Preparation And Application Of Phosphate Group-targeted Molecularly Imprinted Materials

Phosphate groups are widely present in non-living and living forms,and in some drug molecules,the introduction of phosphate groups can well improve the water solubility of drug molecules.In living organisms,phosphate groups are involved in the composition of structures such as nucleic acids and cytoskeletons.The reversible phosphorylation of tyrosines present in proteins during cell signaling plays a key regulatory role in eukaryotic life activities such as cell growth control,cell cycle regulation,gene transcription,apoptosis,motility and metabolism.Therefore,a new method of molecular imprinting technique targeting phosphate groups was developed in this thesis.Molecular imprinting is a bionic recognition technique,using which molecularly imprinted polymers（MIPs）are prepared to specifically recognize target molecules.Oriented surface molecular imprinting is an emerging molecular imprinting technique based on the modification of functional groups on carrier particles,where the template molecules are immobilized on the surface of the carrier particles by binding to the functional groups through interaction force,and on this basis the imprinted layer is polymerized and the template molecules are eluted to obtain MIPs.The epitope imprinting strategy is a new approach based on the oriented surface molecular imprinting technique to identify intact target proteins by imprinting a short section of signature sequence.Magnetic nanoparticles have magnetic separation properties and biocompatibility and are widely used in biological research.In this thesis,by modifying the magnetic nanoparticles with a layer of TiO₂functional groups,the substances with phosphate groups are fixed on the surface of magnetic nanoparticles in a directional manner through electrostatic interaction,and then an imprinted layer is formed in the presence of functional monomer 3-aminopropyltriethoxysilane（UPTES）and cross-linking agent ethyl orthosilicate（TEOS）,and the template molecules elute to obtain MIPs.The MIPs have specific imprinted cavities,which are adapted to the template molecules in shape size and spatial structure,so the MIPs have adsorption specificity for the template molecules.MIPs have specific adsorption properties to the template molecules.Meanwhile,during the adsorption process,the titanium ions in the imprinted cavities bind the phosphate groups of the template molecules through electrostatic interaction,which enhances the adsorption ability of MIPs to the template molecules.The work in this thesis focuses on three aspects.1.Using oriented surface imprinting strategy with the small molecule substance cytidine monophosphate（CMP）as the template molecule,the targeted imprinting conditions were optimized,and it was clear that TiO₂ modification on the surface of magnetic nanoparticles and washing away of excess template molecules after binding of the template to TiO₂ could improve the adsorption capacity of MIPs.The adsorption capacity was enhanced by 2.92-fold after TiO₂ modification on the magnetic nanoparticle surface and 0.70-fold after washing away the excess template molecules after binding the template to TiO₂.2.The above optimized imprinting strategy was applied to prepare dexamethasone sodium phosphate MIPs,and the morphology of MIPs was characterized by transmission electron microscopy（TEM）,and the MIPs were spherical with core-shell structure.The ratio of silylation reagents（TEOS:UPTES）in the preparation of MIPs was optimized,and the effect of imprinting time on the adsorption performance of MIPs was investigated.The highest imprinting factor（IF）of 4.44 was achieved when TEOS:UPTES=9.4:0.6 and the imprinting time was 45 min.The MIPs showed only a 7.31%decrease in enrichment efficiency after five repeated uses,indicating its good reusability,and also only a 13.8%decrease in enrichment efficiency after six months of storage,indicating its good stability.Finally,the enrichment efficiency of MIPs in real samples was tested,and the enrichment efficiency was up to 74.5%when dexamethasone sodium phosphate standards were added to urine and then enriched with MIPs.3.The MIPs were prepared using the epitope phenylphosphonic acid（PPA）,an epitope of tyrosine phosphopeptide,as the template using an epitope imprinting strategy.The MIPs have good selective specificity and can selectively enrich tyrosine phosphopeptide from mixed standard peptide solutions（Tyrosine phosphopeptide:Serine phosphopeptide:Threonine phosphopeptide=1:1:1）.In conclusion,this thesis focuses on the enrichment of phosphate group substances and combines molecular imprinting techniques with magnetic nanoparticles to prepare phosphate group-targeted molecularly imprinted materials,providing a new research method for the selective enrichment and identification of phosphorylated substances.