A New Method For Reversed-phase Microemulsion Orient Surface Imprinting And Its Application In Phosphorylated Peptide Enrichment

PURPOSEMolecular imprinting(MIT)involves the formation of selective sites with template memory in the polymer matrix.In recent years,molecularly imprinted polymers(MIPs)have attracted wide attention due to their required selectivity,physical stability,thermal stability,and low cost.They are widely used in solid phase extraction,chemical sensors and artificial antibodies.Wait for many fields.With its rapid development as a research hotspot,it faces many challenges at the same time,involving biological macromolecular imprinting,binding site heterogeneity,template leakage,incompatibility with aqueous media,low binding ability and slow mass transfer.This limits its application in various aspects,especially the cumbersome steps and inefficiencies of the existing molecular imprinting techniques.In response to this problem,we have developed a new high-efficiency molecular imprinting preparation strategy called: we use the inverse nanoemulsion to provide what we call "nano-reactor" synthetic silicon-coated magnetic nanoparticles and oriented surface imprinting This method is called Reversed Phase Microemulsion Oriented Surface Imprinting(RPM-OSI).Moreover,this strategy can be extended to other imprinting systems.The versatility of this imprinting method is shown,and the emergence of this method provides a new idea for the rapid and efficient preparation of molecularly imprinted materials.METHODFirst,we selected the appropriate functional monomers to synthesize MIPs with adenosine monophosphate(AMP)as imprinting template by RMP-OSI method and Bulk Imprinted respectively.We used AMP imprinted polymer to adsorb AMP solution,and then selected the appropriate eluent.After elution,the absorbance at 264nm(AMP has the largest UV absorption peak at 264 nm),a series of characterization of synthetic AMP-imprinted SENMPs(Silica-Encapsulated Nanomagnetic Particles),and optimization of this new synthesis method.This was followed by a series of comparisons with Bulk imprinted AMP imprinted SENMPs,including cross-reactivity(selecting seven compounds with similar molecular structure to the template)to demonstrate the superiority of the new imprinting method.Secondly,since phosphorylated proteins have great biological significance in organisms,their abundance is too low.Enrichment is a common practice before detection.Molecular imprinting technology is a new enrichment method developed at present.It is also the current research hotspot,aiming at the hot spot of phosphorylated protein detection.We use RMP-OSI method and phosphate as template to synthesize the corresponding imprinted materials,and optimize this method accordingly.The phosphorylated peptide was specifically enriched and combined with matrix-assisted laser desorption Time-Of-Flight mass spectrometry(MALDI-TOF MS)to establish an off-line combination platform for efficient identification of protein phosphorylation.Finally,in eukaryotes,the residues that undergo protein phosphorylation are serine(Ser),threonine(Thr)and tyrosine(Tyr),and the phosphorylation ratio on Ser,Thr and Tyr residues is 1800:200:11.Therefore,it is necessary to selectively enrich the pTyr peptide prior to MS analysis.In order to further expand the application range of RMP-OSI imprinting method,we synthesized the corresponding imprinted silica-coated nano-magnetic particles SENMPs with phenylphosphonic acid(PPA)as template.The tyrosine phosphorylated peptide was specifically enriched for MALDI-TOF MS detection.RESULTSWe optimized the RMP-OSI imprinting method,and the imprinting effect was mainly expressed by the imprinting factor(IF)value.We have found through a series of experiments that the best imprinting conditions are three kinds of silylating reagents in the proportion of TEOS: APBA-GPTES: APTES For 9.8:0.2:0.2,the molar ratio of template molecule to APTES is 1:1.Later,we compared this new method with the traditional Bulk imprint.The former obtained the cross-reactivity of the imprinted material is much lower than the traditional imprinting method,which proves that it has good anti-interference and selectivity,and it is "one.The advantage of the pot method is that it greatly simplifies the experimental steps.Then we modified the SENMPs of the phosphate-imprinted ones in the same way(changing their functional monomers),and optimized the synthesis process.The optimum silylation reagent was added in the proportion of TEOS: UPTES=9.8:0.2,template phosphate The amount of addition is 1:1 with the silicon source UPTES molar ratio to the functional monomer.Then,the ?-casein(?-casein)after trypsin digestion was adsorbed by PO4-imprint SENMPs,and it was found to have excellent adsorption performance to the phosphorylated peptide.Finally,we further expanded the application range of this imprinting method,and synthesized PPA-imprinted SENMPs with phenylphosphonic acid as imprinting template,and enriched tyrosine phosphorylated peptides,because only the template was replaced during the synthesis,other functions such as The monomer and silylation reagents are the same as the synthesis process of the phosphate-imprinted polymer,so the best results are directly optimized by phosphate blotting.