode and the second reference electrode, thereby increasing the variation amplitude of the capacitor value.

Referring to fig. 3, embodiment 3 is proposed as an improvement on embodiment 2, and embodiment 3 provides a method for manufacturing a differential impedance potential type biosensor, comprising the steps of: s1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, and S13. To avoid the above description, embodiment 3 will be described only with respect to the modification steps S8, S9, S10, S11, S12, and S13.

On the one hand, step S8: and a second output electrode is arranged in the signal output area.

It should be noted that the second output electrode may be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the second output electrode can be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material together. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

The second output electrode functions to output a potential signal.

On the other hand, step S9: and a third detection electrode and a fourth detection electrode which have the same space size and reaction ends provided with a biological reaction material and an inert material are arranged in the reaction detection area.

The biological reaction material includes, but is not limited to, enzymes, antibodies, DNA, RNA, and the like.

The reaction end having a bioreaction material means that a bioreaction material for performing a bioreaction with a solution to be measured is provided at the reaction end.

It should be noted that the inert materials include, but are not limited to: protein blocking agents such as Bovine Serum Albumin (BSA) and albumin (OVA); organic blocking agents such as 6-mercaptohex-1-ol (MCH), and 1, 6-Hexanedithiol (HDT); sucrose, glycerol, gelatin, amino acids, polyvinylpyrrolidone, and the like.

The inert end is provided with an inert material, which means that the inert end of the electrode is provided with an inert material that does not undergo biochemical reactions.

It should be noted that the third detection electrode and the fourth detection electrode may be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the third detection electrode and the fourth detection electrode can also be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

The third detection electrode and the fourth detection electrode may be made of the same material or different materials. For example, the third detection electrode is made of a carbon material, and the fourth detection electrode is made of a gold material.

In addition, the size, the spacing and other parameters of the third detection electrode and the fourth detection electrode can be adjusted according to different application situations.

It should be noted that the third detection electrode and the fourth detection electrode respectively perform specific biochemical reactions with the solution to be detected through the biological reaction material at the reaction end thereof.

It should be noted that the third detection electrode and the fourth detection electrode may be disposed in a parallel rectangular structure to form a plate capacitor structure. An interdigital structure or other deformation structures may be provided, but it is required that these deformation structures satisfy that the sizes of the third detection electrode and the fourth detection electrode are the same and the interval between the third detection electrode and the fourth detection electrode is the same.

On the other hand, step S10: and in the reaction reference area, a third reference electrode and a fourth reference electrode which have the same interval size and inert ends of which are provided with inert materials are arranged.

It should be noted that the inert materials include, but are not limited to: protein blocking agents such as Bovine Serum Albumin (BSA) and albumin (OVA); organic blocking agents such as 6-mercaptohex-1-ol (MCH), and 1, 6-Hexanedithiol (HDT); sucrose, glycerol, gelatin, amino acids, polyvinylpyrrolidone, and the like.

The inert end is provided with an inert material, which means that the inert end of the electrode is provided with an inert material that does not undergo biochemical reactions.

It should be noted that the third reference electrode and the fourth reference electrode may be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the third reference electrode and the fourth reference electrode can also be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

In addition, the materials used for the third reference electrode and the fourth reference electrode can be the same or different. For example, the third reference electrode is made of a carbon material, and the fourth reference electrode is made of a gold material.

In addition, the size, the spacing and other parameters of the third reference electrode and the fourth reference electrode can be adjusted according to different application situations.

It should be noted that the third reference electrode and the fourth reference electrode function to have the same change as the third detection electrode and the fourth detection electrode when facing the interfering object by forming the same inert material layer as the third detection electrode and the fourth detection electrode. Further, the influence of the interfering object can be removed by the difference.

It should be noted that the third reference electrode and the fourth reference electrode may be disposed in a parallel rectangular structure to form a plate capacitor structure. An interdigitated structure or other deformed structure may be provided, but it is required that these deformed structures satisfy that the third reference electrode and the fourth reference electrode have the same size and the same pitch between the third reference electrode and the fourth reference electrode.

