| Fe3O4 have great potential in biomagnetic separation and other fields due to their unique properties such as superparamagnetism,high specific surface area,low toxicity,and easy separation under external magnetic field.Magnetic separation has the advantages of time saving,gentleness,and easy automation.Simple application of an external magnetic field removes target compounds directly from crude media.However,in practical applications at present,it is difficult for magnetic nanoparticles to maintain dispersion and stability in complex environments,and the binding efficiency with targets is low.At the same time,low magnetic response sensitivity and self-aggregation caused by large material sizes affect the application of biological separation..Therefore,how to develop Fe3O4 magnetic nanoparticles with simple preparation method,obvious magnetic response behavior and high binding efficiency is of great research value.The synthesis of magnetosome nanoparticles by magnetotactic bacteria through biomineralization in nature has given important inspiration.As an important organelle of magnetotactic bacteria,magnetosomes are composed of phospholipid bilayers on the outer layer and inorganic ferric oxide nanoparticles inside.Compared with iron oxide magnetic materials synthesized by chemical methods,the naturally formed magnetic small Bulk nanoparticles have a more uniform morphology and have multiple advantages in magnetic properties,solution stability,and biocompatibility.The biomineralization process of magnetosomes in magnetotactic bacteria depends on the coordinated regulation of related membrane proteins,from ion transport to crystal nucleation and growth,to form magnetic nanoparticles with complete crystal forms and uniform sizes.At the same time,related core biomineralization proteins have unique structural features and unique sequences,which endow magnetosome nanoparticles with excellent biocompatibility,solution environment stability and monodispersity,and the surface is easy to functionalize.An ideal protein template for the synthesis of magnetic nanoparticles.Therefore,how to use biomineralized protein as a template to regulate and synthesize nanocrystalline particles from the bottom up is of great research value.It is noteworthy that this protein template biomimetic mineralization synthesis method is simple and repeatable,environmentally friendly and harmless,and the synthesized nanoparticles have good biocompatibility and magnetic properties.Based on the above theories,this project selected two representative mineralized proteins,Magnetosome membrane special protein 6(Mms6)and Bovine Serum Albumin(BSA),as templates.Through two different biomimetic synthesis techniques,they explored and obtained minerals with small particle size and high magnetic properties.Iron oxide nanoparticles,used in the field of magnetic separation.The C-terminus of the Mms6 protein of magnetotactic bacteria is rich in hydrophilic acidic amino acids,which play an important role in the bionucleation of magnetosome nanocrystals.BSA is a cage-like protein with broad prospects.Its sequence contains a variety of functional amino acids,such as amino,carboxyl,and sulfhydryl groups.These groups can provide specific binding sites for metal ions,forming a protein cage structure,which is beneficial to nanoparticles.Mineralization regulation.In the first part,the Mms6 protein of magnetotactic bacteria is used to construct a reverse micelle microcapsule system to simulate the magnetosome vesicle system inside magnetotactic bacteria,and biomimetic synthesis of magnetosome-like nanoparticles.The reverse micelle system can effectively control the size of the crystals and significantly improve the biological function of the Mms6 protein in regulating the magnetosomes.The magnetosomes obtained by this method have the crystal morphology similar to the magnetosome nanoparticles of magnetotactic bacteria.And magnetic properties,can effectively identify the external magnetic field.Moreover,after hydrophilization,the magnetosomes have excellent monodispersity and water solubility in aqueous solution.In the in vivo experiments of tumor mice,they have obvious magnetic targeting ability and biological safety.Magnetosome nanoparticles have expanded a broad space in the application of biomagnetic separation.The second part,development and synthesizes ultra-small magnetosome-like nanoparticles(≤10 nm)based on the Mms6 protein template of magnetotactic bacteria.The surface is functionalized by using hydrophilic carboxylated polyethylene glycol(mPEG2000-COOH),then,conjugated to an antibody to the COVID-19 receptor-binding domain(RBD).The resulting magnetosome-like immunomagnetic beads(Mal-IMB)exhibit high magnetic responsiveness comparable to commercial magnetic beads,with a saturation magnetization of 90.6 emu/g.Furthermore,their smaller particle size offers a significant advantage by providing a higher specific surface area,allowing conjugation of higher numbers of RBD single-chain fragment variable(RBD-scFv)antibodies,thereby enhancing immune capture capacity and efficiency.To verify the practicability of Mal-IMBs,we evaluated their performance in recognizing RBD antigens,achieving a maximum capture capacity of 83 μg/mg per unit mass.Furthermore,we demonstrated the binding ability of Mal-IMB to SARS-CoV-2 pseudoviruses using fluorescence microscopy.Mal-IMB effectively enriches pseudoviruses at a low copy concentration of 70 copies/mL,and the ultra-small Mal-IMB exhibits excellent magnetic responsiveness and binding efficiency.By employing a multi-site virus binding mechanism,the enrichment and isolation of SARS-CoV-2 in complex environments has been significantly improved,which facilitates the rapid detection of COVID-19 and helps to take effective measures to prevent its spread.In the third part,the biomineralization protein BSA group was selected as the template for the synthesis of magnetic nanoparticles,and the Fe3O4 nanocrystal and microstructure were successfully regulated,and a high-quality magnetic carbon-based adsorption material with nano-network and ultra-thin nano-layer structure was obtained,BSA The carbon nanolayer formed by protein not only improves the stability and adsorption efficiency of the material,but also has good magnetic responsiveness.BSA protein has a rich and delicate multidimensional structure and many functional groups that bind metal ions.Using it as a template can effectively promote the synthesis of magnetic metal oxides and form a multidimensional network structure.After annealing treatment,the coated BSA protein film finally formed a special protein nanolayer(PNL)structure,which showed a high removal ability for Cu(Ⅱ)and Cr(Ⅵ)in sewage through adsorption and reduction.At the same time,the excellent paramagnetism of Fe3O4@PNL can be easily separated from sewage by an external magnet.This part of the work allows us to further understand and expand the idea of protein templates in the preparation of magnetic carbon-based adsorption materials,and broaden the application range for magnetic separation.These works provide a practical way for the high-level application of magnetic nanoparticles,and provide different research ideas for understanding the mechanism of biomineralization proteins at the organic/inorganic interface. |
