Preparation of Protein Molecular-Imprinted Polysiloxane Membrane Using Calcium Alginate Film

14 Pages·2017·7.3 MB·English

polymers
Article
Preparation of Protein Molecular-Imprinted
Polysiloxane Membrane Using Calcium Alginate
Film as Matrix and Its Application for Cell Culture
DongLiu1,KongyinZhao1,2,* ID,MengQi2,ShuwenLi2,GuoqingXu2,JunfuWei1,2
andXiaolingHe2
1 StateKeyLaboratoryofSeparationMembranesandMembraneProcesses,TianjinPolytechnicUniversity,
Tianjin300387,China;[email protected](D.L.);[email protected](J.W.)
2 SchoolofMaterialScienceandEngineering,TianjinPolytechnicUniversity,Tianjin300387,China;
[email protected](M.Q.);[email protected](S.L.);[email protected](G.X.);
[email protected](X.H.)
* Correspondence:[email protected];Tel.:[phone]
Received:18January2018;Accepted:9February2018;Published:10February2018
Abstract: Bovine serum albumin (BSA) molecular-imprinted polysiloxane (MIP) membrane was
prepared by sol-gel technology, using silanes as the functional monomers, BSA as the template
and CaAlg hydrogel film as the matrix. The stress-strain curves of wet CaAlg membrane and
molecular-imprinted polysiloxane membrane were investigated. We evaluate the adsorption
and recognition properties of MIP membrane. Results showed that the adsorption capacity of
BSA-imprintedpolysiloxaneforBSAreached28.83mg/g,whichwas2.18timesthenon-imprinted
polysiloxane(NIP)membrane. Theadsorptionratewashigherthanthatoftheprotein-imprinted
hydrogel. BSA-imprinted polysiloxane membrane could identify the protein template from
competitive proteins such as bovine hemoglobin, ovalbumin and bovine γ-globulin. In order to
obtainthebiomaterialthatcanpromotecelladhesionandproliferation,fibronectin(FN)-imprinted
polysiloxane (FN-MIP) membrane was obtained by using fibronectin as the template, silanes as
functional monomers, and CaAlg hydrogel membrane as the substrate or matrix. The FN-MIP
adsorbed more FN than NIP. The FN-imprinted polysiloxane membrane was applied to culture
mouse fibroblast cells (L929) and the results proved that the FN-MIP had a better effect on cell
adhesionthanNIP.
Keywords: calciumalginate;polysiloxane;molecular-imprintedpolymer;bovineserumalbumin
adsorption;fibronectin;cellculture
1. Introduction
Biomaterialscaffoldmimicstheextracellularmatrixandservesasatemporaryskeletonforcell
growth[1],migration,proliferation,differentiationandfinallymaturation[2–4]. Biomaterialsthat
identifyspecificmolecularpotentialhaveattractedalotofattentiontoallowadsorptionandadhesion
of proteins that properly accommodate and rebuild the target tissue [5]. The studies on protein
adsorption,proteinrecognitionaswellastheinteractionbetweenproteinsandbiomaterialshavebeen
widelystudied[6–8].
Molecular imprinting technique is an effective method for the establishment of specific
identification sites for target molecules in synthetic materials [9–13]. The obtained
molecularly-imprintedpolymershavecomplementarybindingsitesandtemplate-specificrecognition
cavities,whichcanserveasrepositoriesofdrugmoleculesandimprovedrugresidencetime[14–17].
Molecular imprinting technology has been widely used in various small molecules and made an
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Polymers2018,10,170 2of14
excellentprogress.However,theimprintingofproteinisstillamajorchallengeowingtothecomplexity
ofsurfacestructures,thehighflexibilityofconformationanddiversityofproteinsequences[18,19].
One of the interesting biomaterials is polyacrylamide, which is always used to prepare
cross-linkinghydrogelsexistingproteinrecognitionsites[20–23]. Althoughpolyacrylamidehydrogels
havehighselectivitytotemplateproteins,theyareshortofamplethermostabilityandmechanical
strength. In addition, the diffusion of protein in the hydrogel is quite slow. Surface imprinting
techniquecanbeappliedtosolvetheproblemofproteindiffusion[24–26]. Asfunctionalmonomers,
silanes and its derivatives were employed to synthetize molecular-imprinted polysiloxane on the
surfaceofsolidsubstrate. Organicsilaneiseasytopolymerizebysol-gelprocesstocreatepolysiloxane,
which has high and low temperature resistance, hydrophobicity and physiological inertia. Most
silanescontainfunctionalgroupsthatreactwithproteins. Mosbachetal. discussedthepreparation
of protein molecular imprinting polymers by organic silane monomers [27]. There were multiple
hydrogenbondsbetweenthesilanolgroupsofpolysiloxaneandsurfacepolarresiduesofproteins.
Theresultsshowthatthesilicondioxidecoatedwithpolysiloxanehasaffinityfortheglycoprotein
transferrin. ToruShiomietal.[28]synthesizedproteinmolecular-imprintedpolymeronsilicausing
3-aminopropyltrimethoxysilane and trimethoxypropylsilane as the functional monomers for the
determinationofhemoglobin(Hb). Twokindsoforganicsilaneswerepolymerizedonasurfaceof
poroussilicaaftertheHbtemplatewascovalentlyimmobilizedbyformingiminebonds. Asensitive
silicagelmicrospherecaptureproteinwaspreparedbyKyokoFukazawaetal.[29]. Itwasusedfor
highselectiveidentificationofbovineserumalbumin(BSA).Theimprintedproteinwasimmobilizedto
silicabeadsbyusingaphospholipidpolymercontainingbothactiveestergroupsandsilanecoupling
groups. ZianLinetal.[30]fabricatedamacroporoussilicamonolithforproteinmolecularimprinting.
Inour previous work, BSAmolecular-imprinted polysiloxane(MIP) waspreparedwith silanesas
functional monomers, BSA as templates and calcium silicate containing mesoporous SiO on the
2
surface(CaSiO @SiO )asthematrix[31].However,theabove-preparedMIPparticlesareinconvenient
3 2
insomeapplications,especiallyincellculture.
As one of the biological materials extracted from brown algae, calcium alginate has been
successfully used in food, drug delivery, tissue engineering and beverages because of its
biocompatibility[32].Alginate-basedhydrogelshavebeenwidelyusedforcellmicroencapsulation[33].
BSA-imprintedcalciumalginatemembranehasbeenpreparedforthereleaseofBSAfromthealginate
matrix[34]. OurpreviousworkwastoprepareBSA-imprintedpolymersbasedoncalciumalginate
hydrogelmicrospheres[35]. However,theprotein-imprintedmicrospheresareinconvenientincell
culture,especiallywhenacellsheetisrequired.
Inthispaper,calciumalginate(CaAlg)hydrogelmembranesweresynthesizedbycross-linking
sodium alginate (SA) in CaCl solution. BSA molecular-imprinted polysiloxane polymer was
2
preparedusingβ-methoxyethylenetriethyoxysilane(KH-570)andγ-amidopropyltriethyoxysilane
(KH-550)asfunctionalmonomers, BSAastemplateandCaAlghydrogelmembraneasthematrix.