On the other hand, step S11: and electrically connecting the third reference electrode with the connection point of the input positive electrode and the first detection electrode.

On the other hand, step S12: and electrically connecting the fourth reference electrode, the second output electrode and the third detection electrode in common.

On the other hand, step S13: and electrically connecting the fourth detection electrode with the connection point of the second reference electrode and the input cathode.

It should be noted that the electrical connection may be performed by a wire, in common to the steps S11, S12, and S13. The conducting wire can be made into a single-layer structure by adopting a metal material or a conductive non-metal material. The conductive non-metallic material can be carbon or graphene.

In addition, the conducting wire can be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material.

In addition, the parameters such as the size, the spacing and the like of the conducting wires can be adjusted according to different application situations.

In addition, the relative positions of the signal input area, the signal output area, the reaction detection area and the reaction reference area can be adjusted according to different application situations. The shape of the wires for electrical connection can also be adjusted accordingly.

Referring to fig. 4, embodiment 4 is proposed as an improvement on embodiment 3, and embodiment 4 provides a method for manufacturing a differential impedance potential type biosensor, comprising the steps of: s1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, and S15. To avoid the repeated description, embodiment 3 will explain only the modification steps S14 and S15.

On the one hand, step S14: a fourth reinforcing electrode having a reaction end provided with a biological reaction material and an inert material is provided between the third detection electrode and the fourth detection electrode.

It should be noted that the fourth enhanced electrode may be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the fourth enhanced electrode can also be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

It should be noted that the fourth reinforcing electrode functions as: and increasing the variation amplitude of the dielectric constant of the capacitor formed by the third detection electrode and the fourth detection electrode, thereby increasing the variation amplitude of the capacitor value.

On the other hand, step S15: between the third reference electrode and the fourth reference electrode, a third reinforcing electrode whose inert end is provided with an inert material is disposed.

It should be noted that the third enhanced electrode may be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the third enhanced electrode can also be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

It should be noted that the third reinforcing electrode functions as: and increasing the variation amplitude of the dielectric constant of the capacitor formed by the third reference electrode and the fourth reference electrode, thereby increasing the variation amplitude of the capacitor value.

It should also be summarized that the size and spacing of all electrodes (including the detection electrode, the enhancement electrode, and the reference electrode) in examples 1-4 can be set according to a uniform size standard and spacing standard.

Referring to fig. 5 to 10, another object of the embodiments of the present disclosure is to provide a differential impedance potentiometric biosensor, which achieves the effect of improving the anti-interference capability and sensitivity.

Hereinafter, the disclosed embodiments will present a plurality of disclosed embodiments to the differential impedance potentiometric biosensor.

Referring to fig. 5, embodiment 5 provides a differential impedance potential type biosensor comprising: an insulating substrate 2, an input positive electrode 7, an input negative electrode 8, a first output electrode 9a, a first detection electrode 4a, a second detection electrode 4b, a first reference electrode 5a, and a second reference electrode 5 b.

On the other hand, the insulating substrate 2 is formed by processing an insulating material, and has a longitudinal thickness and a signal input region, a signal output region, a reaction detection region, and a reaction reference region.

It should be noted that the insulating substrate may be a rectangular flat plate with a longitudinal thickness, and may also be a flat plate with other shapes according to specific application, such as a triangular flat plate, a pentagonal flat plate, and other flat plates with other shapes.

The insulating substrate may be made of a polymer such as polyethylene, polypropylene, polyacrylamide, polystyrene, polyvinyl chloride, polyetherimide, or polyethylene terephthalate.

The insulating substrate can also be made of epoxy resin, epoxy glass cloth laminated board, polyimide resin and other resins or composites.

The insulating substrate can also be made of cellulose, glass, ceramics, paper and other insulating materials. In addition, the thickness and size of the insulating substrate may be set according to specific needs.

