Studies On Synthesis And Adsorption Properties Of Magnetic Fe₃O₄-protein Imprinted Polymers

Highly specific protein recognition is one of the most important research topics in life science,which also provides technical basis for works such as proteomics,diagnose and protein isolation and purification.In order to realize highly specific protein recognition,in recent years,a lot of work has been done to develop highly selective adsorption materials.Among which magnetic nanoparticles with selective adsorption ability and wide range of application,has been gaining more and more attention.The selective adsorption of magnetic nanoparticles was realized by surface modification with specific ligands?e.g.antibody and aptamer,etc?.And tedious and long-time multi-steps were often involved for the preparation.In the meantime,those ligands are expensive and difficult to achieve,and the stability and suitability of those ligands hindered further application.Molecularly imprinting technology or MIT can produce artificial receptor with the characteristics of selective adsorption.Due to its low preparation cost,high chemical/thermal stability and wide range of application,molecularly imprinted polymers are the most possible alternative for natural counterparts.The combination of magnetic separation and protein imprinting to construct magnetic protein imprinted compisites for selective adsorption is gaining more and more attention.But current magnetic protein imprinting studies are hindered by issues such as tedious preparation steps,low magnetic response,long binding equilibrium time,low binding capacity and poor binding selectivity.So it is important to develop facile and effective methods to achieve selective adsorption of protein with magnetic protein imprinted composites.Accordingly,four types of magnetic protein imprinting composites were prepared in this thesis and the adsorption properties were evaluated.The sections are listed as below:1.In the preface Chapter,molecular imprinting theory,classification,preparation methods and application were discussed in detail.Novel MIPs with magnetic property and other properties were analyzed.And the development of protein imprinting was focused,which provide the base for further protein imprinting research,and imply the significance of protein imprinting.2.In Chapter 2,Fe₃O₄ nanoparticles were synthesized with different methods,and solvothermal synthesis was chosen as the proper method for our research purpose,which can produce carboxyl functinalized magnetic nanoparticles with one step,and the final product with diameter of about 200 nm,could be well dispersed in water with saturated magnetism of 67.06 emu/g,a very high magnetism which guarantees strong magnetic response.Simplified one step functionalization with polymerizable double bond was achieved for later surface imprinting.Lysozyme?Lyz?was chosen as template,acrylamide?AAm?and N,N'-methylene-bisacrlamide?MBA?were used as functinal monomer and crosslinker,respectively,polymerization were initialize after pre-polymerization step to achieve magnetic protein surface imprinted composites.The saturatin magnetism density was up to 40 emu/g,and fast magnetic separation was presented within 30 seconds,which means a good magnetic response property.Binding experiments were carried out to reveal that the protein imprinting polymers had fast binding kinetics,with only 10 min to achieve binding equilibrium.Binding capacity is 29.3 mg/g,and imprinting factor?IF?is 1.88.Binding kinetics models analysis showed that the adsorption fit well with pseudo-second-order kinetics model.And binding isotherm analysis demonstrated that the adsorption isotherm fit well with Langmuir model,which means homogeneous binding sites on the imprinting polymer surface.Binding selectivity experiments results showed that the selectivity coefficiency for comparative protein BSA and for OVA is 1.69 and 1.73,respectively,which demonstrated the binding selectivity of the prepared imprinting composites.Competitive binding experiments were carried out and results showed the binding specification.Finally,real egg white sample were prepared for adsorption,and results showed specific recognition towards target protein Lyz with quick magnetic separation.Through optimized surface functionalization step and controlled imprinting layer thickness,the as-prepared magnetic protein imprinted composites with strong magnetic response core can be further applied in biosamples for target protein magnetic separation.3.In Chapter 3,template protein was directed restricted onto the surface of carboxyl functionlized Fe₃O₄ magnetic nanoparticles to prepare novel magntic protein imprinted composites.Template protein Lyz was directed restricted onto the surface of carboxyl functionlized Fe₃O₄ magnetic nanoparticles through electrostatic interaction and hydrogen bonding,later acrylamide imprinting polymerization were carried out by free radical initialization to get surface imprinting layer coated on the Fe₃O₄ nanoparticles.The saturatin magnetism density of the magnetic protein imprinted composites was increased to55 emu/g,fast magnetic separation was presented and a good magnetic response was achieved.The introduction of electrostatic interaction and hydrogen bond by carboxyl group into acrylamine imprinting system can enhance imprinting effect.Binding experiments revealed that adsorption equilibrium was reached after 30 min and binding capacity is 52 mg/g.The binding kinetics data fit well with pseudo-second-order kinetics model.Binding isotherm data fit well with Langmuir model.The selective adsorption experiments results showed a better effect.Competitive binding experiments further showed specific recognition ability.Thus with this method,the as prepared magnetic protein imprinted composites demonstrated an enhanced imprinting effect and had potential for practical application.4.In Chapter 4,carboxyl functionlized Fe₃O₄ magnetic nanoparticles were introduced in sol-gel protein imprinting system as doped functional monomer.In this process,Lyz was chosen as template molecule,APTES as functional monomer and TEOS as crosslinker,respectively,carboxyl group functionalized Fe₃O₄ nanoparticles were doped into this pre-polymerization system.And magnetic protein imprinted composites were achieved by sol-gel process.Carboxyl group functionalized Fe₃O₄ nanoparticles act with two roles:one as magnetic response material,the other as functional co-monomer to provide carboxyl group for protein binding,and an improved imprinting effect for protein recognition is expected.The saturatin magnetism density of the as-prepared magnetic protein imprinted composites was up to 10.80 emu/g,and magnetic separation was presented.Binding experiments showed that the binding kinetics were improved,binding equilibrium were reached after 30 min,binding capacity is 91.74 mg/g,and IF is 8.86.Binding selectivity experimental results showed that the SC for both two comparative proteins is beyond 4,which means a good binding selectivity.Competitive binding experiment results demonstrated the specific recognition.In real sample adsorption experiments,the as-prepared magnetic protein imprinted composites can selectively separate target Lyz from egg white sample,and demonstrated the merit of large binding capacity.Thus the magnetic protein imprinted composites prepared with this method have magnetic separation ability and very good binding selectivity towards target protein with wide range of application.5.In Chapter 5,template protein directed restricted onto the surface of carboxyl functionlized Fe₃O₄ magnetic nanoparticles with sol-gel imprinting polymerization to synthesize magnetic protein imprinted composited was presented.Template protein Lyz was directed restricted onto the surface of carboxyl functionlized Fe₃O₄ magnetic nanoparticles through electrostatic interaction and hydrogen bonding,later sol-gel surface imprinting polymerization with APTES and TEOS as functional monomer and crosslinker respectively,were carried out to get imprinting layer coated on the Fe₃O₄ nanoparticles.The saturatin magnetism density of the prepared magnetic protein imprinted composites was up to 43 emu/g,and fast magnetic separation was presented,which means a good magnetic response.The introduction of electrostatic interaction and hydrongen bond into sol-gel process imprinting can enhance imprinting effect.Binding experiments revealed that 80%adsorption equilibrium was reached within 10 min and after 30 min adsorption equilibrium was reached,which means a good binding kinetics.Binding capacity is 43.9 mg/g and IF was 4.99.Binding selectivity experiments demonstrated the good selectivity for the as-prepared imprinting composites.Competitive binding experiments further showed specific recognition ability.In real sample adsorption experiments,the as-prepared magnetic protein imprinted composites can selectively adsorb Lyz in egg white samples.Thus the magnetic protein imprinted composites prepared with this method demonstrated fast magnetic separation ability with good binding selectivity towards target protein,and are worth of application.