The molecular-imprinted polysiloxane (MIP) membrane was analyzed by means of transmission
electronmicroscope(TEM),scanningelectronicmicroscopy(SEM),andFouriertransforminfrared
spectroscopy(FT-IR).TheadsorptionandrecognitionpropertiesofMIPmembranewereevaluated.
In order to obtain the biomaterial that can promote cell adhesion and proliferation, fibronectin
(FN)-imprintedpolysiloxanewaspreparedbyusingFNastemplate,silanesasfunctionalmonomers,
andCaAlghydrogelmembraneasthematrix. TheresultsofcellcultureshowedthattheFN-imprinted
polysiloxanehadabettereffectoncelladhesionthanNIP.
2. MaterialsandMethods
2.1. Materials
Sodiumalginate(SA,chemicalgrade,M =3.1×105)wasboughtfromTianjinNorthernChina
W
MedicalChemicalReagent(Tianjin,China). Oxalicacidandcalciumchloride(CaCl ,analyticalgrade)
2
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was obtained from Shanghai Shen Xiang Chemical Reagent Co., Ltd. (Shanghai, China). Tianjin
Shengbin Chemical Plant (Tianjin, China) supplied β-methoxyethylene triethyoxysilane (KH-570)
and γ-amidopropyl triethyoxysilane (KH-550). Bovine serum albumin (BSA, M 67 kDa, pI 4.9),
W
ovalbumin (Ova, M 43 kDa, pI 4.7), bovine hemoglobin (Hb, M 64.0 kDa, pI 6.9) and bovine
W W
γ-globulin(Glo, M 160kDa, pI7.1)wereboughtfrom LanjiofShanghaiScience andTechnology
W
DevelopmentCompany(Shanghai,China). TheCellularBiologyInstituteoftheChineseAcademy
ofSciences(Shanghai,China)suppliedmousefibroblastcells(L929). Fibronectin(FN,440KD)was
boughtfromQianchenBiologicalTechnologyCompany(Shanghai,China). MTTCellProliferation
CytotoxicityAssayKitwasfromWuhandoctorDEbioengineeringCo.,LTD.
2.2. Apparatus
Ultraviolet spectrophotometer (TU-1901) was offered by Beijing Puxi general instrument Co.,
Ltd.,Beijing,China. Thefulltemperatureoscillationincubator(HZQ-F)wasfromDonglianelectronic
technologydevelopmentCo.,Ltd.,Harbin,China. Electronicbalance(FA2004N)wasachievedfrom
ShanghaiprecisionscienceinstrumentCo.,Ltd.,Shanghai,China. Fieldemissionelectronmicroscope
(S-4800)wasfromHitachiCo.,Ltd.,Tokyo,Japan. Constanttemperaturemagneticstirrer(85-2)was
suppliedbyShanghaiSileinstrumentfactory,Shanghai,China. Theinfraredspectrometer(Nicolet
iS50)wasformThermoScientificCorp. NewYork,NY,USA.Integratedthermalanalyzer(SDTQ600)
wasachievedfromTAInstruments-WatersLtd.,Shanghai,China. Singlefibertensiletestingdevice
(LLY-06F)wassuppliedbyLaizhouelectronicinstrumentCo.,Ltd.,Laizhou,China.
2.3. PreparationofCalciumAlginate(CaAlg)HydrogelMembranes
The calcium alginate (CaAlg) hydrogel membranes were prepared according to the
literature[36,37]. Firstly,differentamountsofSApowders(0.3046,0.4082,0.5128,0.6186and0.7254g)
wereaddedindividuallyintofivebeakersthatcontained20mLwaterandthemixturewasstirred
for2h. ThenSAsolutionswiththeSAcontentof1.5%, 2.0%, 2.5%, 3.0%and3.5%wereobtained
respectively. WeputasidetheSAsolutionsfor12htoeliminatebubblesafterultrasonicprocessing
for5min. Thenwescraped3–10goftheviscousSAsolutionintomembranesonaglassplateusing
aglassrodtwinedcopperwirewiththediameterof0.4mm. TheglassplatewiththeSAsolution
wasputintoCaCl solutionswiththeconcentrationof1.5%, 2.5%, 3.5%and5.0%respectivelyfor
2
cross-linking5h. TheresultingCaAlghydrogelmembraneswerestoredinCaCl aqueoussolution
2
(1%)forfutureuse.
2.4. PreparationofCaAlgHydrogelBasedMIPandNIPMembranes
Approximately0.100g CaAlgmembranewasplacedinvialsand10mLof1.34mg/mLBSA
solutionwasadded. After12h0.050mLKH-570and0.050mLKH-550silaneswereaddedintothe
vials. Thevialswereincubatedat20◦Cfor48hforthehydrolysisandcondensationpolymerizationof
twosilanes. ThenthesupernatantwasremovedandtheCaAlgmembranecoatedwithpolysiloxane
werewashedwith10mLoxalicacidsolution(0.5mol/L)toremovethetemplateBSA.Afterwashing
themembranewithdistilledwaterforremovalofoxalicacid,BSAmolecular-imprintedpolysiloxane
membranewaspreparedandwasnotedasMIP(BSA-MIP).
Simultaneously, non-imprinted polysiloxane was also fabricated according to the above
proceduresandnamedasNIPwhentheBSAsolutionwasreplacedwithdistilledwater.
Fibronectin(FN)-imprintedpolysiloxanemembranewaspreparedbyusingFNasthetemplate
accordingtotheabovemethod,andtheresultingimprintedpolysiloxanewasnamedasFN-MIP.
2.5. Characterizations
TheCaAlgmembranespreparedwith3.0wt%SA,2.5wt%CaCl andtheMIPmembranesby
2
usingtheCaAlgmembranesasthematrixwereusedforcharacterization.Wemeasuredthethicknesses
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of CaAlg and MIP membranes in wet form with a digital display micrometer gauge provided by
ShanghaiLuchuanCo.,LTD.
We examined the morphologies of CaAlg and MIP membranes by using a scanning electron
microscope(SEM).Transmissionelectronmicroscopy(TEM)wasusedtoinvestigatethemicroscopic
morphologyofMIPmembraneat200kVacceleratingvoltage. Epoxyresinembeddingandultrathin
sectioningwereusedtoprepareTEMsamplesonaLeicaUltracutUCTultramicrotomer.
We investigated the chemical structures of CaAlg and MIP membranes by Fourier transform
infraredspectrometry(FT-IR).
WetestedthemechanicalpropertiesoftheCaAlgandMIPmembranesinwetformbyusinga
tensiletestingmachine(LLY-06F).
2.6. AdsorptionofBSAonBSA-MIPandNIPMembranes
About100mgMIPandNIPmembraneswereputintotheglassbottlecontaining0–2.00mg/mL
BSAaqueoussolutiontodeterminetheadsorptionisotherms,respectively. Onordertodeterminethe
adsorptiondynamicsandtheimprintingefficiency(IE)ofMIPandNIPmembranes,1.36mg/mLBSA
aqueoussolutionwasused. Anultravioletspectrophotometerwasusedtomeasuretheconcentration
oftheaboveBSAaqueoussolutionatcertaintimeintervals. AccordingtoEquation(1),theequilibrium
adsorptioncapacity(Q )(mg/g)ofproteinontheMIPandNIPmembranewasdetermined.
e
Q =(C −C )V/W (1)
e 0 e
where V (mL) is the volume of BSA solution, C and C (mg/mL) is the initial and final BSA
0 e
concentration(mg/mL),andW(g)isthemassofdriedMIPorNIPmembrane.
