| Chapter I.Preparation and Characterization of Boric Acid Functionalized NanoparticlesObjectives:Glycoproteins play an important role in many biological processes,and are frequently used as biomarkers for different diseases.Effective separation of glycoproteins is one important step in many research areas and diagnostics.Therefore,it is of great value to investigate new materials to separate and enrich glycoproteins.Boronic acid can form stable ester bonds with cis-diol groups in the carbohydrate site of glycoproteins.In order to gain further insight into the structure-function relationship of nanoparticle-supported boronic acid polymer brushes,a series of nanoparticle-supported boronic acid polymer brushes for affinity separation of glycoproteins were synthesized to study the impact of the polymer structure on glycoprotein binding.Methods:Silica nanoparticles?SiO2?were synthesized before polymer brushes were prepared by surface-initiated atom transfer radical polymerization of glycidyl methacrylate,followed by stepwise modification of the pendant as well as the end functional groups to introduce the boronic acid moieties through click reaction.Two different methods were used and compared:?1?Sulfuric acid treated before azidation:SiO2@PGMA was first treated with sulfuric acid,to open the epoxy ring on GMA polymerized chains,then sodium azide was used to introduce azide groups onto terminal Br active sites of polymer rings.The obtained nanaoparticles were denoted as SiO2@PGMA-OH@N3,with azide groups only on main chains terminals.?2?Direct azidation:SiO2@PGMA was treated with sodium azide,to open epoxy rings on GMA polymer brushes,introducing azide groups both on chains branches and on main chains terminals,obtaining SiO2@PGMA-N3@N3.Click chemistry reaction was applied to modifyalkynylphenylboronicacidtoazidatednanoparticles,obtaining SiO2@PGMA-OH@BA,with boronic acid groups only on main chains terminals,and SiO2@PGMA-BA@BA,with boronic acid groups both branches and main chains terminals.Scanning electron microscopy?SEM?,Fourier Transform infrared spectroscopy?FT-IR?,thermogravimetric analysis?TGA?,and elemental analysis were applied to analyze physicochemical characteristics of the two boronate nanoparticles.Fluorescence analysis based on Alizarin Red S was used to analyze boronate fluorescent properties of nanoparticles,UV spectrophotometry was applied to particles sedimentation analysis.Glycoprotein adsorption analysis of the two composite nanoparticles were carried out and compared.Finally,effects from different boronate properties and particles sedimentations on glycoproteins binding properties of the two nanoparticles were analyzed.Results:FT-IR,TGA,and elemental analysis results showed that two kinds of boronate polymer brushes were successfully modified onto the surface of SiO2nanoparticles.SiO2@PGMA-BA@BA had boronic acid groups both on branches and main chains terminals,was more boronated than SiO2@PMGA-OH@BA,with boronic acid groups only on main chains terminals.But SiO2@PMGA-OH@BA was more dispersible in aqueous solution.Protein adsorption results showed that the adsorption time of boric acid functionalized nanoparticles to glycoproteins was about 2 h.Both of the nanoparticles can be used for glycoproteins adsorption,higher for glycoproteins?ovalbumin and horseradish peroxidase?,while only a little for non-glycoprotein?bovine serum albumin?.Its binding property for ovalbumin was higher than that for horseradish peroxidase,because ovalbumin is of higher glycosylation level.Both of them showed pH dependent adsorption property to ovalbumin,binding changed with pH as followed:pH 9.0>pH 7.4>pH 4.0.The calculated adsorption capacity for ovalbumin of SiO2@PGMA-BA@BA,with boronic acid both on main chains terminals and branches,was 153.40 mg/g,of SiO2@PGMA-OH@BA,with boronic acid only on main chains terminals,was 126.70 mg/g,their adsorption capacities were with no statistical differences.Conclusions:Two kinds of boronic acid functionalized polymer brushes have been successfully modified onto the surface of SiO2 nanoparticles,the obtained particles were with different boronate properties,both of them can be used for glycoproteins adsorption,and both of their glycoprotein binding properties were pH responsive.The boric acid groups on SiO2@PGMA-BA@BA were located both on the polymer brushes branches and on the terminals of brushes main chains.Those of SiO2@PGMA-OH@BA were modified only on terminals of main chains.Thus SiO2@PGMA-BA@BA had more boron groups than SiO2@PGMA-OH@BA,but the former had poorer dispersibility in aqueous solution.Their adsorption of glycoprotein were of no significant differences,indicating that the most important boronic acid groups for glycoproteins adsorption were located on the terminals of polymer chains.Chapter?.Preparation and Characterization of Temperature-responsive Boric Acid Functionalized NanoparticlesObjectives:Environmental responsive nanomaterials are finding extensive applications in areas spanning bioseparations,biosensors,and