Study Of Novel Methods For Phosphorylation And Dephosphorylation Detection

Phosphorylation and dephosphorylation is the key to the cascade signal amplification system in cells. A series of exponential phosphorylation may be induced to answer the physical or chemical signals outside cells, which can further cause the expression of some specific genes. Accordingly, some functional proteins are thus activated. Later, the phosphorylated proteins are dephosphorylated so that the cells can return to normal state. Protein kinase and phosphatase are the main enzymes which catalyze phosphorylation and dephosphorylation, respectively. Abnormal regulation of these enzymes may lead to many serious diseases. In this dissertation, we have proposed two novel electrochemical systems to monitor phosphorylation and dephosphorylation. Compared with the traditional methods, the proposed electrochemical methods offer the advantages of high sensitivity, better selectivity and low cost for the detection. The methods may also be of utility for medical application in the future.1. An electrochemical strategy for monitoring phosphorylation based on rolling circle amplification.A novel electrochemical method to monitor protein phosphorylation and to assay protein kinase activity based on rolling circle amplification is proposed. Firstly, substrate peptide immobilized on a gold electrode can be phosphorylated by protein kinase A. Then, Zr4+ links phosphorylated peptide and DNA primer probe by interacting with the phosphate groups. After the introduction of the template probe and phi29 DNA polymerase, rolling circle amplification is achieved on the surface of the electrode. As the product, a very long DNA strand, may absorb a large number of electrochemical speices, hexaammineruthenium(Ⅲ) chloride, via the electrostatic interaction, localizing them onto the electrode surface, initiated by protein kinase A, a sensitive electrochemical method to assay the enzyme activity is proposed. This method may be a good candidate for monitoring phosphorylation in the future.2. A novel electrochemical method for the detection of dephosphorylation by alkaline phosphatase.A novel electrochemical method for the detection of dephosphorylation by alkaline phosphatase is developed. Two complementary DNA probes (DNA 1 and DNA 2) andλexonuclease are employed. Firstly, the 5'-phosphoryl end of DNA 1 is dephosphorylated by alkaline phosphatase. Then DNA 1 hybridizes with DNA 2, previously modified on a gold electrode surface. In this double-strand DNA, DNA 2 will be promptly cleaved byλexonuclease with its phosphoryl at the 5'end. After the DNA 2 strand is completely digested, DNA 1 will be released from the double strands and then hybridizes with another DNA 2 strand on the electrode surface, thus the cycle of the digestion of DNA 2 and the release of DNA 1 continues. Since the DNA probes may absorb electrochemical species, the removal of DNA 2 strand from the electrode surface, initially activated by alkaline phosphatase, will result in the decrease of the detected electrochemical signal. A sensitive biosensor is thus achieved. The detection of the enzymatic activity in complex biological fluids is also realized. So, the proposed method might become a promising approach for alkaline phosphatase assay.