Research On Spatiotemperal Microchip Electrophoresis System And Boronate Affinity Biomimetic Molecule Recognition

Proteins can interact with other molecules,which constructs the biological foundation for proteins to function and is of vital research significance.The interaction could be characterized through all kinds of instruments to give information on different levels,and meanwhile,various applications based on known interaction systems have been booming all the time,in which the most common are antibody,enzyme and lectin Amongst these researching methods,electrophoresis and chromatography are the most widely applied due to their functions of one-dimensional or multi-dimensional separation and purification,easy simulation of the environment for interactions to happen,and tandem or parallel with other detection formats.To study protein recognition by means of electrophoresis and chromatography,is to involve proteins and their interacting targets in the separation process.After comparing the grams with and without interactions and by theoretical calculations,thermodynamic parameters can be obtained.Usually in electrophoresis and chromatography,post-column single-point detection is applied,that is,the detector situated on the end the of separation column collects the signals of each analyte when passing the detector in orders,and then relect information such as immigration or retention time,peak shape,peak height,peak area and so forth on the time-axis one-dimensional grams.Because the interacting process occurs in the whole separation channel,the post-column single-point detection cannot collect the necessary information comprehensively and promptly.To completely monitor the interacting process in the whole separation channel,the detection format in electrophoresis and chromatography needs to be improved to expand the researching ability of electrophoresis and chromatography to give multi-dimensional information.And this is of vital importance in the field of protein study.Due to sensitivity to environment and easy loss of activity,many kinds of biomimetic materials are explored to replace natural proteins in harsh conditions.Protein glycosylation is one of the most common post-translational modification,which can affect the stability of proteins,binding force of proteins with receptors and other important functions of proteins.Boronate affinity-based materials,due to reversible and pH-controlled binding properties,have been widely applied in the field of protein glycosylation,especially for simulating antibodies and lectins to recognize proteins and sugars.Accordingly the major contents of this thesis are research on novel instrument and new biomimetic materials of protein interaction,including four parts as follows(1)Here we present the concept of spatio-temporally resolved detection(STRD)and a proof-of-the-concept microchip electrophoresis(MCE)-STRD system.The MCE-STRD system was mainly composed of a PDMS microchip,a linear light source and a CCD,which were specialized for spatio-temporal resolving capability by short-distance placement.A significant advantage of STRD over conventional detection schemes is its capability for monitoring the dynamic processes of molecular events occurring in the separation channel in both spatial and temporal resolutions.This was demonstrated through the monitoring of the dynamic processes of protein-DNA and protein-drug interactions in chip isoelectric focusing(chip IEF).The MCE-STRD system provided not only whole pictures of the entire dynamic processes just in a glance but also quantitative kinetic information(dissociation rate constants)of the dynamic processes.The STRD could provide multi-dimensional information and is a promising detection format.(2)A new method called boronate affinity-based oriented surface imprinting was proposed for facile preparation of glycoprotein-imprinted microplates.A template glycoprotein was first immobilized by a boronic acid-modified microplate through boronate affinity binding,and then a thin layer of polyaniline was formed to cover the microplate surface via in-water self-copolymerization.After the template was removed by an acidic solution,3D cavities that can rebind the template were fabricated on the microplate surface.The prepared MIPs exhibited several highly favorable features,including excellent specificity,widely applicable binding pH,superb tolerance for interference,high binding strength,fast equilibrium kinetics and reusability.The MIP-based ELISA method was finally applied to the analysis of AFP in human serum.The result was in good agreement with that by radioimmunoassay,showing promising prospect of the proposed method in clinic diagnostics.(3)We present a new format of enzyme activity assay that can effectively eliminate the effects of sample matrix.The key is a 96-well microplate modified with molecularly imprinted polymer(MIP)on the surrounding walls prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting.The prepared MIP exhibited strong affinity towards the template enzyme as well as superb tolerance for interference.Thus the enzyme molecules in complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay,which eliminated the interference from sample matrix.On the other hand,because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes,the enzyme captured by the MIP was directly used for activity assay.Thus additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided.Assay of ALP in human serum was successfully demonstrated,suggesting promising prospect of the proposed method in real-world applications.(4)A facile and novel method was proposed to establish virtual lectin array based on boronate affinity-based pH-featured encoding for discrimination of monosaccharides by pattern recognition.The dependence of boronate affinity on environmental pH was selected to encode each monosaccharides for feature generation,and the pH-featured encoding was used to construct the virtual lectin array.On the basis of the virtual array,pattern recognition algorithms were applied for data analysis.Monosaccharides were discriminated by principal component analysis and the relations in the virtual lectin array was unraveled by cluster analysis.In this proof-of-concept work,without time-consuming synthesis or preparation,the proposed method was successful in mimicking lectin array and discriminating nine elementary monosaccharides found in the nature,and it was also a new way in expanding the applications of boronate affinity-based materials and methods.