Magnetic Molecular Imprinting-based Construction Of Glycoprotein Electrochemical Sensor And Its Application In AFP And CEA Detection

Glycoprotein biomarkers are important for early diagnosis,treatment,and biomedical research of many diseases.It has been widely used in the clinical diagnosis,treatment,and efficacy evaluation of major diseases.However,low abundance glycoprotein biomarkers coexist with many high abundance protein molecules in the biological system,which brings some challenges to their detection.In this study,cheap ovalbumin（OVA）was used as the representative of glycoprotein.Two electrochemical sensing assays were developed and applied to the sensitive detection of the glycoprotein tumor markers alpha fetoprotein（AFP）and carcino embryonic antigen（CEA）.Molecularly imprinted polymers（MIPs）is a type of synthetic polymer with selective recognition of cavities,complementary in shape,size,and function to the template molecule.They have been widely used in the enrichment and separation of proteins.In this study,Fe₃O₄nanoparticles were used as the magnetic core,glycoprotein as the template molecule,tetraethoxysilane（TEOS）as the cross-linker,and Phenyltrimethoxy silane（PTMOS）as the functional monomer.The glycoprotein was firstly modified on the surface of Fe₃O₄nanoparticles in a targeted manner using the affinity interaction between the boronic acid group and the cis-diol structure on the sugar chain.Then,the magnetic molecularly imprinted nanoparticles（MMIPs）were produced by the sol-gel method.Due to the complementary form and hydrogen bonding between the target protein and the imprinted cavity on the nanoparticle surface,the nanoparticles can efficiently achieve specific capture and separation of glycoproteins.Based on this work,two indirect electrochemical detection methods for glycoproteins were established by combining electroactive substances with the MMIPs via the trapped proteins.Firstly,an indirect electrochemical detection method for glycoproteins was developed by chemical cross-link among glutaraldehyde and tyrosine and glycoproteins.An electroactive tyrosine was immobilized on the surface of the MMIPs nanoparticles.Indirect electrochemical detection of glycoproteins was achieved by measuring the oxidation current of tyrosine on the magnetic electrode surface.Using OVA as a representative of glycoprotein,the construction and testing conditions of the method were optimized.The experimental results showed that the oxidation current showed an excellent linear relationship with the logarithm of OVA concentration in the range of 1.0 ng/m L-500.0 ng/m L.The detection limit was 0.3 ng/m L.The method can be used to detect CEA in the concentration range of10.0 ng/m L-100.0 ng/m L.The detection limit was 3.3 ng/m L.The experimental results of the standard addition method showed that the recovery of CEA assay in human serum was between 90.0%and 102.0%.Secondly,an indirect electrochemical detection method for glycoproteins was developed by coordinating Hg²⁺with amino acids.Using the coordination interaction between Hg²⁺ions and amino groups,Hg²⁺was complexed onto the surface of the MMIPs nanoparticles.Electrochemical detection of glycoproteins was achieved by measuring the anodic solvation voltammetric current of Hg²⁺on the magnetic electrode surface.The construction and testing conditions of the method were also optimized using OVA as a representative of glycoproteins.A good linear relationship between logarithm of peak current and logarithm of concentration was observed for OVA concentrations in the range of 10.0pg/m L-100.0 ng/m L.The lower detection limit was 3.0 pg/m L.The method was used to detect AFP and CEA in the ranges of 10.0 pg/m L-10.0 ng/m L and 50.0 pg/m L-2.5 ng/m L.The detection limits for AFP and CEA were 3.0 pg/m L and 16.0 pg/m L,respectively.The recoveries in serum samples ranged from 92.0%to 129.0%,which proved that the method is sensitive and reliable.By comprehensive comparison,The two methods have many advantages,such as high specificity,good chemical stability,sensitive detection and so on.Therefore,both methods can be applied simultaneously to accurate realize electrochemical detection of glycoprotein biomarkers in practical applications.