Fabrication And Application Of Two Ferrocene Probes In QCM Biosensor Assembly And Regeneration Research

Quartz crystal microbalance(QCM) biosensor based on biotin-avidin system has many advantages compared to other biosensors. QCM has the characteristics of non-labled property and real-time online detection. Biotin can modify a variety of biological molecules and can combine with avidin at fast speed, these make this kind of biosensor be widely used in medicine, food safety and environmental monitoring fields. But quartz wafer has high cost, and it is difficult to compare the results of different wafers because the physical parameters and reaction sites of each biosensor chip are different. So achieving the regeneration of biosensor is impending. At the same time, the monitoring methods in the process of biosensor preparation are not intuitive, therefore new methods are essential to research.Figure1 Biosensor preparation and regeneration schematic diagram.In this thesis,biosensor fabrication and regeneration were achieved from Au-S and biotin-avidin bond. Because of strong electrochemical signal of ferrocene, thiol-labeled and biotin-labeled ferrocene probes were designed and assembled to the gold surface. The assembly and regeneration process were monitored by electrochemical method. At last, experiment of amplifying S.aureus DNA by QCM/LAMP coupling technique was carried out to verify the biosensor regeneration methods.1. Biotin-labeled ferrocene probe Bio-Fc and thiol-labeled ferrocene probe HS-Fc were designed, synthesized and characterized by IR, MS, 1H NMR and 13 C NMR.Figure 2 Synthetic route of ferrocene probe Bio-Fc and HS-Fc.2. HS-Fc modified gold electrode disk was prepared by self-assembly method, and was desorbed by reductive desorption, UV/O3, Piranha, and pickling method(HNO3/HCl/H2O). The experimental results from electrochemical workstation, water contact angle tester and atomic force microscopy showed that HS-Fc modified electrode could be desorbed and regenerated successfully by reductive desorption and UV/O3 method, and partly via Piranha and pickling method. The impedance values of the electrode treatment with reductive desorption had rules to follow, that could help us to predict the experiment result. HS-Fc modified gold electrode could be regenerated at least 9 times by reductive desorption method, 8 times by UV/O3 method.3. Ferrocene modified biosensor was prepared through thiol octylic acid and cysteamine method, and results showed that sensor prepared by cysteamine method had better reproducibility. At the same time, the sensor regeneration was achieved by SDS solution washing and thermal destruction method. The experimental process was characterized by electrochemical workstation, water contact angle tester and atomic force microscopy.4. The experiment of S.aureus DNA amplification by QCM/LAMP coupling technique was carried out to verify the biosensor regeneration methods. The results showed that Au-S bond could be regenerated at least two times by reductive desorption method and at least one time by UV/O3 method; biotin-avidin bond could be regenerated by SDS solution washing and thermal destruction method.