Study On Glycoproteins Specific Recognition And Detection Method Based On Boronic Acid Affinity Magnetic Surface Imprinting And SERS

As an important class of biological molecules,the expression of glycoproteins in humans is closely related to the occurrence and development of a variety of cancers,immune deficiencies,and some diseases of old age.Effective monitoring of the expression levels of relevant glycoproteins in humans is of great medical importance and clinical value.However,glycoproteins in organisms not only have a wide variety,similar structure and low abundance,but also coexist with a large number of disruptors,which brings challenges to the in-depth study of glycoproteins.To improve the accuracy and rapidity in the detection process of real biological samples,there is a great need to develop enrichment means with excellent specificity and highly sensitive detection tools.In this study,two boronic acid modified surface enhanced Raman scattering（SERS）probes were prepared to label glycoproteins according to their characteristics;Single template and double template magnetic surface imprinting materials based on boric acid affinity were designed and prepared for efficient enrichment and separation of glycoproteins;On this basis,a boric acid affinity sandwich method based on SERS was constructed for highly sensitive detection of glycoproteins.The main research contents and results are as follows:1.Two novel boronic acid modified SERS probes,Au@Ag-MPBA and Au-MPBA@Ag-MPBA,were synthesized using the dual identity of 4-mercaptophenylboronic acid（MPBA）as both a Raman reporter and boronic acid,and gold core silver shell bimetallic as the enhancement substrate.The results show that the probe preparation method is simple and convenient with low cost,and the strong SERS signal can meet the requirements of portable Raman spectrometer,which can label all glycoproteins and has wide applicability.Meanwhile,it lays the foundation for the construction of antibody free immunosandwich method and highly sensitive detection of glycoproteins based on SERS.2.A boronic acid affinity magnetic imprinting material was designed and synthesized for the enrichment and separation of glycoproteins in complex matrices.First used Fe₃O₄-NH₂ as the core support material to immobilize glycoproteins after covalently grafting boronic acid via amide reaction,and then hydrolyzed using tetraethyl orthosilicate in an alkaline environment to form a thin SiO₂ imprinted layer.This imprinted material exhibits high binding affinity to target glycoproteins and excellent adsorption selectivity due to the synergistic effect of boronic acid with the imprinted cavity.Combined with the SERS probe,the SERS based boronic acid affinity sandwich assay was realized to detect horseradish peroxidase（HRP）with a wide linear range（0.1 ng/m L～1.0 mg/m L）,low detection limit（0.053 ng/m L）and excellent specificity.The universality of the method was verified by imprinting and detection of acid phosphatase and transferrin.Compared with the industry standard enzyme-linked immunosorbent assay,there was no significant difference in precision and accuracy.The use of portable Raman spectrometers so that this sandwich strategy can meet the requirements of point of care testing.In addition,this sandwich strategy exhibited significant advantages in terms of cost-effectiveness,stability,and speed of detection,enabling its application to the detection of target glycoproteins in serum samples with good clinical applicability.3.Since a disease usually has a variety of glycoprotein biomarkers,this study established a general strategy to prepare dual template magnetic molecularly imprinted polymers（D-MMIPs）to achieve simultaneous selective enrichment and separation of two glycoproteins.Firstly,a layer of SiO₂ was coated on the surface of Fe₃O₄ to increase the surface area of nuclear support materials and improve the dispersion and hydrophilicity of magnetic nanoparticles.Due to the class selectivity of boric acid to glycoproteins,4-formylphenylboric acid（FPBA）was introduced as a functional monomer to realize the simultaneous fixation of HRP and ovalbumin（OVA）.Then,a thin imprinted layer was formed by controlled polymerization of the copolymer of dopamine and 3-Aminophenylboric acid.The imprinted material has excellent adsorption performance and good selectivity.The adsorption capacities of OVA and HRP are 93.61 and 65.52 mg/g respectively.After repeated use for 7 times,the adsorption capacity decreases by only 6.8%,and the economic benefit is high.The boronic acid affinity sandwich strategy was established by combining D-MMIPs with the SERS probe to achieve separation and detection of the two glycoproteins with detection limits of 0.708 and 2.15 pg/m L for OVA and HRP,respectively.This sandwich strategy can be applied to the separation and detection of glycoproteins in real samples,promising a non immunological method for the combined detection of glycoprotein based disease markers.In this study,boronic acid affinity magnetic molecularly imprinted material was used as an alternative to antibodies,not only exhibiting comparable specificity and binding as antibodies,but also being stable,inexpensive,and having rapid separation ability than antibodies.In addition,the advantages of SERS over traditional detection methods are obvious,which,combined with the use of portable Raman spectrometers,allow on-site real-time detection.The SERS based boronic acid affinity sandwich strategy finally established in this study overcomes the deficiencies of traditional immunoassay,achieves the specific separation of glycoprotein disease markers with high sensitive detection,provides a reference for early screening and rapid diagnosis of related diseases such as cancer,and has a promising application in the field of precision medicine analysis.