It should be noted that the signal input region, the signal output region, the reaction detection region, and the reaction reference region are functional region divisions performed on the plate surface of the insulating substrate, and may be used to set corresponding functional structures.

On the other hand, an input positive electrode 7 and an input negative electrode 8 are correspondingly arranged in the signal input area.

On the other hand, the first output electrode 9a is provided in the signal output region.

The input positive electrode 7, the input negative electrode 8, and the first output electrode 9a may be formed in a single-layer structure using a metal material or a conductive non-metal material. The conductive non-metallic material can be carbon or graphene.

In addition, the input anode 7, the input cathode 8 and the first output electrode 9a can be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

The materials used for the input positive electrode 7, the input negative electrode 8, and the first output electrode 9a may be the same or different. For example, the input positive electrode 7 is made of a carbon material, and the first output electrode 9a is made of a gold material.

In addition, the input anode 7, the input cathode 8 and the first output electrode 9a can be adjusted in size, distance and other parameters according to different application situations.

The input positive electrode 7, the input negative electrode 8, and the first output electrode 9a correspond to the potential signal input, and the potential signal output, respectively.

On the other hand, the first detection electrode 4a and the second detection electrode 4b are disposed in the reaction detection region at the same pitch, have the same size, and have a bioreaction material and an inert material at their reaction ends.

The biological reaction material includes, but is not limited to, enzymes, antibodies, DNA, RNA, and the like.

The reaction end having a bioreaction material means that a bioreaction material for performing a bioreaction with a solution to be measured is provided at the reaction end.

It should be noted that the inert materials include, but are not limited to: protein blocking agents such as Bovine Serum Albumin (BSA) and albumin (OVA); organic blocking agents such as 6-mercaptohex-1-ol (MCH), and 1, 6-Hexanedithiol (HDT); sucrose, glycerol, gelatin, amino acids, polyvinylpyrrolidone, and the like.

The inert end is provided with an inert material, which means that the inert end of the electrode is provided with an inert material that does not undergo biochemical reactions.

It should be noted that the first detection electrode 4a and the second detection electrode 4b may be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the first detection electrode 4a and the second detection electrode 4b can be made of a metal material and a conductive non-metal material to form a multi-layer composite structure. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

In addition, the first detection electrode 4a and the second detection electrode 4b may be made of the same material or different materials. For example, the first detection electrode 4a is made of a carbon material, and the second detection electrode 4b is made of a gold material.

In addition, the size, the spacing and other parameters of the first detection electrode 4a and the second detection electrode 4b can be adjusted according to different application situations.

It should be noted that the first detection electrode 4a and the second detection electrode 4b perform specific biochemical reactions with the solution to be detected through the biological reaction material at the reaction ends thereof, respectively.

It should be noted that the first detection electrode 4a and the second detection electrode 4b may be disposed in a rectangular structure parallel to each other to form a plate capacitor structure. An interdigital structure or other deformation structures may be provided, but these deformation structures are required so that the sizes of the first and second detection electrodes 4a and 4b are the same and the intervals between the first and second detection electrodes 4a and 4b are the same.

On the other hand, the first reference electrode 5a and the second reference electrode 5b, inert ends of which are provided with an inert material, are disposed at the reaction reference region at the same size and interval.

It should be noted that the inert materials include, but are not limited to: protein blocking agents such as Bovine Serum Albumin (BSA) and albumin (OVA); organic blocking agents such as 6-mercaptohex-1-ol (MCH), and 1, 6-Hexanedithiol (HDT); sucrose, glycerol, gelatin, amino acids, polyvinylpyrrolidone, and the like.

The inert end is provided with an inert material, which means that the inert end of the electrode is provided with an inert material that does not undergo biochemical reactions.

It should be noted that the first reference electrode 5a and the second reference electrode 5b may be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the first reference electrode 5a and the second reference electrode 5b can be made of a metal material and a conductive non-metal material to form a multi-layer composite structure. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

In addition, the materials used for the first reference electrode 5a and the second reference electrode 5b may be the same or different. For example, the first reference electrode 5a is made of a carbon material, and the second reference electrode 5b is made of a gold material.

