Preparation Of Molecularly Imprinted Materials Based On β-cyclodextrin And Their Application In Protein Detection

Proteins are the basic building blocks of all tissues in living organisms,regulating and controlling almost all life activities,including metabolism,energy production,transport,apoptosis,and signal transduction.With the in-depth development of life sciences,proteomics,and disease diagnosis,the specific identification and quantitative detection of proteins have extremely important biological significance.Traditional protein assay methods are based on antigen-antibody recognition,but antibodies are unstable,expensive,and the use of strict conditions.Therefore,it is of great significance and value to study the synthetic antibody.Molecularly imprinted polymers(MIPs)are polymers with properties similar to antibodies,with imprinted cavities complementary to the shape and size of the target molecule.In the recent years,MIPs have attracted much attention owing to its advantages of facile synthesis,stable performance,and low cost.However,proteins imprinted polymers still have some disadvantages such as difficulty in obtaining some templates,complicated preparation,and low selectivity due to the particularity of the template.It is necessary to develop new imprinting strategies to prepare high-performance imprinted materials.β-cyclodextrin(β-CD)are cyclic oligosaccharides with a cone-like structure.β-CD has both the hydrophobic inner cavity and hydrophilic outer surface.Its cavity is moderate,so it can form stable inclusion complexes with various guest molecules such as azobenzene,ferrocene and adamantane.In view of the special property of “inner hydrophobicity and outer hydrophilicity” of β-CD,it has broad application prospects in the fields of separation and enrichment,biological detection and so on.Molecularly imprinted materials based on β-CD are often used for the identification of small organic molecules,and only a small number of imprinted materials are used for protein enrichment.For example,magnetic β-CD was used as to prepare MIPs for the recognition of bovine serum albumin,and the imprinting materials were prepared by self-assembly of the hydrophilic surface and hydrophobic cavity of acryloyl-modifiedβ-CD with lysozyme,which were used for the enrichment of lysozyme in serum.However,the reported imprinting materials based β-CD have disadvantages such as complex preparation,weak specificity,and poor adsorption performance.It is still need for further development of novel imprinted polymers based on β-CD for sensitive detection of specific protein in real samples.Among the imprinting methods of protein,oriented epitope imprinting is widely used due to its uniform recognition sites and high binding efficiency.The immobilization of epitope templates in oriented epitope imprinting is a critical factor.In recent years,some oriented epitope imprinting methods have emerged.For example,the boronic acid-functionalized substrates for immobilization of glycosylated epitopes and His-tag anchored epitope imprinting method can both be used to prepare oriented imprinted polymers.Although oriented epitope imprinted polymers have made some progress,it is still in its infancy,and there is an urgent need to explore a facile and general method for oriented epitope imprinting.Based on the above analysis,two kinds of molecularly imprinted materials based on β-CD were prepared in this paper,which achieved good molecular recognition on performance and the quantitative determination of transferrin(TRF)and neuron-specific enolase(NSE)in human serum.The concrete studies are as follows:1.Magnetic MIP(MMIP)for selective enrichment of TRF were prepared using β-CD.β-CD was functionalized with-SH and immobilized on the surface of magnetic particles through S-Au bonds.Then,the imprinted polymer was prepared by surface imprinting method using dopamine and m-aminophenylboronic acid.The hydrophilic imprinted layer was obtained after removing the templates.The obtained MMIP was combined with surface Enhanced Raman scattering(SERS)to convert the amount of TRF into Raman signal.The sensor had high sensitivity,wide linear range,and strong specificity,which was used to measure TRF in serum sample from patient with liver disease.2.We developed an oriented immobilization epitope imprinting method(host-guest anchored epitope imprinting).Firstly,C-terminal and N-terminal of protein were modified with azobenzene.Then the obtained epitope was immobilized on β-CD modified substrate based on host-guest interaction.Finally,C-terminal epitope imprinted glass plates and N-terminal epitope imprinted Raman-responsive Ag nanoparticles were prepared by the polymerization of various functional monomers.The prepared double MIPs formed a sandwich complex with the target protein,and the detection method based on MIPs-SERS was constructed.Compared with traditional immunoassays,this MIPs-SERS method had significant advantages such as small sample size,wide linear range,reusability,and low cost.It was used to determine the concentration of NSE in serum of patients with small cell lung cancer.The method can also be extended to other proteins,and has broad development prospects in the fields of protein determination and disease accurate diagnosis.