Theimprintingefficiency(IE)ofMIPmembranewasdefinedasfollows:
IE=Q /Q (2)
MIP NIP
whereQ andQ aretheadsorptioncapacityofMIPmembraneandNIPmembrane,respectively.
MIP NIP
2.7. RecognitionPerformanceofBSA-MIPMembrane
TheMIPandNIPmembraneswereputintoeachglassbottle,whichcontains10mL1.36mg/mL
Hb,Ova,GloandBSAsolutionstoassesstherecognitionperformancesofMIP[26].
2.8. AdsorptionofFNonFN-MIPandNIPMembrane
About 20 mg of FN-MIP or NIP membrane was placed in a glass bottle containing 1 mL
0.10 mg/mL FN solution at room temperature. Then the Bradford method was used for protein
quantitation at specific time intervals [38]. The adsorption capacity (mg/g) and the imprinting
efficiency(IE)ofFN-MIPmembranewascalculatedaccordingtoSection2.6.
2.9. CellCulture
Westerilizedthefibronectin(FN)-imprintedpolysiloxane(FN-MIP)andNIPmembranesby70%
(v/v)ethanolandwashedthemwithsterilewater. TheFN-MIPandNIPmembraneswereplaced
intoaglassbottlecontaining5mL0.2mg/mLFNaqueoussolutionforadsorption2h. ThentheFN
adsorbedFN-MIPandNIPmembraneswereplacedintoacultureplate(96-well). WeseededL929
cellsontheFN-MIPandNIPmembranesatadensityof2×104 cells/well. Thecultureplatewas
culturedunderahumidifiedatmospherecontaining5%CO at37◦C.ThemorphologiesofL929cells
2
culturedonNIPandFN-MIPmembraneswereexaminedbyamicroscopeandtheadhesionbehavior
ofthecellswasinvestigatedaccordingtotheliterature[39].
The growth of cells on the membranes was checked by the MTT assay. The culture medium
wasreplacedwith1mL0.5mg/mLfreshculturemediumcontainingMTT.Theexcessmediumwas
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removedand500µLDMSOwasaddedinordertodissolvetheformazancrystalafterincubatingat
3377 °◦CC ffoorr 44 hh.. TThheenn 115500––220000 µμLLD DMMSSOOs soolulutitoionnssw weererep poouurerdedin itnoto9 69-6w-welellpl lpaltaetseasn adndth tehOe DODva vluaeluseosf
37 °C for 4 h. Then 150–200 μL DMSO solutions were poured into 96-well plates and the OD values
of the each well were determined at 492 nm by a micro plate reader.
theeachwellweredeterminedat492nmbyamicroplatereader.
of the each well were determined at 492 nm by a micro plate reader.
33.. RReessuullttss aanndd DDiissccuussssiioonn
3. Results and Discussion
33..11.. CChhaarraacctteerriizzaattiioonnss ooff MMIIPP MMeemmbbrraanneess
3.1. Characterizations of MIP Membranes
33..11..11.. MMoorrpphhoollooggiieess ooff MMIIPP MMeemmbbrraanneess
3.1.1. Morphologies of MIP Membranes
TThhee ddiiggiittaall iimmaaggeess ooff CCaaAAllgg aanndd MMIIPP mmeemmbbrraanneess iinn wweett ffoorrmm aarree sshhoowweedd iinn FFiigguurree 11.. TThhee
ttrraannssTpphaareree ndncicgyyi tooaffl CCimaaAAagllgge smm oeefmm CbbarrAaannlgee wwanaadss hhMiiggIhhPee mrr ttehhmaannb rMManIIPPes mm inee mmwbberrtaa nnfoeer.. mTTh haeer epp oosllhyyosswiillooexxdaa nninee ooFnnig ttuhhreee s s1uu.rr ffTaahcceee
otorffa MMnsIIpPPa rfifeillnmmc yiin noccfrr eCeaaassAeeddlg t thmheee tmthhibiccrkkannneeess wss oaofsf ththhiegeh mmeerem mthbbarnraa nMneeI (P(ffr rmoomemm 00b.1.r10a02n2e± ±. T00h..00e11 99p ottooly 00s.i.1l1o88x00a ±±n e00 .o.00n22 88th mme mmsu))r faaanncdde
dodfee ccMrreeIaaPss eefiddlm tthh ieen ttcrrraaennasssppedaarr eethnnecc yyth ooiffc kCCnaaeAAssllgg o mmf teehmme bbmrraaennmeeb.. SrSauunrreffaa (ccfeer oSSmEEMM 0. 1iimm02aa gg±ee 0ss. 0dd1iiss9pp tlloaa yy0 .tt1hh8ee0 CC ±aa 0AA.0llgg2 8mm meemmmbb) rraaannndee
((dFFeiicggruuerraees e22daa ))t hwweaa tssr sasmmnsoopooattrhheeenrrc ttyhh oaannf CtthhaaaAtt loogff m MMeIImPP bmmraeenmmebb. rrSaaunnreefa ((cFFeii ggSuuErrMee 22ibbm)).a. gTTehhsee drrioosuupgglahhynn eethsssse ooCffa NNAIIlPPg mmmeeemmmbbbrrraaannneee
ww(Faaigss ussriimme 2iillaaa)rr wttooa stthh saamtt ooooff tMMheIIPPr tmmhaeenmm tbbhrraaatnn oeef bbMeeccIaaPuu msseee tmthhbeeyyra wwneee rr(eeF ibbgoouttrhhe cc2oobaa)tt.ee Tddh wwe iirttohhu ttghhheen ppeoosllsyy ossfiill oNoxxIaaPnn mee.. eFFmiiggbuurrraeen 33e
iwiss attshh ees imTTEEilMMar iitmmo aathggaeet ooofff MMMIIIPPP mmmeeemmmbbbrrraaannneee ,, biiennc awwuhhsieicc hthh memyaa wnnyye rBBeS SbAAo--tiihmm cpporraiitnnettdee ddw ppitoohll yythssiiell oopxxoaalnynesei lppoaaxrrattniiccell.ee sFs iwwguiittrhhe a3a
didsii aathmmeee ttTeeErr Mooff iaamrrooauugnnedd o 22f 00M00––I33P22 m00 nnemmmb wwraeenrreee ,ii ddinee nnwttiihfifiieecddh.. many BSA-imprinted polysiloxane particles with a
diameter of around 200–320 nm were identified.

(a) (b)
(a) (b)
FFiigguurree 11.. DDiiggiittaall pphhoottooss ooff CCaaAAllgg mmeemmbbrraannee ((aa)) aanndd MMIIPP mmeemmbbrraannee ((bb))..
Figure 1. Digital photos of CaAlg membrane (a) and MIP membrane (b).

(a) CaAlg membrane × 10,000 (b) MIP × 10,000
(a) CaAlg membrane × 10,000 (b) MIP × 10,000
Figure 2. SEM images of CaAlg membrane (a) and MIP membrane (b).
