Preparation Of Novel Monolithic Capillary Columns And Their Applications In Capillary Electrochromatography

Capillary Electrochromatography(CEC) is a new micro-separation technique developed in recent years. It has opened up a new way to efficient micro-column separation technique. CEC has been considered as one of the core technologies in nano/micro-separation field, that combines high mass transfer and excellent separation performance, and has attracted the attention of researchers. Up to date, it has been successfully applied in almost all field of life science, including proteomics,metabolomics and pharmaceutical botany etc. Monolithic column is the core of CEC.At present, research works on CEC are mainly focused on both the preparation and application. Though there are many reports on hybrid monolithic column, there still exist some bottle-necks in CEC methodology that seriously limit the application and development of CEC. In this thesis, we are focusing on the preparation of new stationary of CEC, and diversification of monolith technology.1)Based on trimethoxysilylpropanethiol(MPTS)hybrid monolithic columns,post-modification preparation of carboxyl hybrid monolithic column bonded with itaconic acid(IA)was developed. Firstly, MPTS hybrid monolithic column with active thiols was made by the sol-gel reaction of MPTS and TMOS,then itaconic acid was bonded to the monolith via thiol-ene click chemistry to gain the carboxyl hybrid monolithic column. The experimental results show that MPTS-IA hybrid monolith column exhibits high column efficiency, phenols, anilines, and bases could be successfully separated on the column by CEC. This work provided a new way to prepare diverse monolithic column through post-modification. Meanwhile, the column could be a good basement for modifying new material or protein.2)Nowadays, large-scale screening for enzyme discovery, engineering and drug discovery process requires simple, fast and sensitive enzyme activity assay platforms with high integration and potential for high-throughput detection. Herein, a novel Automatic and Integrated Micro-Enzyme Assay(AI?EA) platform was proposed based on a unique micro-reaction system fabricated by a engineered green fluorescence protein(GFP)-functionalized monolithic capillary column, with thrombin as an example. The recombinant GFP probe was rationally engineered to possess a His-tag and a specific substrate sequence of thrombin, which enable it to be immobilized on the monolith via metal affinity binding, and be released afterenzymatic digestion of thrombin. Furthermore, when substituting the thrombin with other enzyme, this strategy could be conveniently extended for detection of other enzymes.3)By taking advantage of the ultrahigh loading capacity of the AI?EA platform and the CE automatic programming set-up, one micro-reaction column was sufficient for many times digestion without replacement. Combined with capillary electrophoresis-laser induced fluorescence(CE-LIF), all the procedures including thrombin injection, on-line enzymatic digestion in the micro-reaction system, and label-free detection of the released GFP were integrated in a single electrophoretic process. The novel micro-reaction system showed significantly enhanced catalytic efficiency, about 30 fold higher than that of the equivalent bulk reaction. Accordingly,the AI?EA platform was highly sensitive with the limit of detection(LOD) down to 1p M of thrombin, about 3 orders of magnitude over the traditional affinity CE-based thrombin assay. Moreover, the AI?EA platform was robust and reliable to detect thrombin in human serum samples and its inhibition by hirudin. Hence, this AI?EA platform exhibits great potential for high-throughput analysis in future biological application, disease diagnostics and drug screening.