Detection Method Of Molecular Interaction On The Surface Of Mammalian Cell Based On SPR Sensing

Molecular biological research in vitro provides the essential information on structure and function of DNA and protein. However, in order to comprehensively understand the life as well as physiological or pathological processes, it is necessary to expand molecular biological study in vitro to that on cells, tissues or organs, and eventually on individual. It has been gradually evolved as a"hot-spot"in the field of biomedical research and apparently, one of the major challenges is to develop novel experimental tools to meet the demands of real-time monitoring the bio-molecular process on living cells with high-sensitivity and label-free or limited labeling techniques. This thesis combined SPR sensing and molecular detection on living cells, and attempted to explore its research possibility for cell molecular biology and drug developing. The results are as follow.Theoretic feasibility of molecular detection on living cells by SPR sensing was analyzed. The correlations between the wavelength of laser and the propagation length of SPW as well as its penetration depth were obtained by numerical stimulation upon Maxwell equations and Fresnel formula. Two models of the cellular molecular reaction process sensed by SPR were developed. Analysis confirmed the possibility of SPR sensing monitoring these processes theoretically.The procedure of in vivo detecting molecular interaction on phage M13 by SPR sensing was explored. Phage M13 with 12-polypeptides displayed on its surface was used to prepare the phage-displayed protein chip and the real time process of interaction between this 12-polypeptides and the corresponding antibody was monitored. The results showed that SPR sensing could detect this molecular reaction, suggesting a possibility of further preparing the living cell chip for specific purpose of cellular molecular research.Model system of SPR sensing monitoring the interaction between membrane protein EGFR on the surface of human gastric cancer cell line BGC823 and its antibody EGFR1 was selected. On a modified experimental apparatus of SPR sensing, the cell chips with both living and fixed BGC823 cells were prepared and the interaction of EGFR and its antibody EGFR1 was monitored by SPR sensing in real time. Compared with the results detected by using immunofluorescence, the capability of SPR sensing detecting the molecular interaction on living cells was confirmed. The detection values corresponding to different responses were obtained by goat-anti-rabbit IgG and EGFR1 interacting with living BGC823 cells respectively, which suggested the specificity of EGFR1's binding. Different cellular responses associated with different concentrations of EGFR1 were also obtained. However, there was a certain admixture between the molecular binding and cellular response in the detection signal. A conception about two-wavelength SPR for layered detection was proposed to further address this question in future.In summary, SPR sensing can be used for detecting the molecular interactions on living cells and the results shown here provides a good basis of experimental procedure for further study. It is also suggested that SPR sensing may potentially be developed as a practical technique for cell detection in future.