Figure 2. SEM images of CaAlg membrane (a) and MIP membrane (b).
Figure2.SEMimagesofCaAlgmembrane(a)andMIPmembrane(b).
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Figure 3. TEM images of MIP membrane.
FFiigguurree 33.. TTEEMM iimmaaggeess ooff MMIIPP mmeemmbbrraannee..
3.1.2. Identification of the Membrane Formation
33..11..22.. IIddeennttiiffiiccaattiioonn ooff tthhee MMeemmbbrraannee FFoorrmmaattiioonn
Figure 4 shows the FT-IR spectra of CaAlg membrane and MIP membrane. The –OH group of
FFiigguurree 44 sshhoowwss tthhee FFTT--IIRR ssppeeccttrraa oof fCCaaAAlglg mmemembbrarnane eanadn dMMIPI Pmmemembrbarnaen. eT.hTe h–eO–HO gHrogurpou opf
the CaAlg exhibited the peak at 3626 cm−1. The strong absorption of –COO− in CaAlg was observed
tohfet hCeaAClagA elxgheibxihteibdi ttehde ptheeakp eaat k36a2t6 3c6m26−1.c Tmh−e 1s.trTonhge astbrsoonrgptaiobns oorfp –tiCoOnOo−f i–nC COaOA−lg iwnaCs aoAblsgerwveads
at 1610~1500 and 1500~1400 cm−1. The strong absorption peak of –COO− from CaAlg was weakened
aotb 1se6r1v0e~d15a0t01 6a1n0d~ 11550000~a1n4d001 5cm00−~1.1 T4h00e csmtro−n1g. Tahbesosrtprotniogna pbesaokrp otfi o–nCpOeOak− forfo–mC OCaOA−lgfr womasC waeAalkgewneads
in the MIP membrane, indicating the form of cross-linked structure between CaAlg and the
wine athkee nMedIPin mtheemMbrIPanme,e minbdriacantei,nign dthicea tifnogrmth eoff ocrrmosos-flcinroksesd- lisntkruecdtustrreu cbteutrweebeent wCeaeAnlCg aaAnldg atnhde
polysiloxane. The MIP membranes showed strong adsorption at 922 and 1044 cm−1, which was
pthoelypsoilloyxsailnoex.a nTeh.eT MheIPM mIPemmebmrabnreasn esshoshwoewd esdtrsotnrogn gadasdosroprtpiotino nata t992222 aanndd 11004444 ccmm−−11, ,wwhhiicchh wwaass
attributed to the Si–O–Si stretching vibration. The significant difference between CaAlg and MIP
aattttrriibbuutteedd ttoo tthhee SSii––OO––SSii ssttrreettcchhiinngg vviibbrraattiioonn.. TThhee ssiiggnniiffiiccaanntt ddiiffffeerreennccee bbeettwweeeenn CCaaAAllgg aanndd MMIIPP
membranes appeared at 922 cm−1, revealing that the polysiloxane of MIP membrane was created on
mmeemmbbrraanneess aappppeeaarreedd aatt 992222 ccmm−−11, ,rreevveeaalilningg tthhaatt tthhee ppoollyyssiillooxxaannee ooff MMIIPP mmeemmbbrraannee wwaass ccrreeaatteedd oonn
the surface of CaAlg membrane.
tthhee ssuurrffaaccee ooff CCaaAAllgg mmeemmbbrraannee..
100
100
90 MIP membrane 1630
90 MIP membrane 1630 922
e 922
enc 80 3460
smittancansmitta787000 3460 CCaaAAllgg mmeemmbbrraannee 11004444
TranTr6600 33662266 116699331535
50 33112277 15351396
50 1396
40
40 3600 3200 2800 2400 2000 1600 1200 800
3600 3200 28W00ave2n4u0m0ber2 /0 0c0m-11600 1200 800
Wavenumber / cm-1

Figure 4. FT-IR spectra of CaAlg membrane and MIP membrane.
FFiigguurree 44.. FFTT--IIRR ssppeeccttrraa ooff CCaaAAllgg mmeemmbbrraannee aanndd MMIIPP mmeemmbbrraannee..
3.1.3. Mechanical Properties of CaAlg and MIP Membrane
3.1.3. Mechanical Properties of CaAlg and MIP Membrane
3.1.3. MechanicalPropertiesofCaAlgandMIPMembrane
Figure 5 shows the stress-strain curves of CaAlg membrane and the MIP membrane in wet
Figure 5 shows the stress-strain curves of CaAlg membrane and the MIP membrane in wet
formF. iTghuer eM5IPs hmoewmsbtrhaenes twreasss -ssttrroaningecru trhvaens CoafAClgaA mlgemmbermanber abneceaaunsed tthhee inMteIrPacmtieomnsb braentweeiennw theet
form. The MIP membrane was stronger than CaAlg membrane because the interactions between the
fpoorlmys.iTlohxeanMeI Panmde malbgriannaetew. aTshset rsoinlagneer tchoaunpClinaAg lgagmenetm KbrHan-5e5b0 emcaoulseecuthleesi nhtaevraec tbioenens bdeitfwfueseendt htoe
polysiloxane and alginate. The silane coupling agent KH-550 molecules have been diffused to
paloglyinsailtoex aannde aanddsoarlbg ionna tCe.aTAhlge ssihlaenlles tchoruopulignhg ealgecetnrtoKstHat-i5c5 i0ntmeroalcetciuolne sbhetawveeebne ethned pifrfoutsoendattoeda lagminiantoe
alginate and adsorb on CaAlg shells through electrostatic interaction between the protonated amino
agnroduapdss oinrb KoHn-C5a5A0 lagnsdh etlhlse tcharrobuogxhyelaletec trgorsotuaptisc iinnt earlagcitnioatne b[e4t0w].e eTnheth ceopnrdoetonnsaattieodna omf inKoHg-r5o7u0p asnind
groups in KH-550 and the carboxylate groups in alginate [40]. The condensation of KH-570 and
KKHH--555500 aoncdcutrhreedca trob ocxoymlaet eingtoro buepisngin aa plgoilnyastielo[x4a0n].eT nheetwcoonrdke onnsa tthioen CoafAKlgH m-57e0mabnrdanKeH su-5r5fa0coec. cSuor rtehde
KH-550 occurred to come into being a polysiloxane network on the CaAlg membrane surface. So the
tMoIcPo mmeeminbtorabneeisn sghaowpoeldy siinlocrxeaanseedn emtwecohraknoicnatlh peroCpaeArltgiems. embranesurface. SotheMIPmembranes
MIP membranes showed increased mechanical properties.
showedincreasedmechanicalproperties.
PPoollyymmeerrss 22001188,, 1100,, x1 7F0OR PEER REVIEW 77 ooff 1144
Polymers 2018, 10, x FOR PEER REVIEW 7 of 14
0.9
0.9
0.8
2
0.8
MPa)Pa)000...677 121... CMCaaIAAP llmgg emmmeebmmrbabnrraaennee 112
s (M0.6 2. MIP membrane
sminal Stress,nal Stress, (000000......345345
Nomi0.2
o0.2
N0.1
0.1
0.0
0.00.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
0.0 0.2 0.4 St0r.e6tch，0λ.8 1.0 1.2 1.4
Stretch，λ
Figure 5. Stress–strain curves of CaAlg membrane and MIP membrane.
