A facile method for grafting of bisphenol A imprinted polymer shells onto

Applied Surface Science 257 (2011) 6704–6710Contents lists available at ScienceDirectApplied SurfaceSciencej o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /a p s u scA facile method for grafting of bisphenol A imprinted polymer shells onto poly(divinylbenzene)microspheres through precipitation polymerizationYongxiang Wang a ,b ,c ,Qiuming Liu a ,Fei Rong a ,c ,Degang Fu a ,b ,c ,∗aState Key Laboratory of Bioelectronics,Southeast University,Nanjing 210096,ChinabSchool of Chemistry and Chemical Engineering,Southeast University,Nanjing 210096,China cSuzhou Key laboratory of Environment and Biosafety,Suzhou 215123,Chinaa r t i c l e i n f o Article history:Received 17September 2010Received in revised form 10February 2011Accepted 21February 2011Available online 26 February 2011Keywords:Core–shell structures GraftingMolecular imprinting Bisphenol APrecipitation polymerizationa b s t r a c tCore–shell structured polymers are usually prepared by “grafting to”or “grafting from”techniques,wherein polymer supports were first modified by vinyl bonds or initiators.Success can be immediate,but more often a learning curve needs to be traversed.In this paper,direct grafting bisphenol A imprinted polymer onto poly(divinylbenzene)microspheres through precipitation polymerization is introduced.The obtained microspheres were characterized by SEM,TEM,FTIR and BET.Firstly,mono-disperse poly(divinylbenzene)microspheres of 3␮m in size were prepared by precipitation polymerization.The solvent for grafting was then optimized and it was found that the mixtures of toluene and acetonitrile (30/70,v/v)were ideal choice from the image and structure analysis of the shells.Shells of different thick-ness (270–840nm),depending on the concentrations of monomers in the precursor,were then directly grafted to the microspheres by a reactive,entropic capture mechanism.The recognition ability of the microspheres was evaluated by solid-phase extraction and clear selectivity showed toward bisphenol A.The leak of bisphenol A was not found after extraction and thus these core–shell structured imprinted polymers are believed to have potential applications in trace analysis area.© 2011 Elsevier B.V. All rights reserved.1.IntroductionMolecular imprinting is a commonly used method for prepar-ing synthetic polymers with predetermined molecular recognition properties and is attracting widespread interest [1–4],especially in solid-phase extraction (SPE)[5–9],chromatographic separations [10,11],sensors [2],and catalysis [12].To optimize the production and performance of MIP particles,suspension [13,14],disper-sion [5]and seeded polymerization [15,16]have been developed.Despite of undoubted value,optimization of reliable experimental protocols can be lengthy,the general applicability is questionable,and residual emulsifier or stabilizer on the surfaces of the par-ticles potentially compromises selective rebinding of molecules to the imprinted material.As an alternative approach,precipita-tion polymerization is a general method for producing high-quality imprinted spherical particles.However,it has proved difficult to produce large imprinted monodisperse microspheres that are suitable for SPE and HPLC.It has been found that matching the sol-ubility parameter of the developing polymer network to that of the porogenic solvent(s)is particularly important,especially when∗Corresponding author at:State Key Laboratory of Bioelectronics,Southeast Uni-versity,Nanjing 210096,China.Tel.:+862583793091;fax:+862583793091.E-mail address:fudegang@ (D.Fu).simultaneous control of polymer morphology is also desirable.The criterion is usually met for copolymerization of divinylbenzene (DVB)in mixtures of acetonitrile and toluene [17].The group of Stöver has done extensive research on precipitation polymeriza-tion of DVB [18–21],the size of the obtained particle could be tuned upward 10␮m by careful optimization [21].Uniform molec-ularly imprinted microspheres and nanoparticles were prepared by Yoshimatsu et al.through precipitation polymerization [22],varying the composition of the cross-linking monomer allowed the particle size of the MIP beads to be altered in the range of 130–2.4␮m.Narrow particle size distribution (9.5±0.5␮m)MIP was obtained using an antiepileptic drug,carbamazepine as tem-plate molecule and DVB as crosslinking agent by Beltran et al.