Summary:

Through the analysis and detection of biological macromolecules, we can understand the mechanism of biological life activities and obtain biochemical information in the life process, which is of great significance for clinical diagnosis and treatment of diseases. Electrochemical biosensors combine the high sensitivity of electrochemical sensors with the high specificity of biomolecular recognition and have the advantages of fast response, high sensitivity, and good selectivity. Miniaturization provides new tools and methods for rapid, sensitive, and efficient detection of these biomolecules. Electrochemical biosensors immobilize enzymes, nucleic acids, antigen antibodies, cells, and other biologically active substances in the limited space on the electrode surface through suitable immobilization methods, and produce biological and chemical reactions with the analyte without hindering the analyte. reaction. Diffused freely, producing an electrochemical signal for detection. Electrode and its surface immobilization technology determine the performance and application value of electrochemical biosensors. Electrodes play an important role in electrochemical reactions and electrical signal transmission. Electrode material, surface modification, and electrode size all greatly affect the detection capability of the electrode. Regardless of the immobilization method used, it is essential to maintain the activity of the biofunctional molecule and the stability of the sensor. Therefore, for electrochemical biosensors, the selection of substrate electrodes and the immobilization of biosensing elements on the electrode surface are the key points for sensor construction. The screen-printed carbon electrode (SPCE) is a single-use electrode with many advantages such as low price, simple and fast preparation, mass production, no need for pretreatment, and easy commercialization. Its use can not only make electrochemical biosensors simpler, and avoid cross-contamination of electrode surfaces, but also increase the possibility of instant detection.

SPCE can provide disposable chips for many applications, so it is receiving more and more attention. In recent years, SPCE has been widely used to develop novel electrochemical sensing platforms and improve sensor performance. In this thesis, several kinds of electrochemical biosensors with excellent performance were constructed mainly by using SPCE as the substrate electrode. Electrodes and their surface modifications, immobilization methods of six sensitive components, and three commonly used electrochemical analysis methods, followed by a review of the application of electrochemical biosensors based on screen-printed electrodes, and finally the background and research of this paper content. In Chapter 2, a simple, disposable electrochemical DNA sensor was constructed for highly sensitive amperometric detection of target DNA via gold nano-induced silver deposition. Using SPCE as the base, first, modify the polydopamine (PDA) film with active groups on the SPCE by electrochemical oxidation polymerization, and then under weak alkaline conditions, the amino-modified DNA probe (pDNA) is passed through a one-step method A sandwich-type DNA sensor was formed by covalently binding to PDA through a Schiff base reaction, and then immobilizing the target DNA and reporter DNA-functionalized (rDNA-) complex on pDNA through a hybridization reaction. Immobilizing on the surface of the electrode can induce the deposition of catalytic silver, amplify the stripping current signal of the deposited silver, and achieve high-sensitivity detection of the target DNA by detecting the amplified current signal.

The detection limit of the method proposed in this chapter can be as low as 0.3 pm, and the DNA sensor has good selectivity for non-complementary DNA. In the third chapter, a disposable electrochemical impedance RNA sensor based on DNA tetrahedral nanoprobes and enzyme-catalyzed signal amplification was prepared. First, a layer was electrochemically deposited on the SPCE substrate electrode, and then the pyramid-shaped DNA tetrahedral nanoprobes self-assembled on the electrode surface through the formation of thiols at three vertices and Au-S bonds. When there is a target, part of the stem-loop structure of the DNA probe hybridizes with the target, and the other part hybridizes with the biotin-labeled reporter DNA. Then, by binding avidin and biotin-labeled horseradish peroxidase (HRP), HRP is immobilized on the electrode surface and enzymatically amplifies the signal.  HRP can induce and catalyze the oxidation of 4-chloro-1-naphthol (CN) by hydrogen peroxide, resulting in the deposition of insoluble matter on the electrode, forming an insulating layer that hinders the transfer of electrons between the electrode and the electrolyte solution. The impedance is greatly increased, thereby realizing the signal amplification detection of RNA. Chapter 4 presents a simple and sensitive electrochemical detection method for phospholipase A2 (PLA2) based on liposomal nanoprobes encapsulated with methylene blue (MB) and single-use SPCE. Liposome nanoprobes were synthesized in one step by encapsulating MB in liposomes and then dropped onto the surface of SPCE simultaneously with the samples. Liposome nanoprobes encapsulated in lipid MB in vivo were released due to hydrolysis of the liposomal phospholipid bilayer by PLA2 in the sample.

Due to the strong adsorption of MB by carbon materials, the released MB was adsorbed and enriched on SPCE. After the electrodes were cleaned, square wave stripping voltammetry (SWV) was performed in PBS bottom solution, and the activity of PLA2 was detected according to the current signal. Therefore, naked SPCE detection of electroactive MB can indirectly detect PLA2 enzyme activity, which can be used for the determination of human serum samples. The method is simple to operate and is expected to be used for the determination of PLA2 in clinical and biological samples. Chapter 5 discusses a simple and sensitive method for the detection of PLA2 based on liposomal nanoprobes and glucometer readouts. The signal substance glucose is wrapped in the inner cavity of the phospholipid liposome vesicle to form a liposome probe, then the phospholipid bilayer membrane is cut off by the target PLA2 hydrolyzed phospholipid, the liposome is destroyed, and the signal substance is released with a blood glucose meter Glucose is detected, and the enzyme activity is determined according to the relationship between the reading signal of the blood glucose meter and the activity of PLA2. This method does not require labeling, is simple, fast, and low-cost, and is expected to be applied to POC detection.

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