controlled gene/drug delivery.On the basis of boric acid functionalized nanoparticles prepared in the first chapter,temperature-responsive monomer,N-isopropyl acrylamide?NIPAm?was added to prepare temperature-responsive boric acid functionalized nanoparticles.The impacts of location of boronic acid ligands,nanoparticles sedimentation,and temperature on glycoprotein binding with the nanoparticle-supported polymer brushes were further compared and studied.analyzed.Methods:SiO2 nanoparticles with initiators were prepared following the same protocol as Chapter I,then NIPAm and glycidyl methacrylate?GMA?were used as co-monomers,using surface-initiated atom transfer radical polymerization?SI-ATRP?method,to polymerize brushes on the surface of SiO2 nanoparticles,obtaining NIPAm-co-GMA co-polymer brushes SiO2@PNCG.SiO2@PNCG was then azide-functionalized in two ways:?1?Sulfuric acid treated before sodium azide treatment,obtaining SiO2@PNCG-OH@N3 with azide groups only on terminals of polymer brushes main chains;?2?Directly sodium azide treated,obtaining SiO2@PNCG-BA@N3 with azide groups both on branches and on main chains terminals.In addition,only NIPAm was used as functional monomer for polymerization of another kind of polymer brushes on SiO2 nanoparticles,was also carried out and azidated,denoted as SiO2@PNIPAm@N3.Click chemistry was used to introduce boronic acid groups onto these three azide-functionalized nanoparticles,obtaining SiO2@PNCG-OH@BA,with boronic acid groups only on main chains terminals,SiO2@PNCG-BA@BA,with boronic acid groups both on main chains terminals and branches,and SiO2@PNIPAm,with boronic acid groups only on main chains terminals.Electron microscopy,infrared spectroscopy,thermogravimetric analysis and elemental analysis were carried out to analyze their physiochemistry properties;Alizarin Red S was applied to analyze boronate fluorescent signals of nanoparticles;UV spectrophotometry was applied to analyze particles sedimentation.Glycoprotein adsorption analysis of the three thermal responsive boronate nanoparticles were carried out for different proteins,under different pH and temperature conditions.Thermal responsive boronate nanoparticles were compared with non-thermal responsive boronate nanoparticles,analyzing effects from boronate level and aqueous sedimentation on nanoparticles glycoproteins binding properties.Results:Infrared spectroscopy,TGA,and elemental analysis results proved that three different amout of borionc acid groups were successfully modified onto the nanoparticles,among which their boronated level was SiO2@PNCG-BA@BA?with boronic acid both on branches and terminals?>SiO2@PNCG-OH@BA?with boronic acid only on terminals?>SiO2@PNIPAm@BA?with boronic acid only on terminals?.Their sedimentations in aqueous solution were of no significant differences,all better than those synthesized in the first chapter,and with temperature-response,having worse sedimentation at 40°C than at 20°C.They all performed adsorption for glycoproteins?ovalbumin and horseradish peroxidase?,much higher than non-glycoprotein bovine serum albumin,among which higher adsorption for ovalbumin than horseradish peroxidase,because ovalbumin is more glycosilated.Their binding to ovalbumin were all thermal responsive,worse at 40°C than at 20°C.Their binding was also pH responsive,pH 9.0>pH 7.4>pH 4.0.Their calculated adsorption capacity Qmax were:SiO2@PNCG-OH@BA?with boronic acid only on terminals??129.64 mg/g?,SiO2@PNCG-BA@BA?with boronic acid both on branches and terminals??159.38mg/g?,and SiO2@PNIPAm@BA?with boronic acid only on terminals??118.37 mg/g?,also no statistical differences.Conclusions:Using this method,three temperature-responsive boric acid functionalized SiO2 nanoparticles were synthesized,all of them can be used for selective glycoproteins adsorption.Their adsorption was both pH-responsive and temperature-responsive.Although the three boronate nanoparticles were modified with different amount of boronic acid groups,they had no significant differences on glycoproteins binding,indicating the most important boronic acids contributing to protein binding are located at the chain termini of the polymer brushes.The temperature-responsive boronic acid-functionalized nanoparticles were more stable in aqueous solutions than those non-temperature-responsive ones,but their glycoprotein binding ablities showed no significant differences.This indicated that sedimentation in aqueous solution was not the main factor affecting glycoproteins binding of these nanoparticles.Chapter?.Temperature Responsive Boroic Acid Functionalized Nanoparticles Applied to Glycoproteins SeparationObjectives:New methods for protein separation that requires minimal alternation of the chemical compostion of a protein solution,with conditions more favourable for final step protein purification were developed using temperature responsive boric acid functionalized nanoparticles.They were applied for glycoproteins separation in complex samples,their separation conditions were optimized to increase