Study And Application Of Protein Imprinting Technique Based On Magnetic Nanoparticles

Molecular imprinted technology (MIT) is a kind of technique used for thesynthesis of tailor-made recognition materials by forming a polymer network aroundtemplate molecules. Compared with natural receptors, molecularly imprinted polymershas advantages like low cost, easy to be synthetic, high stability, reusability, and so on.Therefore it has attracted more and more attention of researchers．So far, molecularimprinted technique with small molecules (such as traditional Chinese medicine(TCM), metal ions, amino acids, etc.) as templates has made significant progress. Butthe imprinting technique of bio-macromolecules particularly protein is still not mature,and developed relatively slow. This is mainly decided by the nature of proteins, such aslarge size, complicated structure and flexible conformation.In this study, modified ferroferric oxide nanoparticles were used as carriers. Withbovine hemoglobin as template, surface imprinting technique was adopted in this work.We investigated the performance of the molecularly imprinted polymers by adsorptionexperiments, and attempted to use it in the real sample. Specifically, this paper mainlyincludes the following contents:(1) The preparation of magnetic molecularly imprinted nanoparticles for therecognition of bovine hemoglobinWith acrylamide and itaconic acid as functional monomers, we prepared bovinehemoglobin (BHb) imprinted nanoparticles on vinyl modified Fe3O4particles using thesurface imprinted technique. The composition and morphology of the resultantmagnetic molecularly imprinted nanoparticles were investigated by Fourier transforminfrared spectrometry (FT-IR), X-ray diffraction (XRD), field emission scanningelectron microscope (FESEM), thermogravimetric anaLyzis (TGA), vibrating samplemagnetometer (VSM). The radio of AAm/IA was investigated and finally we chose thebest recipe:200mg of Fe3O4@MPS,32mg AAm,0.544mg IA,32mg BHb to carryout the subsequent research. The results of the following experiments showed that theadsorption capacity of BHb-imprinted polymers which prepared with the best recipewas as high as77.6mg/g, IF was3.2to4.1, equilibrium adsorption time was2.5h, andit showed an excellent selectivity towards the template protein BHb. In addition, thesaturated magnetic intensity of Fe3O4@MIPs was32.35emu g-1. It means the paticlescan be separated from the reaction medium quickly under the reaction of an externalmagnetic field. All of these results showed that this method may be applicable to the selective separation of target protein in real samples.(2) The synthesis of bovine hemoglobin imprinted polymer on amino-modified magnetic nanoparticles and its recognition performance studyWith acrylamide and itaconic acid as functional monomers, amino-modifiedmagnetic nanoparticles as carriers, we prepared bovine hemoglobin (BHb) imprintednanoparticles and investigated its adsorption performance. The experimental resultsshowed that we successfully formed imprinting cavities on the surface ofBHb-imprinted particles through the polymerization process. The equilibriumadsorption time of BHb-imprinted particles was5h, the saturated adsorption capacityof the template protein can reach176.2mg/g, which is very considerable. The result ofselective adsorption experiment revealed that the BHb-imprinted particles had smalladsorption capabilities of bovine serum albumin (BSA) and ovalbumin (OVA), butshowed a relatively high adsorption capacity of Lysozyme (Lyz), there are two maincauses for this. Firstly, the size of Lyz is far less than that of bovine hemoglobin;Secondly, the isoelectric point of Lyz is greater than6.2, so Lyz is positively chargedat pH6.2. Since MIP nanoparticles were negatively charged, the electrostatic forcebetween Lyz and the carboxyl group of the nanoparticles chains is impetus. These tworeasons make Lyz easy to distribute into the imprinted cavities. Based on the aboveresults, the imprinted particles may be used to separate a series of proteins whoseisoelectric point is greater than6.2.(3) The preparation of magnetic bovine hemoglobin imprinted polymer byatom transfer radical polymerization and its recognition performance studyA new type of magnetic BHb-imprinted polymer was prepared by atom transferradical polymerization, with using N-isopropyl acrylamide, acrylamide andα-methylacrylic acid as functional monomers, initiator modified Fe3O4particles ascarriers. We synthesized BHb-imprinted polymers in different pH buffer solutions,namely4.0,5.4,6.8,8.2. And we investigated the adsorption performance of theBHb-imprinted nanoparticles prepared in the optimal pH buffer solution throughthermodynamics adsorption experiment, dynamic adsorption experiment and selectiveadsorption experiment. The results showed that the adsorption capacity ofBHb-imprinted polymers prepared at pH4.0was found to be39.9mg/g. The Langmuirmodel was employed to represent the rebinding isotherms, the fitting results show thatthe theoretical adsorption capacity of MIPs is43.08mg/g. Dynamic adsorptionexperiments showed that the equilibrium adsorption time of MIPs was about4h. Inselective adsorption experiment, MIPs showed an excellent selectivity towards the template protein BHb, IF was1.81. We can know, BHb-imprinted polymer prepared byatom transfer radical polymerization has a good recognition performance of templateprotein BHb, this is of a certain reference significance for bovine hemoglobinimprinting technology.