In addition, the size and spacing of the first reference electrode 5a and the second reference electrode 5b can be adjusted according to different applications.

It is also noted that the first reference electrode 5a and the second reference electrode 5b function to have the same change as the first detection electrode 4a and the second detection electrode 4b when facing the interfering object by forming the same inert material layer as the first detection electrode 4a and the second detection electrode 4 b. Further, the influence of the interfering object can be removed by the difference.

It should be noted that the first reference electrode 5a and the second reference electrode 5b may be disposed in a rectangular structure parallel to each other to form a plate capacitor structure. An interdigitated structure or other deformed structure may be provided, but it is required that these deformed structures satisfy that the sizes of the first reference electrode 5a and the second reference electrode 5b are the same and the pitch between the first reference electrode 5a and the second reference electrode 5b is the same.

On the other hand, the input positive electrode 7 is electrically connected to the first detection electrode 4a, for example, by a lead wire 3 a.

The second detection electrode 4b, the first reference electrode 5a, and the first output electrode 9a are electrically connected in common, for example, by a wire 3 b.

The second reference electrode 5b is electrically connected to the input cathode 8, for example, by a wire 3 c.

It should be noted that the electrical connection may be made by a wire.

In addition, the conducting wire can be made into a single-layer structure by adopting a metal material or a conductive non-metal material. The conductive non-metallic material can be carbon or graphene.

In addition, the conducting wire can be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material.

In addition, the parameters such as the size, the spacing and the like of the conducting wires can be adjusted according to different application situations.

In addition, the relative positions of the signal input area, the signal output area, the reaction detection area and the reaction reference area can be adjusted according to different application situations. The shape of the wires for electrical connection can also be adjusted accordingly.

Referring to fig. 6, example 6 is proposed as an improvement on example 5, and example 6 provides a differential impedance potential type biosensor comprising: the detection electrode assembly comprises an insulating substrate 2, an input positive electrode 7, an input negative electrode 8, a first output electrode 9a, a first detection electrode 4a, a second detection electrode 4b, a first reference electrode 5a, a second reference electrode 5b, a first reinforcing electrode 6a and a second reinforcing electrode 6 b. To avoid the description, embodiment 6 will be described only with respect to the first reinforcing electrode 6a and the second reinforcing electrode 6b, which are improved features.

On the other hand, the first reinforcing electrode 6a, which has a reaction end provided with a biological reaction material and an inert material, is disposed between the first detection electrode 4a and the second detection electrode 4 b.

The first reinforcing electrode 6a may be made of a metal material or a conductive non-metal material to have a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the first enhanced electrode 6a can also be made of a metal material and a conductive non-metal material to form a multi-layer composite structure. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

It should be further noted that the first enhancing electrode 6a functions as: the amplitude of change in the dielectric constant of the capacitance formed by the first detection electrode 4a and the second detection electrode 4b is increased, thereby increasing the amplitude of change in the capacitance value.

On the other hand, the second reinforcing electrode 6b, the inert end of which is provided with an inert material, is disposed between the first reference electrode 5a and the second reference electrode 5 b.

It should be noted that the second enhanced electrode 6b may be made of a metal material or a conductive non-metal material to have a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the second enhanced electrode 6b can also be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

It should be noted that the second reinforcing electrode 6b functions as: the magnitude of change in the dielectric constant of the capacitance formed by the first reference electrode 5a and the second reference electrode 5b is increased, thereby increasing the magnitude of change in the capacitance value.

