Preparation Of Metal-chelated Anchored Epitope Imprinting Materials And Their Adsorption Properties For Glycoproteins

Glycoproteins are common proteins in the human body and play important roles in molecular recognition,cell adhesion,immune response and signal transduction.Many glycoproteins are important biomarkers and therapeutic targets in medical diagnosis and play important roles in the diagnosis and treatment of cerebrovascular diseases,kidney diseases,cancers,as well as certain hereditary diseases.Therefore,the determination of glycoprotein content in the body is of great significance to the study of the occurrence and development of certain diseases.However,due to the low abundance of glycoproteins and severe matrix interference in complex biological samples,the detection of such biomolecules remains a great challenge.Therefore,samples need to be pretreated and glycoproteins need to be enriched before detection.Molecular imprinting technology（MIT）has attracted much attention for its specificity,cost-effectiveness and controllable structure among various recognition technologies for glycoproteins.The molecularly imprinted polymers（MIPs）obtained through MIT are known as artificial antibodies or plastic antibodies.Compared with natural antibodies,MIPs have the advantages of easy preparation,low cost,good stability,good reusability and tolerance to harsh conditions.For traditional protein imprinting,protein recognition is still challenging due to its large molecular size,complex three-dimensional structure and conformational instability in organic solvents.Epitope molecular imprinting technology（EMIT）is a promising tool for specific protein recognition.Unlike proteins,epitope templates do not have secondary and tertiary structures,so epitope is less sensitive to external environmental factors.Compared to whole protein templates,epitope templates are more stable during the imprinting process and can be easily obtained by artificial synthesis,even for rare proteins.In the present work,two kinds of epitope molecularly imprinted polymers（EMIPs）with immobilized epitope based on metal chelation were prepared and applied to the specific recognition of Ovalbumin（OVA）in egg white and Transferrin（TRF）in human serum.The specific research is as follows:1.Magnetic EMIP for OVA selective enrichment was prepared using caffeic acid（CA）as functional monomer.Using His-tagged ovalbumin Ig E binding epitope as template,the template was immobilized on Ni²⁺-modified magnetic material by metal chelation interaction between histidine and Ni²⁺.A novel polycaffeic acid coated epitope molecular imprinted polymer was prepared by the autooxidation of CA in the presence of hexamethylenediamine（HMDA）in the aerobic environment.The results of characterization and adsorption reflect that the prepared EMIP has good binding affinity and specificity for OVA,good reusability and good adsorption potential of glycoproteins in actual samples.2.Magnetic EMIP for selective TRF enrichment was prepared using norepinephrine（NE）and m-aminophenylboronic acid（APBA）as functional monomers.Using His-tagged transferrin C-terminal nonapeptide as template,a layer of imprinted coating with controllable thickness formed by the self-copolymerization of norepinephrine and m-aminophenylboronic acid in water was deposited on the substrate surface after the template was immobilized by metal chelating.After the template was removed by eluent,a three-dimensional cavity with complementary molecular shape of template was formed in the imprinted layer.The results show that the prepared epitope imprinted magnetic material has good adsorption performance for TRF,and it is actually applied to the enrichment of TRF in human serum.