US-12625104-B2 - Method for improving stability of electrochemical sensor

US12625104B2US 12625104 B2US12625104 B2US 12625104B2US-12625104-B2

Abstract

The present disclosure provides a method for improving stability of an electrochemical sensor. The method includes the following steps: S 1 , manufacturing an electrochemical sensor; S 2 , immobilizing a biosensitive molecular enzyme on a working electrode of the electrochemical sensor; S 3 , setting a immobilization agent on a surface of the biosensitive molecular enzyme; and S 4 , adding a protective film on a surface of the immobilization agent, such that the protective film is deposited on the working electrode to improve the stability of the electrochemical sensor. In the present disclosure, glutaraldehyde (GA), polyvinyl alcohol (PVA), and polyethylene glycol (PEG), or polyaniline (PANI), the GA, the PVA, the PEG, and polyurethane (PU) are arranged on a surface of the biosensitive molecular enzyme. Therefore, the surface of the biosensitive molecular enzyme forms a composite protective film, which reduces a probability of direct exposure of an enzyme layer to the air.

Inventors

Yue Cui

Assignees

PEKING UNIVERSITY

Dates

Publication Date: 20260512
Application Date: 20230727
Priority Date: 20220729

Claims (9)

1 . A method for improving stability of an electrochemical sensor, comprising the following steps: S 1 , manufacturing an electrochemical sensor, wherein a process for manufacturing the electrochemical sensor comprises micro/nanofabrication and screen printing; and wherein the electrochemical sensor is a three-electrode system comprising a reference electrode, a working electrode, and a counter electrode, or a two-electrode system comprising the working electrode and the reference electrode, or a two-electrode system comprising the working electrode and the counter electrode; and the working electrode is one or a combination of more selected from the group consisting of gold, platinum, and carbon; S 2 , immobilizing a biosensitive molecular enzyme on the working electrode of the electrochemical sensor; S 3 , setting an immobilization agent on a surface of the biosensitive molecular enzyme with the immobilization agents of polyaniline (PANI) and glutaraldehyde (GA); and S 4 , adding a protective film with a multilayer structure consisting of polyurethane (PU), polyvinyl alcohol (PVA), and polyethylene glycol (PEG) on a surface of the immobilization agent, such that the protective film is deposited on the working electrode to improve the stability of the electrochemical sensor.
2 . The method for improving stability of an electrochemical sensor according to claim 1 , wherein the process for micro/nanofabrication of the electrode system is selected from the group consisting of: a process for the micro/nanofabrication of the three-electrode system comprises: (1) preparation of the working electrode and the counter electrode: conducting metal evaporation or sputtering by the micro/nanofabrication to obtain a nano-gold layer or a nano-platinum layer, and then conducting electroplating on a surface of the nano-gold layer or the nano-platinum layer to form a Prussian blue layer, thereby obtaining a gold/Prussian blue electrode or a platinum/Prussian blue electrode; and (2) preparation of the reference electrode: conducting sputtering or metal evaporation to form a silver electrode, and immersing the silver electrode in a ferric chloride solution, such that some silver generates silver chloride through a chemical reaction, thereby obtaining a silver/chloride silver electrode; and a process for the micro/nanofabrication of the two-electrode system comprises: (1) preparation of the working electrode: conducting metal evaporation or sputtering by the micro/nanofabrication to obtain a nano-gold layer or a nano-platinum layer, and then conducting electroplating on a surface of the nano-gold layer or the nano-platinum layer to form a Prussian blue layer, thereby obtaining a gold/Prussian blue electrode or a platinum/Prussian blue electrode; and (2) preparation of the reference/counter electrode: conducting sputtering or metal evaporation to form a silver electrode, and immersing the silver electrode in a ferric chloride solution, such that some silver generates silver chloride through a chemical reaction, thereby obtaining a silver/chloride silver electrode.
3 . The method for improving stability of an electrochemical sensor according to claim 1 , wherein the process for screen-printing the electrode system is selected from the group consisting of: a process for the screen printing of the three-electrode system comprises: (1) preparation of the working electrode and the counter electrode: conducting screen printing with a gold composite paste, or a platinum composite paste, or a carbon composite paste that generally comprise comprising an electronic mediator selected from the group consisting of Prussian blue; and (2) preparation of the reference electrode: conducting screen printing with a silver/silver chloride composite paste; and a process for the screen printing of the two-electrode system comprises: (1) preparation of the working electrode: conducting screen printing with a gold composite paste, or a platinum composite paste, or a carbon composite paste; and (2) preparation of the reference/counter electrode: conducting screen printing with a silver/silver chloride composite paste.
4 . The method for improving stability of an electrochemical sensor according to claim 1 , wherein the biosensitive molecular enzyme is an enzyme or an enzyme mixture that is uricase for a uric acid sensor, or a mixture of creatinine amidohydrolase, creatine amidinohydrolase and sarcosine oxidase for a creatinine biosensor, or glucose oxidase for a glucose biosensor, or cholesterol oxidase for a cholesterol biosensor, or a mixture of lipase, glycerol kinase, and glycerol phosphate oxidase for a triglyceride biosensor.
5 . The method for improving stability of an electrochemical sensor according to claim 1 , wherein polyaniline (PANI) is added as an immobilization agent before immobilizing the biosensitive molecular enzyme.
6 . The method for improving stability of an electrochemical sensor according to claim 5 , wherein the protective film has a principal component of polyvinyl alcohol (PVA).
7 . The method for improving stability of an electrochemical sensor according to claim 1 , wherein glutaraldehyde is added as an immobilization agent.
8 . The method for improving stability of an electrochemical sensor according to claim 7 , wherein the protective film is a composite film prepared from PVA and polyethylene glycol (PEG).
9 . The method for improving stability of an electrochemical sensor according to claim 1 , wherein polyurethane is added after setting the immobilization agent.