Referring to fig. 7, example 7 is proposed as an improvement on example 6, and example 7 provides a differential impedance potential type biosensor comprising: the detection electrode assembly comprises an insulating substrate 2, an input positive electrode 7, an input negative electrode 8, a first output electrode 9a, a first detection electrode 4a, a second detection electrode 4b, a first reference electrode 5a, a second reference electrode 5b, a first enhancement electrode 6a, a second enhancement electrode 6b, a second output electrode 9b, a third detection electrode 11a, a fourth detection electrode 11b, a third reference electrode 10a and a fourth reference electrode 10 b. To avoid the above description, embodiment 7 will be described only with respect to the second output electrode 9b, the third detection electrode 11a, the fourth detection electrode 11b, the third reference electrode 10a, and the fourth reference electrode 10b, which are improved features.

On the one hand, the second output electrode 9b is disposed in the signal output region.

The second output electrode 9b may be made of a metal material or a conductive non-metal material to have a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the second output electrode 9b can be made of a metal material and a conductive non-metal material to form a multi-layer composite structure. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

The second output electrode 9b functions to output a potential signal.

On the other hand, the third detection electrode 11a and the fourth detection electrode 11b have the same size, and the reaction ends thereof are provided with the bioreaction material and the inert material, and are disposed at intervals in the reaction detection region.

The biological reaction material includes, but is not limited to, enzymes, antibodies, DNA, RNA, and the like.

The reaction end having a bioreaction material means that a bioreaction material for performing a bioreaction with a solution to be measured is provided at the reaction end.

It should be noted that the inert materials include, but are not limited to: protein blocking agents such as Bovine Serum Albumin (BSA) and albumin (OVA); organic blocking agents such as 6-mercaptohex-1-ol (MCH), and 1, 6-Hexanedithiol (HDT); sucrose, glycerol, gelatin, amino acids, polyvinylpyrrolidone, and the like.

The inert end is provided with an inert material, which means that the inert end of the electrode is provided with an inert material that does not undergo biochemical reactions.

It should be noted that the third detection electrode 11a and the fourth detection electrode 11b may be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the third detection electrode 11a and the fourth detection electrode 11b can be made of a metal material and a conductive non-metal material to form a multi-layer composite structure. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

The third detection electrode 11a and the fourth detection electrode 11b may be made of the same material or different materials. For example, the third detection electrode 11a is made of a carbon material, and the fourth detection electrode 11b is made of a gold material.

In addition, the size and spacing of the third detecting electrode 11a and the fourth detecting electrode 11b can be adjusted according to different applications.

It should be noted that the third detection electrode 11a and the fourth detection electrode 11b perform specific biochemical reactions with the solution to be detected through the biological reaction material at the reaction ends thereof, respectively.

It should be noted that the third detection electrode 11a and the fourth detection electrode 11b may be disposed in a rectangular structure parallel to each other to form a plate capacitor structure. An interdigital structure or other deformation structures may be provided, but it is required that these deformation structures satisfy that the sizes of the third and fourth detection electrodes 11a and 11b are the same and the interval between the third and fourth detection electrodes 11a and 11b is the same.

On the other hand, the inert ends of the third reference electrode 10a and the fourth reference electrode 10b are provided with an inert material and disposed in the reaction reference region.

It should be noted that the inert materials include, but are not limited to: protein blocking agents such as Bovine Serum Albumin (BSA) and albumin (OVA); organic blocking agents such as 6-mercaptohex-1-ol (MCH), and 1, 6-Hexanedithiol (HDT); sucrose, glycerol, gelatin, amino acids, polyvinylpyrrolidone, and the like.

The inert end is provided with an inert material, which means that the inert end of the electrode is provided with an inert material that does not undergo biochemical reactions.

It should be noted that the third reference electrode 10a and the fourth reference electrode 10b may be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the third reference electrode 10a and the fourth reference electrode 10b can be made of a metal material and a conductive non-metal material to form a multi-layer composite structure. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

In addition, the materials used for the third reference electrode 10a and the fourth reference electrode 10b may be the same or different. For example, the third reference electrode 10a is made of a carbon material, and the fourth reference electrode 10b is made of a gold material.

In addition, the size and spacing of the third reference electrode 10a and the fourth reference electrode 10b can be adjusted according to different applications.