FFiigguurree 55.. SSttrreessss––ssttrraaiinn ccuurrvveess ooff CCaaAAllgg mmeemmbbrraannee aanndd MMIIPP mmeemmbbrraannee..
3.2. Adsorption of MIP Membrane Prepared with Different CaCl2 and SA Concentrations
33..22.. AAddssoorrppttiioonn ooff MMIIPP MMeemmbbrraannee PPrreeppaarreedd wwiitthh DDiiffffeerreenntt CCaaCCll2 aanndd SSAA CCoonncceennttrraattiioonnss
2
Figure 6 shows the adsorption capacity and the IE of MIP membrane prepared with different
Figure 6 shows the adsorption capacity and the IE of MIP membrane prepared with different
CaCl2F aignudr eSA6 schoonwtesnttsh. eTahdes aodrspotiropntiocnap caacpiatyciatyn doft MheIPIE anofdM NIIPP mmeemmbbrraannee pinrcerpeaarseedd wwiitthh dthifef erraeisnet
oCCfaa CCthlle22 aaSnnAdd SScAoAn cccooennnttteeranntttisso.. nTT hhbeee caaaddussosoerr ppottifio ontnh ceca apfpoaarccmitityayto iooffnM M oIPIfP am annoddrNe N IpPIoPml ymesmielmobxrbaarnnaeen eion nicn rcethraeesae ssdeudwrf iwatchietth ho etfh rCea iarsaAeislogef
of the SA concentration because of the formation of more polysiloxane on the surface of CaAlg
mtheemSAbrcaonnec epnrterpaatiroend bweciathu sae ohfitghheefro rSmAa tcioonntoefnmt. oTrheep ocloynsicleonxtarnaetioonn tohfe sCuarCfalc2 edoifdCn’atA wlgomrke monb rathnee
ampdreesmpoarbprretaidonnwe iqpthureaapnhatiirtgeyhd eo rfw SMiAthIPc oa an nhteding Nth.eITPrh SmeAceo mncobcnernatentrneatst.. i oTTnhhoee fMcCoaInPCc lem2ndetrmiadtbnior’tannw eoo fra kdCsoaonCrbtlh2e edda imddnsoo’rtre p wptirooorntkeq iuonna tn httahitney
adsorption quantity of MIP and NIP membranes. The MIP membrane adsorbed more protein than
tohfeM NIPIPa mndemNbIPramnee mdbidr aanneds. tThhe eIEM rIePamcheemd borvaenre 2a.d0.s oHrobwedevmeor,r ethper ointefilnuetnhcaen othf ethNe IcPomnceemntbrraatnioend oidf
the NIP membrane did and the IE reached over 2.0. However, the influence of the concentration of
SaAnd atnhde ICEarCeal2c hoend tohvee rIE2. 0w. Haso wnoetv esri,gtnhieficinanfltu. eBnaceseodf tohne ctohne cmenetcrhatainoincaolf SpAerfaonrdmCanacCel 2oofn CthaAeIlgE
mSwAea msanbnordat nsCiegsan,C i2fil.25c a ownntt . t%Bha eCs eIadEC olw2n aatnshd en 3mo.t0e cwshigta nn%iifc iSacAlanp wte.r efBorear msuesaden dco enfoo rft hCthea eAm plegrecmhpaaenrmaictbiaorlan np oeefsr ,fCo2ar.5mAwlagnt ac%en dCo aft hCCel2 aMAanIlPgd
mm3.0eemmwbbtrr%aannSeeAss ,i wn2. e5thr ewe utf os%elld oCwfoaiCrntlgh2 esatnpudrde i3pe.sa0.r wa tito %n oSfAC waAerlge aunsdedt hfeorM thIPe mpreempbarraatnieosni noft hCeaAfollglo awnidn gthsetu MdiIePs.
membranes in the following studies.
50 6 50 6
4550 MIP membrane 6 4550 MIP membrane 6
y (mg/g)mg/g)334344050505 NIMNIEEIIIPPP mmmeeemmmbbbrrraaannneee 4545 y (mg/g)mg/g)334344050505 NIMNIEEIIIPPP mmmeeemmmbbbrrraaannneee 4545
Adsorption capacitAdsorption capacity (1122112235050505050 123123IEIE Adsorption capacitAdsorption capacity (1122112235050505050 123123IEIE
5 5
0 0 0 0
01.0 1.5 2.0 2.5 3.0 3.5 4.00 01.0 1.5 2.0 2.5 3.0 3.5 4.00
1.0 1.5The co2n.c0entrati2o.n5 of SA3 .(0wt%)3.5 4.0 1.0 1.5The co2n.c0entrati2o.n5 of SA3 .(0wt%) 3.5 4.0
Th(ea c)o n1c.e5n trwatito n% o fC SaAC (wl2t %) Th(eb co) n2c.e5nt rwatito n% o fC SaAC (wl2t %)
50 (a) 1.5 wt % CaCl2 6 50 (b) 2.5 wt % CaCl2 6
4550 MIP membrane 6 4550 MIP membrane 6
45 NMIIPP mmeemmbbrraannee 5 45 NMIIPP mmeemmbbrraannee 5
y (mg/g)mg/g)3343405050 INIEEIP membrane 445 y (mg/g)mg/g)3343405050 INIEEIP membrane 445
n capacitapacity (22230550 33IEIE n capacitapacity (22230550 33IEIE
AdsorptioAdsorption c11112505050 1212 AdsorptioAdsorption c11112505050 1212
5 5
0 0 0 0
01.0 1.5 2.0 2.5 3.0 3.5 4.00 01.0 1.5 2.0 2.5 3.0 3.5 4.00
1.0 1.5The co2n.c0entrati2o.n5 of SA3 .(0wt%) 3.5 4.0 1.0 1.5The co2n.c0entrati2o.n5 of SA3 .(0wt%) 3.5 4.0
Th(ec c)o n3c.e5n twrattio %n o Cf SaAC (lw2 t%) Th(ed c)o n5c.e0n twrattio %n o fC SaAC (lw2 t%)
(c) 3.5 wt % CaCl2 (d) 5.0 wt % CaCl2
FFiigguurree 66.. AAddsosorprptitoino ncacpaapcaictyit yanadn dIEI Eof otfheth MeIMP ImPemmebmrabnrea npereppraerpeadr ewditwh idthiffdeirfefnert eCnatCCl2a Canld aSnAd
cFoignuceren t6ra. tAiodnsso. rption capacity and IE of the MIP membrane prepared with different CaCl2 an2d SA
SAconcentrations.
concentrations.
