| The immunoassay for the detection of protein plays a very important role in life sciences. At present, detection methods such as RIA, ELISA, Western Blot play important roles in protein detection. But, With the development of the life sciences, the disadvantage of the routine methods has already been revealed which made them limited in some fields requiring high sensitivity such as early diagnosisof diseases. Then, it is very necessary to study the new analysis methods of high sensitivity, simple operation and universal applicability. Obviously, the enhancement of immunoassay signal is very important to improve the sensitivity of immunosensor. Electrochemical apparatus gain more and more attention because they were simple, sensitive and easy to be minisized. So it is undoubted that the study of new electrochemical immunoassay with signal enhancement can accelerate the development of immunoassay. Therefore, this research focuses on the enhancement of the signal of electrochemical immunoassay. This research consists of three following parts:In the first part, a novel electrosensor based on aptamer recognization and biocatalytic metal deposition were proposed for the sensitive detection of platelet-derived growth factor BB (PDGF-BB). In this method, the capture antibody was immobilized onto the electrode first, then, antigen and biotinylated Aptamer were dropped onto the electrode to form sandwich immunocomplex. Following with a avidin-biotin reaction, ALP which can initiate the hydrolysis of ascorbic acid 2-phosphate(AA-p) to produce ascorbic acid was immobilized onto the gold electrode. The later, in turn, reduces silver ions on the electrode surface, leading to deposition of the metal onto the protein modified electrode surface. The deposited silver metal was detected by linear sweep voltammetry (LSV). Based on the high sensitivity of biocatalytic metal deposition, this method showed a good linearity wich the concentration of PDGF in the range of 1 to 1000 ng/mL with a detection limit of 0. 8ng/mL.In the second part, an electrochemical immunasensor has been developed based on functionlized si nanoparticles (Si NPs) for the detection of prostate specificity antigen (PSA). In this method, Si NPs functionlized with detection antibody and alkaline phosphatase (ALP) were for the first time been synthesized and used in electroimmunoassay for the detection of PDGF-BB. The capture antibody was first immobilized onto a gold electrode via a self-assembled layer. Then, ALP was bound to the electrode through a sandwich immunoreaction. Following with the catalytic hydrolysis of AA-p conduced by ALP, silver ions was reduced and deposited on the electrode. The amount of silver deposited on the electrode was proportional to the amount of antigen captured on the electrode. For his method used Si NPs as enzyme carrier can enhanced the sensitivity of the immunosensor dramatically, a linear relationship between antigen and peak current was obtained in the range of 1 to 35 ng/mL with a detection limit of 0.76ng/mL.In the third part, an ultrasensitive immunosensor based on enzyme-encapsulating liposome and biocatalytic metal deposition for the detection of PSA was described. The feasibility of the proposed immunoassay method was investigated using a sandwich immunoassay format with human PSA as the analyte. Detection antibody, antigen and detection antibody modified liposome were dropped onto the gold electrode in turn to form immunocomplex. Then, Bound liposomes were lysed with surfactant to release the encapsulated ALP which was used to initiate the deposition of silver nanoparticles on the electrode. The utilization of the liposome as enzyme carrier and high catalysis activity of enzyme enhanced the sensitivity in immunoassay. this method showed a good linearity with the concentrations of PDGF in the range of 0.01 to 100 ng/mL with a detection limit of 0. 007ng/mL. |
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