Study Of Novel Phosphopeptides Biosensors Based On Nanomaterials

Phosphorylation is a ubiquitous post-translational modification of protein and polypeptides that regulates various cellular activities including cellular growth, division and signal transduction. The aberrant phosphorylation may lead to various diseases, such as cancer, leprosy and alzheimer. Therefore, the identification and quantification of phosphopeptides as well as the related enzyme activities are vital for the study of biological processes and the development of diagnostic and therapeutic for diseases. Electrochemical detection phosphorylation often involves two aspects: one is the analysis of kinase activity with which we could determine the degree of phosphorylation and screen related kinase inhibitor; another is the quantitative detection of phosphopeptides. In this thesis, we focus on the quantitation of phosphopeptides and proposed two novel electrochemical sensor systems basing on the coordination-chelation between metal oxide and phosphare groups to detect phosphopeptides quickly and accurately. The main researches are listed as below:1. Graphene-Ti O₂（RGO-Ti O₂） nanocomposites modified electrode for t he detection of phosphopeptidesGraphite Oxide（GO） was firstly prepared by the modified Hummers method, and the RGO-Ti O₂ nanocomposites were obtained by one-step hydrothermal method. With the RGO-Ti O₂ nanocomposites as a capture surface for phosphopeptides, potassium ferricyanide as electrochemical prote, we reported a simple but sensitive electrochemical method for the detection of phosphopeptides. From the experiment, before and after the adsorption of phosphopeptides, the peak current changed significantly, while the nophosphopeptides cannot cause this change. So, the proposed method might effectively distingush phosphopeptides from nophosphopeptides. And with the increase of concentration of P-peptides, the peak current decreases gradually. As a result, the decreased peak current is linear proportional to the logarithm of the concentration of P-peptides within the scope of 1⁵00 μM. The regression equation is I=89.676-20.150x（x is the logarithm of the concentration P-peptidss）, and the detection limit is 0.953 μM, with a linear coefficient 0.947（S/N=3）.2. Competitive approach to the electrochemical detection of phosphopeptides on a porous Zr O₂ thin film electrodeA competitive adsorption strategy has been designed to electrochemical ly detect phosphopeptides（P-peptides）. A porous Zr O₂ thin film electrodeposited by hydrogen bubble template method was used as a capture surface for P-peptides. Basing on the coordination-chelation between Zr O₂ and phosphate group, P-peptides could replace the electroactive Fc-peptides previously adsorbed on the modified electrode. The results reveal that the decreased peak current is linear proportional to the logarithm of th e concentration of P-peptides from 0.1 μM to 500 μM with a detection limit of 0.0934 μM（S/N=3）, showing the high sensitivity of the competitive biosensor. It is found that the adsorption of Fc-peptides on the modified electrode follows the first-order kinetics. The binding rate constant kb and the dissociation rate constant kd are calculated as 49.6 M^-1s^-1 and 1.97×10-3s^-1, respectively.