Label-free Detection Of Some Bcl-2 Family Proteins

Modern analytical chemistry has now gone into a so-called stage of analytical science and monitoring in life activity process and detection of biomacromolecules are one of important research directions. Apoptosis is a programmed cell-death process. Drug-resistance of tumor cells mediated by apoptosis regulating factors is one of widely approved mechanisms. Bcl-2 family proteins are important regulators of cell apoptosis and their expression change is closely related to drug-resistance of tumor cells. The detection of Bcl-2 proteins and Bax proteins, which are all members of Bcl-2 protein family, was performed by using three kinds of label-free methods in this thesis. Some classic labels in biological detection including fluorescence dye molecules, fluorescence proteins and radioactive isotopes were not involved in the relevant research process that displayed such characteristic as simplicity and easy operability. The presented work may widen the application range of modern analytical chemistry in life science researches. The main contents are listed as follows:1. Nanocomposites consisted of Au NPs and Bcl-2 monoclonal antibodies and the glassy carbon electrode modified with Bcl-2 proteins were successfully prepared and characterized, respectively. A sensitive method for the quantitative detection of tumor cells was developed and the investigation on the difference of Bcl-2 protein expression on human breast cancer MCF-7 cells and adriamycin-resistant human breast cancer MCF-7 cells(MCF-7/ADR) was performed based on the competitive immunoreaction using electrochemical AC impedance spectroscopy. Under the optimal conditions, the charge-transfer resistance change(?Rct) was proportional to the logarithm of the cells concentration from 5?102 to 1.6?106 cell m L-1 with a detection limit of 170 cell m L-1. The expression of Bcl-2 protein on MCF-7/ADR cells was found to be significantly higher than that on MCF-7 cells.2. Ferrocene-chitosan composite(Fc-CS) gel with excellent electrochemical activity and biocompatibility was prepared and it was electrodeposited on the glassy carbon electrode(GCE) surface. The GCE modified with the electrochemical probe and the antibodies was then obtained by immobilizing Bax monoclonal antibodies using gold nanoparticles(Au NPs). The specific reaction between Bax proteins and their antibodies could inhibit the electron transfer between the electrode and electrolyte solution due to the steric hindrance effect, resulting in the decrease of the peak current in DPV measurement. Under the optimal conditions, the peak current change(?I) displayed a linear response in the range of the Bax protein concentration(c Bax) from 0.5-500 ng m L-1 with a detection limit of 0.15 ng m L-1. So a simple, sensitive, and label-free electrochemical method for the detection of Bax proteins was developed.3. Poly(neutral red) was prepared on the quartz crystal microbalance(QCM) gold electrode by electrochemical polymerization and Bax monoclonal antibodies was immobilized using glutaraldehyde. A simple, sensitive and label-free piezoelectric method for the detection of Bax proteins was developed based on the specific reaction between antigens and antibodies by measuring the frequency change of quartz crystal in air before and after immunoreaction.