3.3. Adsorption Kinetics of BSA on MIP and NIP Membrane
3.3. Adsorption Kinetics of BSA on MIP and NIP Membrane
Polymers2018,10,170 8of14
Polymers 2018, 10, x FOR PEER REVIEW 8 of 14
3.3. AdsorptionKineticsofBSAonMIPandNIPMembrane
FFiigguurree 77 sshhoowwss tthheeB BSSAAa addsosorprptitoionnd dynyanmamicisccsu cruvrevseosn oMn MIPIaPn adnNd INPImPe mmebmrabnrea.nWe.i tWhiinth5i.n5 5h.,5t hhe,
tahdes oadrpstoiropntiroante rwataes whaigs hhiagnhd atnhde ethqeu ielqiburiiluibmriuadms oardpstoiornptcioapna ccaiptyac(Qitye) (rQeae)c rheeadch7e5d% 7f5o%rb footrh bNotIhP NanIPd
aMnIdP MmIePm mbreamnbe.raTnhee. aTdhseo arpdtsioornprtaioten orfatteh eofB tShAe- BimSApr-iinmtepdripnoteldys piloolxyasnileoixnanthei sinp tahpiesr pwapaserm wuacsh mfaustcehr
ftahsatnert hthaatno fthtahte oBf StAhe- iBmSpAr-iinmtepdrihnyteddr ohgyedl,rowgheilc, hwthoiochk 2to4ohk t2o4 rhe atoch rethacehe tqhuei leibqruiiulimbriaudmso ardpstioornpt[i3o7n] .
[A3f7t]e. rA5f.t5erh 5.t5h eh Qthee oQfeM ofI PMmIPe mmebmrabnreanwea wsa2s.1 28.1t8im tiemsehs ihgihgehretrh tahnanN NIPIPm meemmbbrraannee,,w wiitthh aa vvaalluuee ooff
2288..8833 mmgg//gg. .TThhee aaddssoorrppttioionn ccaappaacciittyy ooff BBSSAA bbyy NNIIPP mmeemmbbrraannee aatt 55..55 hh wwaass 1133..5522 mmgg//gg.. TThhee hhiigghheerr
aaddssoorrppttiioonn ccaappaacciittyy ooff BBSSAA oonn MMIIPP wwaass dduuee ttoo tthhee ffoorrmm ooff tthhee rreeaacctteedd ssiitteess aanndd tthhee ccoommpplleemmeennttaarryy
ccaavviittiieess bbeettwweeeenn BBSSAA aanndd tthhee MMIIPP mmeemmbbrraannee.. BBeeccaauussee tthhee ssiitteess aanndd tthhee iimmpprriinnttiinngg ccaavviittiieess wweerree oonn
tthhee ssuurrffaaccee ooff tthhee MMIIPP mmeemmbbrraannee aanndd BBSSAA ccoouulldd sspprreeaadd mmoorree eeaassiillyy,, ssoo tthhee MMIIPP mmeemmbbrraannee hhaadd aa
ffaasstteerr aaddssoorrppttiioonn rraattee ffoorr iittss tteemmppllaattee..
40
g) 35 MIP membrane
g/
m NIP membrane
y ( 30
acit 25
p
a
c
n 20
o
orpti 15
s
d
A 10
5
0
0 5 10 15 20 25
Adsorption time (h)
Figure 7. Adsorption dynamics of BSA on MIP and NIP membrane.
Figure7.AdsorptiondynamicsofBSAonMIPandNIPmembrane.
3.4. Adsorption Thermodynamics of BSA on MIP and NIP Membrane
3.4. AdsorptionThermodynamicsofBSAonMIPandNIPMembrane
Figure 8 shows the adsorption thermodynamics of BSA on MIP and NIP membrane. With the
Figure8showstheadsorptionthermodynamicsofBSAonMIPandNIPmembrane. Withthe
increase of initial BSA concentration, the adsorption capacity increased rapidly and gradually
increaseofinitialBSAconcentration,theadsorptioncapacityincreasedrapidlyandgraduallytended
tteonbdaeldan tcoe .bTahlaenecqe.u Tilhiber ieuqmuialidbsriourmpt iaodnscoarppaticointy c(aQpa)coitfyB (SQAe)o onf MBSIPAm oenm MbIrPan meewmabsrmanoer ewthasa nm2o.0re5
e
than 2.05 times than that of NIP membrane. When the concentration of BSA is low, the amount of
timesthanthatofNIPmembrane. WhentheconcentrationofBSAislow,theamountofBSAisnot
BsuSfAfi cisie nnottt osufifflilctiehnet stpoe fciilfil tchbe isnpdeicnigfich boilnedainndg hcaovleit ay,ndso ctahveityQ, soin tchree aQsee sinwcriethastehse winitchr ethaese inocfreBaSsAe
e
of BSA concentration. The adsorption capacity of MIP film tends to be stable when almost all the
concentration. TheadsorptioncapacityofMIPfilmtendstobestablewhenalmostalltheimprinting
imprinting sites are occupied.
Psiotlyemsearsr e20o1c8,c 1u0p, xie FdO.R PEER REVIEW 9 of 14
The rebinding curves of NIP and MIP membrane can be followed by the Freundlich model
equation: 50
Qe = Qf · Ce1/n (3)
MIP membrane
g)40 NIP membrane
where Qf is the rough g/rebinding capacity (mg/g), Ce is the equilibrium concentration of BSA
m
(mg/mL), and 1/n is the ay(dsorption intensity. The linearized plots of lnQe versus lnCe for the NIP and
MIP membrane are showacited3 0in Figure 9. The lnQe versus lnCe all exhibited good linearity with an R >
p
0.98. ca
n 20
o
pti
or
s
d10
A
0
0.0 0.5 1.0 1.5 2.0 2.5
The initial concentration of BSA(mg/ml)
Figure 8. Curves of adsorption thermodynamics of MIP and NIP membrane.
Figure8.CurvesofadsorptionthermodynamicsofMIPandNIPmembrane.
4.0
3.6 R=0.9977
3.2
2.8
2.4
Qe R=0.9863
n 2.0
l
1.6
1.2
0.8 MIP membrane
NIP membrane
0.4
0.0
-2.5-2.0-1.5-1.0-0.5 0.0 0.5 1.0 1.5 2.0
lnC
e

Figure 9. Curves of lnQe to lnCe of MIP and NIP membrane.
3.5. Specific Adsorption of BSA on MIP and NIP Membrane
Figure 10 shows the Qe of MIP and NIP membrane for BSA and the competitive protein Ova,
Hb and Glo, respectively. It is demonstrated that the BSA-imprinted membrane showed good
adsorption selectivity for the template BSA. The Qe of BSA on the MIP membrane was 2.2 times as
much as that of Hb or Ova, and 5.4 times as much as that of Glo. The higher BSA adsorption capacity
of the MIP membrane is due to the generation of BSA affinity sites and the complementary cavities
in the polycondensation of functional silanes. The recognition sites of BSA were not complementary
to the Hb, Ova and Glo. So the competitive proteins (Ova, Hb and Glo) were less likely to be
adsorbed on the BSA-imprinted membrane. In contrast, the NIP membrane adsorbed much less BSA
than MIP membrane because there were no specific recognition sites on NIP membrane in the
absence of template proteins. The molecular volume of Glo was larger than that of BSA and the other
competitive proteins, so the Qe of γ-Glo on MIP membrane was much lower than that of any other
proteins.
