Label-free Detection Of Biomolecular Interaction By Oblique-incidence Reflectivity Difference Method

Biological macromolecules research is one of the most important topics in life science.So far fluorescent label detection still plays a pivotal role in the field of biologicalmacromolecules interactions. However, with the development of the bio-chip technology,label detection shows some deficiencies. The tagging process is costly and cumbersome, andthe marker may affect the structure and even the function of the target. Surface PlasmaResonance and Ellipsometry have also been used to label-free detection of molecularinteractions, but they cannot meet the current needs in life science due to their throughputlimits. Therefore, exploring and developing techniques of label-free detection of bio-chips isstill one of the most crucial steps for the life Science breakthroughs. In this dissertation, weuse a new method, which is called Oblique-Incidence Reflectivity Difference, for label-freeand high throughput detection of bio-chips and real-time monitoring the interaction dynamicsof biomolecules. The main experimental results are as follows:1. Bio-chip prepared with Rabbit IgG solution with different concentrations wasdetected by OIRD method and clear2D image was obtained, suggesting that this method canlabel-free distinguish the concentrations of molecules while label method can not make it.2. Specific interaction between Rabbit IgG with three different concentrations and GoatAnti-Rabbit IgG based on bio-chip was detected by OIRD and fluorescent label methodsrespectively. OIRD results show perfect consistency with that of fluorescent label method,indicating the feasibility of OIRD method to detect bio-chips.3. Detecting each step of fabricating rabbit IgG chip with300spots and specifcinteraction between Rabbit IgG chip and Goat Anti-Rabbit IgG was successfully conductedby using OIRD method, demonstrating the feasibility of OIRD method in tracking detectionof bio-chips fabricating and interaction processes.4. Bio-molecule microarrays with10,000biological samples were detected within200minutes with OIRD method, suggesting the OIRD method can be used to label-free and high-throughput detection of bio-chips;5. Real-time monitoring specific interactions between Human IgG and GoatAnti-Human IgG, Rabbit IgG and Anti-Rabbit IgG, and Mouse IgG and Goat Anti-Mouse IgGusing OIRD method was carried out. Binding reaction time and the corresponding reactionkinetic curves were obtained. These results revealed the feasibility of OIRD method inreal-time monitoing dynamics processes of biomolecular interactions.6. Biomolecular interactions between Human IgG and Goat Anti-Human IgG, RabbitIgG and Anti-Rabbit IgG, and Mouse IgG and Goat Anti-Mouse IgG, each of them containsthree different concentrations, were successfully real-time monitored with OIRD method. Thekinetic curves corresponding to different concentrations were all obtained. This paves theway for further studying and obtaining reaction kinetics parameters.The above results demonstrate the feasibility of OIRD method in label-free andhigh-throughput detecting bio-chips and real-time monitoring biomolecular intecteractionsprocesses. Therefore, OIRD has very promising applications in the field of life science...