Preparation Of Self-assembling Peptide Functionalized Micro/nano Materials And Its Application In Separation And Enrichment

In nature,nonspecific adsorption of proteins from solution to various biochemical engineering and micro-nano materials interfaces is fundamental in many natural and industrial processes and has attracted extensive attention in various fields of immunosensing,electrophoresis separation,sample enrichment analysis,medical tissue materials,and antifouling engineering materials,etc.Especially when analyzing complex biological samples(such as blood,tissue fluid,extracellular fluid,etc.),it is particularly important to investigate and explore the nonspecific adsorption of proteins on a substrate.The main reason is that the nonspecific interaction of protein molecules at the interface may lead to low device performance,substantial sample loss,and low detection signal.Although a great deal of the researches on the nonspecific adsorption of proteins on the interface have been made,most of the studies on the proteins adsorption on the substrates still stay in the investigation of the qualitative research or macroscopic phenomena.However,there is still much controversy about the understanding of the adsorption mechanism of the proteins at the interface of materials at the molecular level.But,the investigation of the adsorption mechanism of protein molecules at the interface not ouly significantly facilitates our comprehension of protein adsorption on the materials,but also lays a foundation for solving the nonspecific proteins adsorption on a solid surface.Purthermore,it ean promote further integration among different fields in developing and searching various biological antifouling materials.In view of this,we select biochemical materials(including microfluidic chip materials,commonly used biomaterials,micro-nano magnetic materials)as the substrates surfaces,and systematically investigate the spontaneous adsorption,ie self-assembly behavior,of peptides and proteins on the surface of substrates.Combined with the advantages of the substrates in the field of separation and enrichment fields,we initially established universal approaches for the fabrication of biochemical materials based on basic amino acid-containing self-assembling peptides for complex biological samples separation and enrichment analysis.The full text mainly includes the following chapters:Chapter 1 IntroductionFirstly,the application of peptide self-assembly is summarized.Secondly,the microfluidic chip materials and magnetic nanoparticles used in the research and their functionalization and application studies are introduced.Finally,the research content and purpose of this thesis are clarified.Chapter 2 Study on adsorption mechanism of protein/peptide on solid surfaceOur previous studies have preliminarily demonstrated that the ionic complementary peptide of EAR16-?[(Ala-Glu-Ala-Glu-Ala-Arg-Ala-Arg)2]can form a complete amphiphilic self-assembled coating mainly composed of ?-sheets on the surface of hydrophilic and hydrophobic polydimethylsiloxane(PDMS)surfaces,which has good biocompatibility and efficiently suppress nonspecific adsorption of standard proteins or proteins from human whole blood.The results clearly indicated the critical role of the ionic hydrogen bonding betvreen the ?-amino group(?-NH2)in the basic amino acid residues and the negative charges on the PDMS surfaces in the strong adsorption of peptides and proteins on the surfaces.In order to verify this hypothesis,we designed and synthesized the ionic complementary peptides of EAK16-? and its quatemized derivative QEAK16-?,and systematically studied their self-assembly on the commonly used microfluidic chip of polymethyl methacrylate(PMMA)under physiological pH conditions.The characterization techniques including atomic force microscopy(AFM),water contact angle(WCA),X-ray photoelectron spectroscopy(XPS),and attenuated total reflection Fourier transform infrared spectroscopy(ATR-FTIR)were performed,and the protein-repelling and blood compatibility measurements are also utilized to verifiy the adsorption mechanism.Chapter 3 Versatile functionalization of microfluidic chips and biomaterial surfaces based on self-assembled peptidesBased on the above works,in order to further understand the adsorption mechanism of peptides and proteins on the surface at the molecular level,we provide a versatile,low-cost,and environmentally friendly functional method for different substrates.In this chapter,we designed small molecule peptides with hydrophobic amino acids(alanine,A)and hydrophilic amino acids(lysine,K)alternately arranged,ie AK-?,AK-?,and AK-?.We select commonly used microfluidic chip of PDMS and biochemical materials