Polymers 2018, 10, x FOR PEER REVIEW 9 of 14
50
MIP membrane
g)40 NIP membrane
g/
m
y(
Polymers2018,10,170 cit30 9of14
a
p
a
c
on 20
The rebinding curvesorptiof NIP and MIP membrane can be followed by the Freundlich
s
modelequation: Ad10
Q =Q ·C 1/n (3)
e f e
0
whereQfistheroughrebinding0c.0apacity0.(5mg/g)1,.0Ceisth1e.5equili2b.r0iumco2.n5centrationofBSA(mg/mL),
and 1/n is the adsorption intensity. TThhee inlitiinale caonrcizenetrdatiponl ooft sBSoAf(lmngQ/ml)versus lnC for the NIP and MIP
e e
membraneareshowFiegdurien 8.F Ciguurvrees 9o.f aTdhsoerlpntiQone tvheerrmsuosdylnnaCmeiacsl loef xMhIPib aintedd NgIPo omdemlibnreanaer.i tywithanR>0.98.
4.0
3.6 R=0.9977
3.2
2.8
2.4
Qe R=0.9863
n 2.0
l
1.6
1.2
0.8 MIP membrane
NIP membrane
0.4
0.0
-2.5-2.0-1.5-1.0-0.5 0.0 0.5 1.0 1.5 2.0
lnC
e

FigFuirgeu9re. C9. uCruvrevseso fofl nlnQQee ttoo llnnCCee oof fMMIPIP anadn dNINP ImPemmebmrabnrea. ne.
3.5. Specific Adsorption of BSA on MIP and NIP Membrane
3.5. SpecificAdsorptionofBSAonMIPandNIPMembrane
Figure 10 shows the Qe of MIP and NIP membrane for BSA and the competitive protein Ova,
Figure10showstheQ ofMIPandNIPmembraneforBSAandthecompetitiveproteinOva,Hb
Hb and Glo, respectiveely. It is demonstrated that the BSA-imprinted membrane showed good
andGadlos,orrepstipoenc tsievleeclyti.vIittyi sfodre tmheo tnesmtrpaltaetde BthSaAt. tThheeB QSeA o-fi mBSpAr ionnte tdhem MemIPb mraenmebsrhanoew wedasg 2o.2o dtimadess oarsp tion
selectmivuicthy afso trhtaht eoft Hemb oprl aOtveaB, aSnAd. 5T.4h teimQees aosf mBuScAh aosn ththate oMf GIlPo. mTheem hbigrhaenre BwSAa sad2s.o2rtpitmioens caapsamcituyc h as
thatooff Hthbe MorIPO mvae,mabnrdan5e. 4is tdimuee tsoa tshme guecnheraastiothna otf oBfSGAl aof.fiTnhitey hsiitgesh eanrdB SthAe caodmsoprlepmtieonntacrayp caacviittyieso fthe
MIPmin ethme bproalnyceoinsddeunseattioonth oef gfuenncetiroantiaol nsiloafnBesS. AThaef firencoitgynsitiitoens sainteds othf eBScAo mwperlee mnoetn ctoamryplceamveintiteasryin the
to the Hb, Ova and Glo. So the competitive proteins (Ova, Hb and Glo) were less likely to be
polycondensationoffunctionalsilanes. TherecognitionsitesofBSAwerenotcomplementarytothe
adsorbed on the BSA-imprinted membrane. In contrast, the NIP membrane adsorbed much less BSA
Hb, Ova and Glo. So the competitive proteins (Ova, Hb and Glo) were less likely to be adsorbed
than MIP membrane because there were no specific recognition sites on NIP membrane in the
on the BSA-imprinted membrane. In contrast, the NIP membrane adsorbed much less BSA than
absence of template proteins. The molecular volume of Glo was larger than that of BSA and the other
MIPmembranebecausetherewerenospecificrecognitionsitesonNIPmembraneintheabsenceof
competitive proteins, so the Qe of γ-Glo on MIP membrane was much lower than that of any other
tempplartoetepinros.t e ins. ThemolecularvolumeofGlowaslargerthanthatofBSAandtheothercompetitive
protePionlysm,esros 2t0h18e, 1Q0,e x oFfORγ -PGEElRo RoEnVIMEWIP membranewasmuchlowerthanthatofanyotherpro1t0e ionf 1s4.
40
36
g/g)32 MIP membrane
m NIP membrane
y (28
cit24
a
p
ca20
g
n16
di
n
bi12
e
R 8
4
0
BSA Ova Hb Glo
Proteins used in specific adsorption experiment
Figure 10. Adsorption capacity of MIP membrane and NIP membrane for BSA, Ova, Hb and Glo,
Figure 10. Adsorption capacity of MIP membrane and NIP membrane for BSA, Ova, Hb and
respectively.
Glo,respectively.
3.6. Rebinding Behavior of FN on FN-MIP and NIP Membrane
Fibronectin (FN) can promote cell growth, increase cell wall rate and enhance cell metabolism.
If a material can selectively bind FN, then it has better biocompatibility. However, FN is very
expensive. FN molecularly imprinted polymers (FN-MIP) was prepared according to the optimum
condition of BSA-imprinted polymer. The adsorption dynamic curves of FN on FN-MIP and NIP
membrane are shown in Figure 11. It is found that the adsorption capacity almost reached
equilibrium after 3.0 h. The FN-MIP adsorbed more FN than NIP, and the IE reached 2.3. The
adsorption capacity of FN was much lower than that of BSA, because its initial concentration is only
one tenth of BSA. The molecular weight of FN is 440 kD, which is significantly greater than BSA (6.8
kD). In the rebinding process, the hole in the material is more difficult to allow FN in and out. The
adsorption happened mainly on the surface. So the time of adsorption equilibrium was shorter than
that of BSA.
4.0 6
FN-MIP membrane
3.5
g) NIP membrane 5
mg/3.0 IE
city (2.5 4
a
p
n ca2.0 3IE
o
orpti1.5 2
ds1.0
A
1
0.5
0.0 0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Adsorption time (h)
Figure 11. The adsorption dynamic curves of FN on FN-MIP and NIP membrane.
3.7. Cell Culture on FN-MIP and NIP Membranes
Fukazawa et al. [41] provided a new molecular imprinting method using fibronectin (FN) to
capture cells as a template. In this paper, FN-imprinted polysiloxane membrane was prepared
according to the optimum conditions of BSA molecular imprinting. After adsorption of FN, L929
cells were cultured on the FN-MIP and NIP membrane, and Figure 12 shows the morphologies of
L929 cells on the FN-MIP and NIP membranes after 4 h. The amount of cells adhered to FN-MIP
membrane was much more than the cells adhered to the NIP membrane, indicating that FN-MIP
membrane was more appropriate for culturing cell than NIP membrane.
Polymers 2018, 10, x FOR PEER REVIEW 10 of 14
40
36
g)32 MIP membrane
g/
m NIP membrane
y (28
cit24
a
p
a20
c
g
n16
di
n
bi12
e
R 8
4
0
BSA Ova Hb Glo
Proteins used in specific adsorption experiment
Figure 10. Adsorption capacity of MIP membrane and NIP membrane for BSA, Ova, Hb and Glo,
Polymerress2p0e1c8t,iv10e,ly17. 0 10of14
3.6. Rebinding Behavior of FN on FN-MIP and NIP Membrane
3.6. RebindingBehaviorofFNonFN-MIPandNIPMembrane
Fibronectin (FN) can promote cell growth, increase cell wall rate and enhance cell metabolism.