It should be noted that the third reference electrode 10a and the fourth reference electrode 10b function to have the same variation as the third detection electrode 11a and the fourth detection electrode 11b when facing the interfering object by forming the same inert material layer as the third detection electrode 11a and the fourth detection electrode 11 b. Further, the influence of the interfering object can be removed by the difference.

It should be noted that the third reference electrode 10a and the fourth reference electrode 10b may be disposed in a parallel rectangular structure to form a plate capacitor structure. An interdigitated structure or other deformed structure may be provided, but it is required that these deformed structures satisfy that the third reference electrode 10a and the fourth reference electrode 10b are the same size and the pitch between the third reference electrode 10a and the fourth reference electrode 10b is the same.

On the other hand, the third reference electrode 10a is electrically connected to a connection point between the input positive electrode 7 and the first detection electrode 4 a.

The fourth reference electrode 10b, the second output electrode 9b, and the third detection electrode 11a are electrically connected in common.

And a fourth detection electrode 11b electrically connected to a connection point of the second reference electrode 5b and the input cathode 8.

It should be noted that the electrical connection may be made by a wire. The conducting wire can be made into a single-layer structure by adopting a metal material or a conductive non-metal material. The conductive non-metallic material can be carbon or graphene.

In addition, the conducting wire can be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material.

In addition, the parameters such as the size, the spacing and the like of the conducting wires can be adjusted according to different application situations.

In addition, the relative positions of the signal input area, the signal output area, the reaction detection area and the reaction reference area can be adjusted according to different application situations. The shape of the wires for electrical connection can also be adjusted accordingly.

Referring to fig. 8, example 8 is proposed as an improvement on example 7, and example 8 provides a differential impedance potential type biosensor comprising: the detection electrode assembly comprises an insulating substrate 2, an input positive electrode 7, an input negative electrode 8, a first output electrode 9a, a first detection electrode 4a, a second detection electrode 4b, a first reference electrode 5a, a second reference electrode 5b, a first enhancement electrode 6a, a second enhancement electrode 6b, a second output electrode 9b, a third detection electrode 11a, a fourth detection electrode 11b, a third reference electrode 10a, a fourth reference electrode 10b, a third enhancement electrode 6c and a fourth enhancement electrode 6 d. To avoid the description, embodiment 8 will be described only with respect to the third reinforcing electrode 6c and the fourth reinforcing electrode 6d, which are improved features.

On the other hand, the fourth reinforcing electrode 6d, which has a reaction end provided with a bioreaction material and an inert material, is disposed between the third detection electrode 11a and the fourth detection electrode 11 b.

It should be noted that the fourth enhanced electrode 6d may be made of a metal material or a conductive non-metal material to form a single-layer structure. The conductive non-metallic material can be carbon or graphene.

In addition, the fourth enhanced electrode 6d can also be made of a metal material and a conductive non-metal material to form a multi-layer composite structure. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

It should be noted that the fourth reinforcing electrode 6d functions as: the magnitude of change in the dielectric constant of the capacitance formed by the third detection electrode 11a and the fourth detection electrode 11b is increased, thereby increasing the magnitude of change in the capacitance value.

On the other hand, the third reinforcing electrode 6c, the inert end of which is provided with an inert material, is disposed between the third reference electrode 10a and the fourth reference electrode 10 b.

It should be noted that a single-layer structure can be made of a metal material or a conductive non-metal material. The conductive non-metallic material can be carbon or graphene.

In addition, the third enhanced electrode 6c can also be made into a multilayer composite structure by adopting a metal material and a conductive non-metal material. For example, a silver electrode is formed by screen printing, and then a carbon electrode is formed on the silver electrode.

It should be noted that the third reinforcing electrode 6c functions as: the magnitude of change in the dielectric constant of the capacitance formed by the third reference electrode 10a and the fourth reference electrode 10b is increased, thereby increasing the magnitude of change in the capacitance value.

It should also be summarized that the size and spacing of all electrodes (including the detection electrode, the enhancement electrode, and the reference electrode) in examples 5-8 can be set according to a uniform size standard and spacing standard.

