Development And Applications Of Boronate Affinity-Anchored Epitope Controllable Oriented Surface Imprinting

As the material basis of life,protein is the main undertaker of life activities and participates in almost all processes of life.The study of protein structure and function will directly clarify the mechanism of life under physiological or pathological conditions,and is one of the most challenging areas of life science research in the post-genome era.However,many proteins with important physiological functions and clinical value are low in content,complex in structure and dynamic.At the same time,biological samples are complex in composition and there are a large number of interfering substances.Therefore,the specific recognition and separation of target proteins have become a bottleneck in protein research.Classical protein research methods are based on antigen-antibody recognition,and have played an important role in protein isolation,purification,recognition and detection.However,the antibodies used are mainly derived from animal immunization,and suffer from complex preparation processes,high costs,long screening cycles,and sometimes even unavailable.In addition,the stability and reproducibility of antibodies are also problematic.Therefore,finding alternatives to antibodies is not only of great scientific significance,but also of important application value.Molecularly imprinted polymers(MIPs)are chemically synthesized materials that mimic the recognition of antigens by antibodies.MIPs have antibody-like binding properties to the template molecule due to the presence of nanoimprinting cavities that are highly complementary to the shape,size and functional groups of template molecule.MIPs are simple to prepare,inexpensive,more stable than antibodies,and can tolerate various pH,solvents and temperatures.In recent years,many molecular imprinting techniques have emerged for imprinting of proteins.In particular,the boronate affinity imprinting method developed by our group has effectively solved the imprinting of cis-diol-containing compounds such as glycoproteins and glycopeptides.It has the advantages of simplicity,versatility and high efficiency,and has shown good application prospects in the fields of disease diagnosis,biological imaging and single cell analysis.However,this method is only applicable to the imprinting of glycoproteins and glycopeptides,but not to non-glycoproteins and non-glycopeptides.Based on the previously established boronate affinity controllable-oriented surface imprinting method in our group,two controllable imprinting methods for various protein types(including non-glycoprotein,non-glycoprotein,glycoprotein and glycoprotein)have been successfully developed by introducing epitope imprinting technology.The validation of several proteins proves that the proposed method is efficient,convenient and universal.The prepared MIPs have been successfully applied to the specific recognition of proteins,disease diagnosis of protein biomarkers,and targeted recognition and imaging analysis of tumor cells.Firstly,we proposed a boronate affinity-anchored epitope controllable oriented surface imprinting for imprinting of protein.The C-terminal nonapeptide of a protein was selected as an epitope.A lysine is introduced at C-terminal of the epitope,and then glycated with fructose.The glycated epitope template can be easily immobilized on the boronic acid functionalized substrates.A variety of silylating reagents that interact with the epitope sequence for polymerization were selected as monomers,which not only provides multiple interactions and improves affinity,but also can accurately control the thickness of the imprinted layer by adjusting the imprinting time.The prepared MIPs have the advantages of high specificity,strong affinity,fast mass transfer rate and high imprinting efficiency,and can specifically bind to the target protein and its epitope in complex samples.Therefore,the proposed protein imprinting method is simple,versatile and efficient,and has broad application prospects in protein recognition and biomedical applications.Secondly,we developed dual MIP-based plasmonic immunosandwich assay(duMIP-PISA)for the detection of protein biomarkers in complex biological samples with high specificity and ultrasensitivity.We first extended the boronate affinity-anchored epitope controllable oriented surface imprinting to the imprinting of N-terminal epitopes.Then C-terminal and N-terminal epitopes of a protein biomarker were glycated as templates to prepare C-terminal epitope-imprinted gold nanoparticles self-assembled monolayer coated-glass slide as a plasmonic substrate for specific extraction of target protein and N-terminal epitope-imprinted Raman-responsive Ag@SiO2 nanoparticles as nanotags for specific labeling of captured protein,respectively.The formed MIP-protein-MIP sandwich-like complexes could produce significantly enhanced surface-enhanced Raman scattering(SERS)signal.The dual MIP-based recognitions ensured high specificity of the assay,while SERS detection provided ultrahigh sensitivity.The duMIP-PISA has successfully detected neuron-specific enolase(NSE)in human serums,and the difference in expression levels of NSE can be used to distinguish small cell lung cancer patients from healthy individuals.As compared with traditional immunoassay,the duMIP-PISA exhibited multiple merits,including simpler procedure,faster speed,lower sample volume requirement and wider linear range,and can be easily modified and extended to other protein biomarkers.Therefore,the duMIP-PISA will play an important role in the fields of disease diagnosis,biochemical research and signaling pathway study.Finally,we propose boronate affinity-anchored epitope controllable oriented surface dual imprinting for the preparation of protein imprinted polymers with higher specificity and stronger affinity.The C-terminal or N-terminal dodecapeptide of a protein was selected as an epitope and glycated,and the glycated epitope is immobilized on boronic acid functionalized substrates by boronate affinity interaction.The first nine amino acid sequences were carried out first oriented imprinting through the polymerization of various silylating reagents,and the resulting imprinted cavity has multiple interactions with the epitope peptides,thereby producing high affinity.Then the last three amino acid sequences were carried out second oriented imprinting through the polymerization of tetraethyl orthosilicate,and the formed imprinted layer covered the non-specific adsorption sites produced by the first oriented imprinting outside the imprinted cavity,thus reducing the non-specific adsorption on the non-imprinted surface.Through the traditional single-imprinting method,the highest imprinting factor was 6.2,and the lowest dissociation constant was 10-7 M.As a comparison,through the dual-imprinting method,the highest imprinting factor was improved to 16.6,and the lowest dissociation constant was reduced to 10-9 M,and affinity is two orders of magnitude higher than the traditional single-imprinting method.According to the properties of amino acids,the imprinting conditions optimized for C-terminal or N-terminal epitope of the protein have proved to be equally applicable to the imprinting of other proteins.The prepared MIPs exhibit excellent specificity at both peptide and protein levels.Meanwhile,the prepared molecularly imprinted fluorescent nanoparticles achieved the targeted recognition and imaging analysis of transferrin receptor on the surface of tumor cells.Therefore,this method provides more advanced molecular imprinting materials for biomimetic recognition of proteins,and is expected to be widely used in biomedicine.