Description

CROSS REFERENCE TO RELATED APPLICATION This patent application claims the benefit and priority of Chinese Patent Application No. 202210913635.5, filed with the China National Intellectual Property Administration on Jul. 29, 2022, the disclosure of which is incorporated by reference herein in its entirety as part of the present application. TECHNICAL FIELD The present disclosure relates to the technical field of electrochemical sensors, in particular to a method for improving stability of an electrochemical sensor. BACKGROUND Biosensors are composed of two main parts, namely biometric components and signal converters. The biometric component refers to a bioactive substance that has molecular recognition ability and can specifically react with a substance to be tested. The biometric component includes enzymes, antigens, antibodies, nucleic acids, cells, and tissues. The signal converter mainly converts a biometric function into a detectable signal. Currently, commonly used detection methods include electrochemical methods, optical methods, thermal methods, and mass analysis methods. The electrochemical method is the most ideal detection method. Electrochemical biosensors adopt a solid electrode as a basic electrode, and biosensitive molecules are immobilized on a surface of the basic electrode. Through the specific recognition between biomolecules, the biosensitive molecules can selectively recognize and capture target molecules on the surface of the basic electrode. The basic electrode acts as a signal conductor to derive a recognition reaction signal generated on its surface and convert into a measurable electrical signal, thereby achieving quantitative or qualitative analysis of an analyte. Various types of electrochemical biosensor can be formed by combining various biomolecules (such as antibodies, DNAs, enzymes, microorganisms, or whole cells) with electrochemical transducers (including current type, potential type, capacitive type, and conductivity type). According to different biosensitive molecules immobilized on an electrode surface, the electrochemical biosensors can be classified into electrochemical immunosensors, electrochemical DNA sensors, electrochemical enzyme sensors, electrochemical microbial sensors, and electrochemical tissue cell sensors. The electrochemical enzyme sensor refers to the indirect determination of a concentration of the analyte by recording changes through a transducer after the chemical change of biomolecules occurs under the catalysis of an immobilized enzyme. However, enzymes have a poor storage stability at room temperature, and the stability of enzymes during long-term storage is extremely important for the practical application of sensors. In order to improve the storage stability of enzymes at room temperature, this application proposes a method for improving stability of an electrochemical sensor. SUMMARY In order to solve the above problems, the present disclosure provides a method for improving stability of an electrochemical sensor. The foregoing technical objective of the present disclosure is achieved by the following technical solutions: the present disclosure provides a method for improving stability of an electrochemical sensor, including the following steps: S1, manufacturing an electrochemical sensor;S2, immobilizing a biosensitive molecular enzyme on a working electrode of the electrochemical sensor;S3, setting an immobilization agent on a surface of the biosensitive molecular enzyme; andS4, adding a protective film on a surface of the immobilization agent, such that the protective film is deposited on the working electrode to improve the stability of the electrochemical sensor. Further, a process for manufacturing the electrochemical sensor includes micro/nanofabrication and screen printing; the electrochemical sensor is a three-electrode system including a reference electrode, a working electrode, and a counter electrode, or a two-electrode system including the working electrode and the reference electrode, or a two-electrode system including the working electrode and the counter electrode; andthe working electrode is one or a combination of more selected from the group consisting of gold, platinum, and carbon. Further, a process for the micro/nanofabrication of the three-electrode system includes: (1) preparation of the working electrode and the counter electrode: conducting metal evaporation or sputtering by the micro/nanofabrication to obtain a nano-gold layer or a nano-platinum layer, and then conducting electroplating on a surface of the nano-gold layer or the nano-platinum layer to form a Prussian blue layer, thereby obtaining a gold/Prussian blue electrode or a platinum/Prussian blue electrode; and(2) preparation of the reference electrode: conducting sputtering or metal evaporation to form a silver electrode, and immersing the silver electrode in a ferric chloride solution, such that some silver generates silver chloride through a chemical reac