Next, the operation principle of the differential impedance potential type biosensor will be described with reference to fig. 6 and 9, taking the differential impedance potential type biosensor shown in fig. 6 as an example.

First, an equivalent analysis of the total impedance ZD at the detection terminal is formed.

The first detection electrode 4a is connected to the input positive electrode 7 through the wire 3a, and thus the potential of the first detection electrode 4a is the same as the input positive electrode 7 and can be represented by V +.

In the electrolyte, a double-layer capacitor C1 and an electron transfer resistor R1 connected in parallel with the double-layer capacitor C1 are formed equivalently at both interfaces where the solid first detection electrode 4a contacts the liquid electrolyte. Wherein the electron transfer resistor R1 is connected in series with a diffusion-controlled Warburg impedance ZW 1.

The second detecting electrode 4b has an equivalent circuit similar to that of the first detecting electrode 4a, and includes a double-layer capacitor C3, an electron transfer resistor R3, and a Warburg impedance ZW 2.

The first detection electrode 4a and the second detection electrode 4b constitute a plate capacitor structure.

Wherein C2 is the capacitance of the plate capacitor structure.

R2 is the electrolyte resistance between the first detection electrode 4a and the second detection electrode 4 b.

The first detecting electrode 4a, the second detecting electrode 4b and the first enhancing electrode 6a are detecting ends of the biosensor, form a total impedance ZD, and are composed of C1, C2, C3, R1, R2, R3, ZW1 and ZW 2.

Secondly, an equivalent analysis of the reference terminal total impedance ZR is formed.

The second reference electrode 5b is connected to the input cathode 8 by a lead 3c, so that the potential of the second reference electrode 5b is the same as the input cathode 8, which can be denoted by V-.

In the electrolyte, a double-layer capacitor C4 and an electron transfer resistor R4 connected in parallel with the double-layer capacitor C4 are formed equivalently at both interfaces where the solid first reference electrode 5a contacts the liquid electrolyte. Wherein, the electron transfer resistor R4 is connected in series with the diffusion control Warburg impedance ZW 3.

The second reference electrode 5b has an equivalent circuit similar to that of the first reference electrode 5a, and includes a double-layer capacitor C6, an electron transfer resistor R6, and a Warburg impedance ZW 4.

The first reference electrode 5a and the second reference electrode 5b form a plate capacitor structure. C5 is the capacitance of the plate capacitor structure. R5 is the electrolyte resistance between first reference electrode 5a and second reference electrode 5 b.

The first reference electrode 5a, the second reference electrode 5b and the second enhancing electrode 6b form a reference terminal of the biosensor, and the total impedance is ZR, and is composed of C4, C5, C6, R4, R5, R6, ZW3 and ZW 4.

In addition, when the liquid to be detected is added, the first detection electrode 4a, the second detection electrode 4b, the first enhancing electrode 6a, the first reference electrode 5a, the second reference electrode 5b, and the second enhancing electrode 6b are all immersed and react to equilibrium.

The substance to be detected in the solution to be detected can perform specific biochemical reaction with the biological reaction material B1 on the first detection electrode 4a, the second detection electrode 4B and the first enhanced electrode 6a at the detection end of the biosensor.

The effect of signal amplification can be achieved by using various labels. Such as horseradish peroxidase and other enzyme markers, such as avidin and biotin and other specific binding markers, such as gold nanoparticle and other nanoparticle markers, such as methylene blue and other electroactive markers.

Specific biochemical reactions lead to an increase or decrease in ZD. The change in impedance resulting from a specific biochemical reaction is represented by Δ Zd. And the interferents in the solution to be detected can also perform nonspecific adsorption or other reactions on the detection end at the same time, so that the impedance change of the detection end is recorded as delta Zb.

Therefore, after the first detection electrode 4a, the second detection electrode 4b and the first reinforcing electrode 6a at the detection end react and balance with the solution to be detected, the total impedance ZD becomes ZD + Δ Zb.

