Preparation Of Functionalized Monolithic Cap Illary Column And Their Applications In Isolation And Enzyme Immobilization

Capillary monolithic column is a kind of new column prepared by in-situ polymerization method,with the advantages of simple preparation method,excellent mass transfer performance,uniform structure and good permeability,and it plays a crucial role in many fields.According to matrix materials,capillary monolithic columns can be divided into organic polymer monolithic columns,inorganic silica monolithic columns and organic-inorganic hybrid monolithic columns.Although monolithic columns is widely used,there are still many problems existed.For example,instability and functional monomers have greatly restricted the sustainab le development of monolithic columns.Therefore,it is very necessary to find suitable polymer functional monomers.The purpose of this paper is to develop new types of capillary monolithic column,and then to rich the diversity of the research and application of monolithic column.1.1,3,5-trisacryloylhexahydro-1,3,5-triazine?TAT?is a chemical crosslinking agent with high reactivity and can be used to produce high crosslinking patterns.Based on this,we used TAT as a cross-linking agent and methacrylic acid?MA?as a hydrophilic functional monomer.After optimization,a monolithic column functionalized with uniform structure and high stability was prepared.Then the separation performance of the monolithic column was evaluated.The results showed that the monolithic column can achieve baseline separation for polar molecules such as amides,bases,nucleosides,and anilines.2.We prepared a monolithic column with strong hydrophilicity.The monolithic column used ethylene glycol dimethacrylate?EDMA?and dimethylaminoethyl methacrylate?DMAEMA?as cross-linking agents,and bromoacetic acid as functional monomer.After optimization,the monolithic column obtained has homogeneous structure,good permeability,large specific surface area and strong hydrophili city,and it can separate many substances,such as amides,bases,nucleosides,and phenols.The minimum height of the plate was 9.04?m.The number of theoretical plates was100548 N/m.In addition,the monolithic column also performed well in the separati on of modified nucleosides and environmental pollutants chlorophenols.This work provides a certain idea for development of new and excellent monolithic columns.3.Large-scale enzyme discovery and drug discovery requires simple,rapid and sensitive screening platforms.We developed a new automated multil-functional dual enzyme detection platform based on the functionalized green fluorescent proteins?GFPs?.These functionl GFPs are composed of 3 parts,a GFP protein,a his-tagged at its N-terminal,and enzyme-specific substrate recognition sequences between GFP and the tag.They are immobilized on the monolithic column through his-tag/Ni²⁺interaction.After the enzyme digestion,GFP was released and the fluorescence signal is determined by capillary electrophoresis-laser induced fluorescence?CE-LIF?.With2 GFPs containg different charges and enzyme recognition sites,two enzymes activity can be detected.The label-free platform enables simultaneous online detection of thrombin and Matrix metalloproteinase-2?MMP-2?.