If a mFiabtreorniaelc tcinan(F sNel)eccatnivpelryo mbointed cFelNlg, rtohwenth i,ti nhcarse abseetcteerll wbiaolclormatpeaatnibdileitnyh. aHncoewceevllemr, eFtaNb oilsi smve.rIyf
eaxmpeantesriivael.c FaNn smeloecleticvuellayrlbyi nimdpFrNin,ttehde npiotlhyamsebrest t(eFrNb-iMocIoPm) wpaatsib pilrietyp.aHreodw aecvceorr,dFiNngi stov etrhye eoxppteimnsuivme.
cFoNndmitoiolenc uolfa rBlySAim-imprpinritnedtedp oplyomlyemrser(.F NTh-Me aIPd)sowrapstiporne pdayrneadmaiccc ocrudrivnegs toof tFhNeo opnti mFNum-McIoPn adnitdio NnIoPf
mBSeAm-bimrapnrei natered psholoywmne ri.nT hFeigaudrseo r1p1t.i oInt disy nfaomunicdc uthrvaet stohfeF NadosonrFpNtio-Mn IcPaapnadcitNy IPalmmeomstb rraenacehaerde
esqhuowilinbriinumFi gauftreer1 31..0I thi.s Tfohuen FdNth-MatIPth eadasdosrobrepdti omnocraep FaNcit ythaalnm oNsItPr,e aacnhde dtheeq uIEil irberaiuchmeda ft2e.3r.3 T.0hhe.
aTdhseoFrpNt-iMonI Pcaapdascoirtyb eodf mFNo rweaFsN mthuacnh NloIwP,ear nthdatnh ethIaEt roefa BchSeAd, 2b.e3c.aTuhsee aitds sionriptitailo cnocnacpeanctirtaytioofnF iNs ownalys
omnuec thenlothw oerf tBhSaAn. tThhate omfoBlSeAcu,lbaerc wauesieghitts oinf iFtNial icso 4n4c0e nktDra, twiohnicihs oisn slyigonnifeictaenntthlyo gfrBeSaAte.r Tthhaenm BoSlAec u(6la.8r
kwDei)g. hInt othfeF Nrebisin4d40inkgD p,rwocheiscsh, itshsei ghnoilfie ciann tthlye gmreaatteerriatlh iasn mBoSrAe (d6i.f8fikcDul)t. tIon athlleowre bFiNnd iinn ganpdro ocuests. ,Tthhee
ahdosleoripnttihoen mhaaptepreianleids mmaoirneldy ioffinc tuhlet tsouarflalocwe. FSoN tihne atnimdeo ouft .aTdhsoerapdtsioonrp etqiounilihbarpiupmen wedasm sahionrlyteor nthtahne
tshuartf aocfe B.SSAo.t hetimeofadsorptionequilibriumwasshorterthanthatofBSA.
4.0 6
FN-MIP membrane
3.5
g) NIP membrane 5
mg/ 3.0 IE
y ( 4
cit 2.5
a
p
ca 2.0 3IE
n
o
orpti 1.5 2
ds 1.0
A
1
0.5
0.0 0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Adsorption time (h)
FFiigguurree 1111.. TThhee aaddssoorrppttiioonn ddyynnaammiicc ccuurrvveess ooff FFNN oonn FFNN--MMIIPP aanndd NNIIPP mmeemmbbrraannee..
3.7. Cell Culture on FN-MIP and NIP Membranes
3.7. CellCultureonFN-MIPandNIPMembranes
Fukazawa et al. [41] provided a new molecular imprinting method using fibronectin (FN) to
Fukazawa et al. [41] provided a new molecular imprinting method using fibronectin (FN) to
capture cells as a template. In this paper, FN-imprinted polysiloxane membrane was prepared
capture cells as a template. In this paper, FN-imprinted polysiloxane membrane was prepared
according to the optimum conditions of BSA molecular imprinting. After adsorption of FN, L929
accordingtotheoptimumconditionsofBSAmolecularimprinting. AfteradsorptionofFN,L929cells
cells were cultured on the FN-MIP and NIP membrane, and Figure 12 shows the morphologies of
wereculturedontheFN-MIPandNIPmembrane,andFigure12showsthemorphologiesofL929cells
L929 cells on the FN-MIP and NIP membranes after 4 h. The amount of cells adhered to FN-MIP
ontheFN-MIPandNIPmembranesafter4h. TheamountofcellsadheredtoFN-MIPmembranewas
membrane was much more than the cells adhered to the NIP membrane, indicating that FN-MIP
muchmorethanthecellsadheredtotheNIPmembrane,indicatingthatFN-MIPmembranewasmore
membrane was more appropriate for culturing cell than NIP membrane.
appropriateforculturingcellthanNIPmembrane.
Polymers 2018, 10, x FOR PEER REVIEW 11 of 14

(a) FN-MIP, 4 h (b) NIP, 4 h
Figure 12. Photographs of adhesive L929 cells on MIP and NIP after cultured for 4 h.
Figure12.PhotographsofadhesiveL929cellsonMIPandNIPafterculturedfor4h.
Figure 13 shows cell-viability cultured with FN-MIP membrane, NIP membrane and tissue
culture polystyrene board (TCPS) after 1, 3 and 5 days in culture, respectively. L929 cells on FN-MIP
membrane grew more rapidly than those on NIP membrane during 5 days of incubation. Statistical
analysis showed that the live cells on FN-MIP membrane were higher than those on NIP membrane
because the FN-MIP membrane could adsorb more FN than NIP membrane, which made it was
more suitable for the adhesion and growth of cells. Pan et al. [42,43] introduced the cell-adhesive
peptide (RGDS) onto a thermo-responsive cell culture substrate. The substrate could be used as a
highly efficient novel system. The FN-imprinted polysiloxane based on CaAlg membrane is low cost
and easy to be prepared, which would show its great potential applications in cell sheet technology.
1.0
0.9 FN-MIP membrane
0.8 NIP membrane
TCPS
2) 0.7
9
n (4 0.6
sio 0.5
p
or 0.4
s
d
A 0.3
0.2
0.1
0.0
1 day 3 day 5 day
Culture time
Figure 13. MTT assay of L929 cell-viability on FN-MIP membrane, NIP membrane, and TCPS (*p <
0.05).
4. Conclusions
Bovine serum albumin (BSA) molecular-imprinted polysiloxane (MIP) membrane was
successfully synthesized using β-methoxyethylene triethyoxysilane (KH-570) and γ-amidopropyl
triethyoxysilane (KH-550) as functional monomers, BSA as template and CaAlg hydrogel film as the
matrix. The strength of MIP membrane was higher than that of CaAlg membrane. The stable
adsorption capacity of BSA on MIP membrane was 2.18 times higher than that of non-imprinted
polysiloxane (NIP) membrane, with a value of 28.83 mg/g. BSA-imprinted polysiloxane membrane
could recognize BSA by using bovine hemoglobin (Hb), ovalbumin (Ova) and bovine γ-globulin
(Glo) as competitive proteins.
Fibronectin (FN)-imprinted polysiloxane membrane was prepared by using FN as the template
according to the optimum conditions of BSA molecular imprinting. The FN-MIP adsorbed more FN
than NIP, and the IE reached 2.3. After adsorption of FN, L929 cells were cultured on the FN-MIP