[23].For application in SPE,especially challenged by trace analysis,a common drawback for all the methods introduced above is the problem of incomplete template removal or template bleeding,which can occur even after exhaustive washing by different sol-vents or buffers and this greatly impedes the application of MIP,especially for trace analysis when the template is also the targeted molecule(s)[24].This considerably affects the quality of data gen-erated.To solve this problem,target analogues were usually used as templates [25].Recently,several research groups have begun to explore alternative approaches for the development of molec-ular imprinting nanotechniques.Nanosized,imprinted materials are expected to improve the binding capacity,binding kinetics,0169-4332/$–see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.apsusc.2011.02.105Y.Wang et al./Applied Surface Science257 (2011) 6704–67106705site accessibility of imprinted materials and all of templates can be completely removed from the highly cross-linked matrix[26].Core–shell structured particles with nanosized imprinted poly-mers shells can be expected to tackle the problem of incomplete template removal or template bleeding from average imprinted polymers.These particles are usually prepared by“grafting to”or“grafting from”techniques,wherein polymer supports were first modified by vinyl bonds or initiators[27,28].In this paper, we report a simple,effective and reproducible approach of preparing core–shell structures by grafting nanoscale molecularly imprinted polymer onto DVB-based microspheres through precip-itation polymerization without modifying the polymer supports. The as-prepared microspheres presented selective recognition of bisphenol A(BPA)and the leak of BPA was not found after 0.1g microspheres packed in columns were washed by10mL methanol/acetic acid(9/1,v/v)continuously,thus these core–shell structured imprinted polymer are believed to have potential appli-cations in trace analysis area.2.Experimental2.1.MaterialsAll the reagents were used as received.Azobisisobutyronitrile (AIBN)and acrylamide(AA)were purchased from Experimen-tal Reagents,Ltd.(Shanghai,China).Divinylbenzene80(DVB-80) were purchased from Sigma–Aldrich(St.Louis,USA).Phenol was purchased from Lingfeng Reagents Co.Ltd.(Shanghai,China). Diethystilbestrol(DES)and bisphenol A(BPA)were kindly provided by School of Public Health from Southeast University.Acetonitrile and methanol for HPLC were purchased from Fisher Scientific(New Jersey,USA).Water for HPLC was obtained from a Heal Force ultrapure water unit(Shanghai,China).2.2.Synthesis of BPA imprinted polymer grafted core–shellsThe investigated samples are core–shell structures,they have been fabricated by the route as described in detail elsewhere[20]. The modified steps were the monomer loadings and DVB,relative to the solvent,were4vol%and1–2vol%for the core and the shell portion preparations,respectively.Conditions for preparation of imprinted and control core–shells are listed in Table1.For preparation of core,typically,2mL DVB-80were added into a100mLflask containing50mL neat acetonitrile and AIBN(2wt% to DVB-80).After ultrasonic treatment and removal of oxygen by purging of nitrogen for5min,respectively,the precursor was sealed and heated at50◦C for2h and70◦C for24h.The micro-spheres were collected by ultrafiltration and washed by acetone,toluene and acetyl alcohol successively.Then they were dried in a vacuum oven at60◦C over night.For preparation of imprinted polymer core–shells,typically, 0.3mL DVB-80were added into a50mLflask containing30mL sol-vent,100mg poly(DVB-80)micro-spheres,101.3mg AA,54.8mg BPA and AIBN(2wt%to total of DVB-80and AA).The solvent for grafting was then optimized and different ratios of toluene to ace-tonitrile(0/100,15/85,25/75,30/70,35/65,40/60,v/v)were tried. The precursor was sealed and heated at50◦C for2h and60or 70◦C for24h.The core–shell micro-spheres were then collected by ultrafiltration and washed by acetone,toluene and acetyl alco-hol successively.Then they were dried in a vacuum oven at60◦C over night.2.3.CharacterizationThe structure and morphology of the obtained core–shell structured particles were characterized by Fourier transform infrared spectroscopy(FTIR)(Nicolet6700),scanning electron microscopy(SEM)(ZEISS ULTRA plus system),transmission elec-tron microscopy(TEM)(JEM-2100)and automated gas adsorption system(ASAP2020M+C).Solid-phase extraction(SPE)was carried out on a VacMaster (Uppsala,Sweden)vacuum manifold,which was equipped with a vacuum pump and a10-well platform.100mg dry core–shell par-ticles prepared were packed between two frits in1mL SPE columns and washed by∼10mL methanol/acetic acid(9/1,v/v)continuously until no BPA was found in the eluent.Acetonitrile solution con-taining0.2mM phenol,0.1mM BPA and0.1mM DES was prepared for loading.After activated by5mL methanol and1mL acetoni-trile,each column was loaded1mL solution mentioned above.The columns were washed by0,1or2mL acetonitrile before eluted by 0.5mL×2mL methanol and analyzed by HPLC directly.Theflow rate of the solvent/solution was maintained at about1mL min−1.All chromatographic evaluations were performed using a Shimadzu LC-20A(Kyoto,Japan)instrument equipped with a qua-ternary pump,a manual sampler,a diode array detector,and an LCsolution workstation.3.Results and discussionJeffrey et al.have uncovered the growth mechanism of poly(divinylbenzene)microspheres in precipitation polymeriza-tion,which indicated that polymerization of divinylbenzene inTable1Conditions for preparation of imprinted and control core–shells.Name AA(mg)DVB(mL)Toluene Con.