separation efficiency.Methods:The temperature responsive boric acid functionalized nanoparticles prepared in Chapter 2 were used to bind glycoproteins in mixture of horseradish peroxidase and bovine serum albumin.A temperature cycling experiment was first performed to test the adsorption of glycoproteins in the mixed samples;then glucose was added and the temperature cycling experiment was carried out again,to evaluate the effects of temperature and fructose to nanoparticles-glycoproteins binding.The elution conditions were optimized using elution buffer pH,elution temperature and adding fructose.The optimal conditions were used to separate and purify glycoproteins in complex sample.Finally,the adsorption properties of nanoparticles with glycoproteins in egg white samples were investigated,using sodium dodecyl sulfate-polyacrylamide gel electrophoresis?SDS-PAGE?.Results:Temperature cycling experiments demonstrated that by simply changing the solution temperature,glycoproteins bound to boric acid functionalized nanoparticles can be eluted,and this binding-elution can be recycled.Fructose and glycoprotein were competitively adsorbed on the boronic acid functionalized nanoparticles,adding fructose could decrease the adsorption of nanoparticles and glycoproteins.The combination conditions of heating,using low pH elution buffer and adding fructose were more efficient on eluting glycoproteins adsorbed on nanoparticles.SDS-PAGE results had confirmed that boronic acid functionalized nanoparticles can be used for selectively adsorption of glycoproteins in mixed samples of horseradish peroxidase and bovine serum albumin.Adsorption and elution of glycoproteins in egg white samples were also confirmed.Conclusions:For the thermo-responsive polymer brushes,we demonstrated that it is possible to use temperature to control the glycoprotein binding and release.The reversible characteristic of the boronate ester bonds involved in the protein separation process has been revealed through temperature recycling experiments.Glycoprotein bound on boronate affinity particles can be eluted by simply varying the solution temperature.When combined with acidic buffer,the new thermo-desorption provided thehighestproteinrecovery.Thenewboronicacid-functionalized,nanoparticle-supported polymer brushes hold interesting potential to be exploited not only for protein separation,but also other applications including controlled drug delivery and release.Chapter?.Preparation and characteriazation of glycoproteins imprinted polymers based on click reactionObjectives:Click reaction was applied to imprint glycoprotein,synthesizing molecularly imprinted polymer?MIP?on silica nanoparticles,with selectivity to glycosyl sites of template glycoprotein,increacing the adsorption selectivity of boronic acid functionalized nanoparticles for glycoproteins.Methods:?1?Alkynylbenzeneboronic acid was binded with template glycoprotein ovalbumin,obtaining boronate glycoprotein complex,then dialysed to get rid of the unbound boronic acid;?2?Alkynylbenzeneboronic acid combined with ovalbumin was linked onto SiO2@PGMA-N3@N3 via alkynyl-azido click reaction,obtaining glycoprotein-boronic acid-nanoparticles complex;?3?Ovalbumin was eluted,obtaining SiO2@MIP,with ability of selectively recognizing ovalbumin glycosyl patterns.The controlled non-imprinted particle SiO2@NIP was synthesized following the same protocol,except no ovalbumin was added during preparation.Electron microscopy,infrared spectroscopy,boronate functionalization were carried out,adsorption analysis of SiO2@MIP and SiO2@NIP were investigated and compared.Results:Ovalbumin imprinted boronic acid-functionalized nanoparticle SiO2@MIP,and its non-imprinted control group SiO2@NIP,were prepared.Boronate fluorescence response signal could be detected on the surface of SiO2@MIP,but not SiO2@NIP.SiO2@MIP had significant higher adsorption property for glycoprotein ovalbumin,followed by glycoprotein horseradish peroxidase,then non-glycoprotein bovine serum albumin;SiO2@NIP had low adsorption for all the proteins,without similar binding properties.The binding capacity(Qmax)of ovalbumin for SiO2@MIP was 110.03 mg/g,significantly higher than 11.31 mg/g for SiO2@NIP.For SiO2@MIP,the imprinting factor?IF?of template OVA was 8.12,significantly higher than BSA?IF=2.88?,also higher than HRP?IF=5.17?,because the glycosyl sites for HRP is different from OVA.This indicated that SiO2@MIP can selectively adsorb the template protein ovalbumin.For SiO2@MIP,the specificity coefficient?K?of ovalbumin was1.73,higher than that for SiO2@PGMA-BA@BA from Chapter I?K=1.32?.These results showed that,by preparing MIP for certain glycoprotein on silica nanoparticles,the selectivity of boronic acid functionalized nanoparticles can be increased.Conclusions:Using click reaction,MIP with selective recognition property for template glycoprotein glycosyl sites was synthasized.This novel imprinting method can offer new ways for glycoprotein imprinting. |
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