Manufacture And Application Of Electrochemical Biosensor Based On Nanoporous Gold Electrode

Electrochemical biosensor possesses the advantages of specific recognition of biological molecular, biocompatibility and amplification of nanoparticals and response enhancement of electrochemical detection. It has become the main stream of modern biochemical analysis with high sensitivity, good selectivity as well as being easy to miniaturization and automation.This thesis studied a novel electrochemical biosensor based on nanoporous gold electrode and nanoprobe and its application in assay of C-reactive protein (CRP), hepatitis B surface antigen (HBs Ag), K562 cells and P-gp on the membrane of K562 cells. Chemiluminesece was carried out based on isoluminol chemiluminescent nanoprobe to detect the concentration of GSH and free thiols in K562 cells and achieved satisfying results . This thesis mainly consists of the following issues:1 An electrochemical immunosensor based on NPG electrode with HRP-label antibody-AuNPs bioconjugates for highly sensitive detection of protein was established. As a platform model, measurements of CRP were performed in the range of 0.01 to 0.5 ng mL-1 with detection limit of 6.85 pg/mL. The presence of AuNPs enhanced the immobilized amount of HRP-anti-CRP, which improved the sensitivity of the immunoassay. This ultrasensitive protocol promising the determination of CRP with minimum sample amount, and the dilute of the sample can reduce even eliminate the disturbances of other proteins in serum to improve the selectivity of the immunoassay of CRP.2 A new electrochemical biosensor was built based on nanoporous gold (NPG) electrode and HRP labeled antibody modified Au nanopaticles to detect the concentration of hepatitis B surface antigen (HBs Ag) in the serum through sandwich-type immunoassay. The big effective surface area of the NPG electrode and good biocompatibility and amplification of Au nanoparticles enhanced the volume of HRP labeled antibody, causing high sensitivity of the target protein. Results showed that he linear range of HBs Ag was from 0.01 to 1.0 ng/mL and the detection limit was 2.3 pg/mL. The results of some human serum samples showed that the new method compared with the ELISA method very well. What is more, this method is 100 times more sensitive than the ELISA analysis indicating that this method could be used to detect the hepatitis B surface antigen in human serum samples with accuracy, simplicity and sensitiveness.3 A novel electrochemical cytosensor was designed based on the specific recognition of mannosyl on cell surface to concanavilin A (ConA) immobilized on the NPG electrode. The incubation with P-gp monoclonal antibody nanoprobe introduced CdS NPs onto the cell surface immobilized on the modified electrode. The cell number and the amount of P-glycoprotein (P-gp) on cell membrane were quantified by electrochemical stripping analysis of the cadmium dissolved from CdS NPs on the cell surface. Since a single Au NP could be loaded with hundreds of CdS NPs through the DNA linker, a significant amplification for the detection of target cell was obtained. With the use of K562 leukemic cells (K562 cells) as a model, the electrochemical response was proportional to the logarithm of cell concentration in the range from 1.0×102 to 1.0×105 cells mL-1, showing very high sensitivity. The signal amplification could be further used to evaluate the P-gp on the cell surface, and there are about 3.144×103 P-gp molecules on a single living K562 cell. The amount of P-gp on the K562/ADM cell membrane was ten times of that on the K562 cell. This strategy presents a promising platform for highly sensitive cytosensing and convenient evaluation of surface P-gp on living cells.4 A new strategy of photoelectrochemical (PEC) analysis with light source free for the determination of physiological thiols in cells was conducted for the first time. A chemiluminescence (CL) probe prepared in the present method with disulfide bonds could be cleaved by the thiols. The isoluminol labeled polystyrene microspheres (PSMs) cleaved from the the CL probes was collected for the CL reaction to release the luminescence as exciting light for the PEC detection. With GSH as a model, a working curve was constructed for GSH determination in the range of 1.0×10-10 to 1.0×10-8 M and the detection limit (3σ) was 42 pM. The proposed method was successfully applied to the extracts of Ramos cell for intracellular thiols detection.