(vol%)a BPA(mg)Amount of cores(mg)Porogen amount(mL)T(◦C)Poly(DVB-80)020005070 SPM0T%202.50.60109.51003070 SPM15T%202.50.615109.51003070 SPN15T%202.50.61501003070 SPM25T%202.50.625109.51003070 SPN25T%202.50.62501003070 SPM30T%202.50.630109.51003070 SPN30T%202.5063001003070 SPN35T%202.5063501003070 SPN40T%202.5064001003070 SPN1%101.30.33054.81003060 SPM1.5%101.30.33001003060 SPN1.5%159.00.453082.21003060 SPM2%159.00.453001003060 SPN2%202.50.630109.51003060 SPN1%202.50.63001003060 SPM1.5%101.30.33054.81003060a Solvent for preparation of polymer grafted microspheres was mixtures of toluene and acetonitrile(0/100,15/85,25/75,30/70,v/v).6706Y.Wang et al./Applied Surface Science257 (2011) 6704–6710Fig.1.Representative SEM image of poly(DVB-80)particles.acetonitrile occurs by an entropic precipitation mechanism where soluble oligomeric species are captured from solution by reaction with vinyl groups on the particle surface [19].For preparation of core–shell structures,polymer supports of poly(DVB-80)micro-spheres were synthesized by precipitation polymerization in this study.Fig.1illustrates the SEM images of these uniformly sized,mono-disperse poly(DVB-80)core particles with sizes around 3␮m.As for precipitation polymerization,matching the solubility parameter of the developing polymer network to that of the porogenic solvent(s)is particularly important,especially when simultaneous control of polymer morphology is also desirable.Mix-tures of toluene and acetonitrile (25/75,v/v)were usually utilized for synthesis of big sized poly(DVB-80)or other functional poly-mer microspheres mainly composed of DVB [17–23],since near theta solvents such as acetonitrile and mixtures of acetonitrile and toluene are required for distinct poly(DVB-80)microspheres.In this study,different ratios of toluene to acetonitrile were tried for optimization.Fig.2illustrates the overall results of core–shell structures pro-duced in different solvents at 70◦C.It can be found that when the concentration of toluene in the solvent was low (0or 15%),secondary particles formed inevitably for either imprinted or con-trol core–shell structured polymers,which should be attributed to the solubility parameter of acetonitrile.This solvent was not in favor for the formation of large sized beads.When the concen-tration of toluene in the solvent was high (30%)the imprinted core–shell structured beads as-prepared were large,the unifor-mity and monodispersity of the beads were also well maintained,which should be attributed to the solubility parameter of toluene which contributed to the formation of large sized beads.Although monodispersity of the beads was well maintained,the unifor-mity of control core–shell structured polymer beads were not so well maintained in the same conditions.The differencebetweenFig.2.Representative SEM image of core–shell particles (insets are TEM images of the corresponding particles).(a)SPM 0T%,(b)SPM 15T%,(c)SPM 25T%,(d)SPM 30T%,(e)SPN 15T%,(f)SPN 25T%,(g)SPN 30T%,(e)SPN 35T%and (i)SPN 40T%.Y.Wang et al./Applied Surface Science257 (2011) 6704–67106707Fig.3.Pore size distributions for poly(DVB-80)particles and core–shell particles. imprinted and control core–shell structures is interesting when the concentration of toluene in the solvent was30%and this should only be caused by the template(BPA).It can be speculated that BPA bridged the grafting“gap”between the shells and polymer supports through molecular interactions between BPA molecules and aromatic rings of DVB molecules or poly(DVB-80).When the concentration of toluene in the solvent was higher(30%or40%), particles agglomerated and macro-gel type polymer was obtained.The solvent also played an important part on the eventual pore volume,pore size distribution,and specific surface area of the parti-cles.Results are shown in Fig.3and Table1.It can be found that pore size,pore volume and specific surface area of the obtained particles increase with the ascending concentration of toluene in the solvent. The total pore volumes and surface areas of imprinted core–shell structured polymer spheres were up to0.43cm3/g and655m2/g, respectively when concentration of toluene in the solvent was30% in preparation.Table2Size and pore structures