of polyphenylene ethylene(PS)as substrate models.The self-assembly of small peptides on the PDMS and PS surfaces was studied under physiological pH conditions.The protein-repelling properties and biocompatibility of small peptides were investigated.In addition,we also compared the inhibitory efficiency of BSA and AK-? used as blocking reagents on the nonspecific adsorption of antigens and detection antibodies in the carcinoembryonic antigen(CEA)enzyme-linked immunosorbent assay(ELISA).The experimental results show that AK-? exhibits better blocking effect than BSA,which not only reduces the nonspecific adsorption of antigen and detection antibody,but also provides a lower background noise,a lower detection limit,and a wider linear range in CEA measurement.This study laid the foundation for solving the nonspecific protein adsorption problem of PDMS-based engineering materials and PS-based biomaterials,and provided a novel and universal method for the development of various antifouling materials.Chapter 4 Fabrication of C18-functionalized magnetic graphene oxide based on self-assembled peptide for efficient enrichment of peptidesPeptides in human serum have biomarkers associated with some diseases and are therefore of great importance for the isolation and enrichment in serum.In this experiment,we designed and synthesized a self-assembling peptide of(C18-VKVKVK,C18VK-?)containing C18 alkane chain at the nitrogen(N)terminal,and investigated its self-assembly behavior on the surface of magnetic graphene oxide(Fe3O4@GO).Through the molecular interaction between the C18 chains and the peptides,the peptides in the myoglobin hydrolysate and serum are efficiently separated and enriched.Chapter 5 Fabrication of magnetic carbon materials based on self-assembled peptide for the Fe3+ immobilization for selective enrichment of phosphorylated peptidesIn this chapter,self-assembly method for the environmentally friendly functionalization of different magnetic carbon materials based on the self-assembled peptides of EPAKAKAK,EPAK-?,with hydrophilic and hydrophobic residues alternately arranged,was developed for the immobilization of Fe3+ and selective enrichment of phosphorylated peptides.Considering the studying of the adsorption mechanism of peptides and proteins on the solid surfaces in our previous works,the aim of designing this peptide is listed as following:we anticipate that AK-? can act as an anchor adsorbed stably on the magnetic carbon materials via the ionic hydrogen bonding between the ?-NH2 group in K and the negative charged surfaces.In order to achieve Fe3+ fixation,we designed the acidic amino acid glutamic acid(E)and hydrophobic amino acid proline(P)at the N terminal of the AK-? chain,in which the role of P is to control the space structure of the peptide so that the E may own stereoscopic structure and could act as connecting arm of Fe3+.Through one-step self-assembly method,EPAK-? can easily adsorb on the Fe3O4@C and Fe3O4@GO surfaces,in which the affinity of the material for Fe3+ can be enhanced,and on the other hand,high selectively enrichment of the phosphorylated peptides can be achieved due to the protein-repelling properties of the self-assembling peptides.The experimental results show that the current work can high selectively enrich the phosphorylated peptides in standard proteins and practical samples.Chapter 6 Preparation of thiol functionalized Fe3O4@C based on self-assembled peptide for efficient Cu(?)ions removal from aqueous solutionsPeptides are kinds of biomolecules with special structures and multifunctional groups(such as amino group,thiol,hydroxyl group and carboxyl group)and have been widely used in various fields such as biosensing,drug carrier,catalyst,and adsorbent.Based on the above verification of the adsorption mechanism of the peptides and proteins on the surfaces,we designed the thiol(SH-)-rich peptide of Cys-Lys-Cys-Lys-Cys-Lys(CK-?)and investigated its self-assembly on the surface of Fe3O4@C nanoparticles.In this system,ionic hydrogen bonding was formed between the ?-NH2 groups in the CK-? and the negative charges on the Fe3O4@C surfaces so that-SH can exposed outside the materials and could provide active sites for selective chelation of Cu(?)ions in solutions.The atomic absorption spectroscopy(AAS)was used to measure the concentration of residual Cu(?)ions in the solution before and after adsorption.The experimental conditions such as solution pH,adsorption and elution time,elution solvent,and adsorption capacity were optimized.The Cu(?)ions in practical samples of tap and lake water were measured using standard addition method.Chapter 7 ConclusionSummarize the full text and then point out drawbacks of the whole paper.