Preparation And Characterization Of Lysozyme Molecularly Imprinted Polymers On The Surface Of Carbon Nanotubes

Molecular imprinting technique（MIT）,deriving from imitation of the interaction between antigens and antibody,is a novel technique to prepare polymers with specific recognition capacity to template molecules by artificial synthesis methods.Compared with biological counterparts,molecular imprinting polymers（MIPs）have significant advantages,including conformation predetermination,specific recognition,mechanical/chemical stability,ease of preparation,re-usability and low cost.Surface imprinting technique is a new method which is achieved by attaching the template-monomer complexes on the surface of a substrate to form MIPs thin film by polymerization reaction between monomers and cross-linking agent.As a consequence,the imprinting binding sites are located at the surface of the polymer layer,which solves the problems of deep embedding of the imprinting sites,poor imprinting effect,and restricted mass transfer among protein MIPs.In this work,the lysozyme（Lyz）imprinted polymers were prepared through surface molecular imprinting technique using Lyz as template,acrylamide modified carbon nanotubes as substrate,1-allyl-3-methylimidazolium hexafluorophosphate（[AMIM][PF₆]）,1-allyl-3-methylimidazoliumtrifluoromethanesulfonate（[AMIM][CF₃SO₃]）,acrylamide（AAM）,N-isopropylamide（NIPAM）and N-ter-butylacrylamide（TBAM）as functional monomers,NNMBA as crosslinking agent,ammonium persulfate（AP）as initiator and N,N,N’,N’-tetramethyl ethylene diamine（TEMED）as catalyst respectively.The influence of the synthesis conditions on the imprinting performance were studied,and the associated traits of the polymers,including specific surface area,surface morphology,thermal stability,adsorption thermodynamics,imprinting effect and selective adsorption capacity were evaluated.The main research contents are as follows:1.The Lyz-MIPs were prepared on the surface of carbon nanotubes by surface imprinting technique using TBAM,TBAM/AAM,TBAM/[AMIM][PF₆],[AMIM][PF₆],[AMIM][PF₆]/NIPAM,[AMIM][CF₃SO₃]as functional monomers under pH 7 PBS buffer,respectively.The results indicated that the MIPs developed by both single monomer and mixed monomers had no remarkable recognition ability for Lyz although the contents of functional monomers and cross-linking agent have been optimized in detail.This is maybe attributed to the weak interaction between Lyz and the functional monomers and the instability of the template-monomer complexes under pH 7 PBS buffer.2.The Lyz-MIPs were prepared by surface imprinting technique using polar[AMIM][PF₆]as functional monomer under pH 8.9 Tris-HCl buffer.The amount of functional monomers and the cross-linking agent were optimized,and the optimal synthesis conditions were as follows:18 mg of Lyz,400 mg of[AMIM][PF₆]（1.369mmol）,100 mg of NNMBA.The Lyz-MIPs synthesized under the optimal conditions have the maximum adsorption amount of 13.14 mg/g and the imprinting factor of 1.51.Under pH 8.9 Tris-HCl buffer,the stability of the template-monomer complexes were enhanced by the strong electrostatic,hydrogen bonding andπ-πinteraction between the Lyz and[AMIM][PF₆]relative to pH 7 PBS buffer,which resulted in formation of effective imprinting sites with specific adsorption capacity for Lyz on the surface of the carbon nanotubes.3.The Lyz-MIPs were prepared by surface imprinting technique using polar[AMIM][PF₆]and hydrophobic TBAM（total 1.369 mmol）as mixed monomers under pH8.9 Tris-HCl buffer.The optimal synthesis conditions were:18 mg of Lyz,1:1 monomer ratio of[AMIM][PF₆]and TBAM,150 mg of NNMBA.The Lyz-MIPs synthesized under these conditions have the maximum adsorption amount of 22.25 mg/g and the imprinting factor of 1.84,and the selective factor for Cytochrome C（Cyt C）of 1.36.The MIPs prepared with mixed monomers display higher adsorption capacity,better imprinting effect and superior selective adsorption ability compared with that prepared with single monomer.It is mainly because the binding sites increased and the interaction strengthened between Lyz and the mixed functional monomers.Besides the electrostatic interaction,hydrogen bonding andπ-πinteraction,the hydrophobic interaction provided by the introduction of TBAM also plays an important role in imprinting of Lyz.