of BPA-imprinted(SPM)and control(SPN)polymer core–shells.Name Size(␮m)Surfacearea(m2/g)Pore volume(cm3/g)Pore size(nm)Poly(DVB-80) 2.61376.19970.185441 1.97173 SPM0T% 2.41353.71630.143946 1.62781 SPM15T% 3.58398.23220.203222 2.04124 SPN15T% 2.52520.79470.282934 2.17309 SPM25T% 4.52538.52140.299893 2.22753 SPN25T% 2.69560.97520.300550 2.14306 SPM30T% 5.50655.41960.438412 2.67561 SPN30T% 2.41590.46600.3508922.37705Fig.5.Shell thickness of imprinted polymer grafted core–shell(SPM)and con-trol polymer grafted core–shell(SPN)depended on the monomers loading in the precursor.Heating temperature:60◦C.From the optimization above,mixtures of toluene and acetoni-trile(30/70,v/v)seem ideal solvent for grafting BPA imprinted polymer to the poly(DVB-80)supports.Therefore,we prepared BPA imprinted polymer shells next were all in this solvent.In ordertoFig.4.Representative SEM image of core–shell particles of different thickness prepared in mixtures of toluene and acetonitrile(30/70,v/v)(insets are TEM images of the corresponding particles).(a)SPM1%,(d)SPN1%,(b)SPM1.5%,(e)SPN1.5%,(c)SPM2%,(f)SPN2%.6708Y.Wang et al./Applied Surface Science257 (2011) 6704–6710Fig.6.Particle size distributions for poly(DVB-80)particles and the subsequent core–shell particles.study the role of temperature on the synthesis of the core–shell structures,60◦C was tried instead of 70◦C in the later grafting of BPA imprinted polymer.Fig.3demonstrates the imprinted and control shells of different thickness depend on the monomers load-ing in the precursor.The uniformity,mono-dispersity of the beads were better maintained at 60◦C compared with 70◦C.When the precursor solution was heated at 60◦C,the polymerization took place between the vinyl end groups of poly(DVB-80)particles and AA/DVB monomers at a slow rate in the beginning,a thin layer of oligmers started to form at the surface of the polymer supports and the resulting polymer preferentially nucleated and grew on the sur-face of poly(DVB-80)particles with polymer oligomers,leading to the formation of uniform BPA-MIP shells [26].Whereas,the poly-merization took place between the vinyl end groups of poly(DVB-80)particles and AA/DVB monomers and self-agglomerating of oligomers in solution were both at a fast rate in the beginning when the precursor solution was heated at 70◦C,this resulted in grafting and secondary particle initiation at the same time (Table 2).The shell thickness depended on the monomer loading in the precursor,the relationship between thickness of the shells and DVB-80concentration (from 1vol%to 2vol%)in the precursor is shown in Figs.4and 5.Obviously,we can also control the thickness of the shell by tuning the time of polymerization.Fig.6demon-strates the diameter distribution of the polymer supports and the core–shell structures of different shell thickness prepared at 60◦C.It is clearly showed that the uniformity and mono-dispersity of the beads were well maintained after grafting,and they seem no obvious secondary initiation.The BPA templates in the shell can be fully removed by defined volume of methanol/acetic acid (9/1,v/v)even when the thickness was ∼800nm through determina-Y.Wang et al./Applied Surface Science 257 (2011) 6704–67106709Fig.7.FT-IR spectra of (a)poly(DVB-80),(b)SPM 1%,(c)SPN 1%,(d)SPM 1.5%,(e)SPN 1.5%,(f)SPM 2%and (g)SPN 2%.tion of the eluent in the end of extraction,this may be due to the extraordinarily large pore volumes and surface areas of imprinted core–shell structured polymer spheres compared with average imprinted polymers.In order to find whether or not AA was incorporated in the shell during grafting process,these core–shell structures were characterized by FTIR.The core–shell structures of different thick-ness displayed the relatively strong band of carbonylic groups at 1682cm −1,as indicated with arrows in Fig.7.For solid-phase extraction,imprinted and control core–shells were prepared at 60◦C and DVB-80in the precursor was 2vol%in grafting process.Imprinting effects of the core–shell structured polymers were assessed by solid-phase extraction (SPE)of mixtures of 0.2mM phenol,0.1mM BPA and 0.1mM DES from acetonitrile.DES and phenol were structure analogues of BPA and used for com-parison.Results demonstrated in Fig.8shows a clear imprinting effect toward BPA when different volume of acetonitrile was uti-lized as solvent in washing step,while phenol was not found in the elution solution when either 1or 2mL of acetonitrile was utilized in washingstep.Fig.8.Extraction recoveries for DES,BPA and phenol on SPM 2%and SPN 2%using a washing step of (left)0mL,(middle)1mL and (right)2mL acetonitrile.4.ConclusionCompared by conventional “grafting to”or “grafting from”tech-niques,the method 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