At the reference end of the biosensor, the first reference electrode 5a, the second reference electrode 5B and the second enhancing electrode 6B are not provided with the bio-reaction material B1, but are provided with the inert material B2, so that no specific biochemical reaction occurs, and further, no impedance change caused by the biochemical reaction occurs. However, non-specific adsorption of interfering substances or other reactions may occur on the first reference electrode 5a, the second reference electrode 5b, and the second enhancing electrode 6 b. Since the first detection electrode 4a, the second detection electrode 4b, and the first enhancement electrode 6a are identical to the first reference electrode 5a, the second reference electrode 5b, and the second enhancement electrode 6b in size and structure, the interfering impedance changes formed at the first reference electrode 5a, the second reference electrode 5b, and the second enhancement electrode 6b at the reference end are also identical to Δ Zb.

Further, a voltage signal V + is input to the input positive electrode 7, and a voltage signal V-is input to the input negative electrode 8. V + may be ground or a dc or ac signal, and the ac signal may have various waveforms, such as sine wave, triangular wave, square wave, etc. V-can be ground or a direct current or alternating current signal, and the alternating current signal can be various waveforms, such as sine wave, triangular wave, square wave and the like. According to the principle of circuit voltage division, the initial potential of the first output electrode 9a is

Adding the solution to be detected for balancing.

The concentration of the substance to be detected can be obtained by analyzing the potential change of the first output electrode 9 a.

Different signal analysis purposes can be achieved by adjusting parameters such as the ratio of ZR to ZD, the values of V + and V-, the waveform, the phase, the frequency and the like.

For example, when the reactive end and the inert end are prepared, the values of ZD and ZR can be controlled by adjusting parameters such as the thickness and density of the reactive end and the inert end so that ZD and ZR are equal to each other, and Vo = (V + + V-)/2.

One of V +, V-is typically grounded, or V + = -V-. If V + = -V-, the first output electrode 9a is initially at a potential of 0V. When the concentration of the analyte in the solution to be detected is zero, Δ Zd = 0 after the equilibrium because there is no impedance change due to the specific biochemical reaction. In formula (2), Vo' = 0V. When the concentration of the substance to be detected is not zero.

Typically 2ZD is made much larger than 2 Δ Zb + Δ ZD, so Vo' is approximately equal to. The detection voltage and the impedance change are in a linear relation, and then a linear relation graph of the detection signal and the concentration of the object to be detected can be obtained.

In addition, referring to fig. 10, fig. 10 shows an equivalent circuit of a differential impedance potential type biosensor having two detection terminals and two reference terminals, including two input poles, two output poles, and four equivalent impedances ZD, ZR, Z3, and Z4. The operation is the same as above, the output signals are Vo2-Vo1, and larger voltage signal output can be realized, which is not described in detail herein.

In summary, the configuration has strong anti-interference capability, and can further realize higher sensitivity through signal amplification.

The differential impedance potentiometric biosensor and the manufacturing method thereof according to the present disclosure are provided by forming an insulating substrate 2 having a longitudinal thickness and having a signal input region, a signal output region, a reaction detection region and a reaction reference region by processing an insulating material, disposing an input positive electrode 7 and an input negative electrode 8 in the signal input region, disposing a first output electrode 9a in the signal output region, disposing a first detection electrode 4a and a second detection electrode 4b having the same pitch size and a bio-reactive material and an inert material at the reaction end in the reaction detection region, disposing a first reference electrode 5a and a second reference electrode 5b having the same pitch size and an inert material at the inert end in the reaction reference region, and electrically connecting the input positive electrode 7 and the first detection electrode 4a to connect the second detection electrode 4b and the reaction reference region, The first reference electrode 5a and the first output electrode 9a are electrically connected together, and the second reference electrode 5b is electrically connected with the input cathode 8, so that the biosensor with strong anti-interference capability and high sensitivity is manufactured, and the effects of improving the anti-interference capability and the sensitivity are achieved.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.