Macromolecular Nanotechnology Tailored interfaces for biosensors and cell-surface interaction studies via activation and derivatization of polystyrene-block-poly(tert-butyl acrylate) thin films Chuan Liang Feng a,1 , Anika Embrechts a , Ilona Bredebusch b , Anita Bouma c , Ju ¨ rgen Schnekenburger b , Marı ´a Garcı ´a-Parajo ´ c,2 , Wolfram Domschke b , G. Julius Vancso a , Holger Scho ¨ nherr a, * a University of Twente, MESA + Institute for Nanotechnology and Faculty of Science and Technology, Department of Materials Science and Technology of Polymers, P.O. Box 217, 7500 AE Enschede, The Netherlands b Gastroenterologische Molekulare Zellbiologie, Medizinische Klinik und Poliklinik B, Westfa ¨ lische Wilhelms-Universita ¨t, Domagkstr. 3A, 48149 Mu ¨ nster, Germany c University of Twente, MESA + Institute for Nanotechnology and Faculty of Science and Technology, Optical Techniques Group, P.O. Box 217, 7500 AE Enschede, The Netherlands Received 20 January 2007; accepted 6 March 2007 Available online 18 March 2007 Abstract Thin spin-coated films of polystyrene-block-poly(tert-butyl acrylate) (PS 690 -b-PtBA 1210 ) on various substrates are intro- duced as versatile, robust reactive platform for the immobilization of (bio)molecules for the fabrication of tailored bioin- terfaces. The films are characterized by high stability and (bio)reactivity due to the presence of a glassy PS and a reactive PtBA block, respectively. The selective deprotection of the tert-butyl-ester groups in the PtBA skin layer by hydrolysis under acidic conditions, the activation with N-hydroxysuccinimide and the subsequent derivatization with amino function- alized (bio)molecules were investigated. Based on contact angle, FTIR spectroscopy and XPS, fluorescence microscopy and AFM data, it was shown that the (bio)molecules were coupled covalently to the polymer films and that high molecular coverages up to 2.4 poly(ethylene glycol) (PEG) molecules per nm 2 (M n = 500 g/mol) were obtained. Organic dyes, pol- yamidoamine dendrimers, polypeptides, proteins and amino end-functionalized DNA were efficiently and homogeneously immobilized on the PS-PtBA platforms. Grafting of x-amino functionalized PEG afforded surfaces with substantially reduced non-specific adsorption of proteins and DNA. Owing to the glassy nature of PS and the covalent amide linkages, the derivatized films showed excellent stability under a broad range of processing conditions. Finally, the viability of PS 690 -b-PtBA 1210 platforms as versatile biointerfaces was demonstrated in DNA hybridization experiments, as well as cell-surface interaction studies using pancreatic cancer and K562 cells. Ó 2007 Elsevier Ltd. All rights reserved. 0014-3057/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2007.03.021 * Corresponding author. Tel.: +31 53 489 3170; fax: +31 53 489 3823. E-mail address: [email protected](H. Scho ¨ nherr). 1 Present address: Max-Planck-Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany. 2 Present address: NanoBioEngineering Laboratory, Barcelona Scientific Park (PCB), Josep Samitier 1-5, 08028 Barcelona, Spain and Institucio ´ Catalana de Recerca i Estudis Avanc ¸ats, ICREA, 08010 Barcelona, Spain. European Polymer Journal 43 (2007) 2177–2190 www.elsevier.com/locate/europolj EUROPEAN POLYMER JOURNAL MACROMOLECULAR NANOTECHNOLOGY
Transcript
EUROPEAN
European Polymer Journal 43 (2007) 2177–2190
www.elsevier.com/locate/europolj
POLYMERJOURNAL
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Macromolecular Nanotechnology
Tailored interfaces for biosensors and cell-surfaceinteraction studies via activation and derivatization
of polystyrene-block-poly(tert-butyl acrylate) thin films
a University of Twente, MESA+ Institute for Nanotechnology and Faculty of Science and Technology, Department of Materials Science
and Technology of Polymers, P.O. Box 217, 7500 AE Enschede, The Netherlandsb Gastroenterologische Molekulare Zellbiologie, Medizinische Klinik und Poliklinik B, Westfalische Wilhelms-Universitat,
Domagkstr. 3A, 48149 Munster, Germanyc University of Twente, MESA+ Institute for Nanotechnology and Faculty of Science and Technology, Optical Techniques Group,
P.O. Box 217, 7500 AE Enschede, The Netherlands
Received 20 January 2007; accepted 6 March 2007Available online 18 March 2007
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Abstract
Thin spin-coated films of polystyrene-block-poly(tert-butyl acrylate) (PS690-b-PtBA1210) on various substrates are intro-duced as versatile, robust reactive platform for the immobilization of (bio)molecules for the fabrication of tailored bioin-terfaces. The films are characterized by high stability and (bio)reactivity due to the presence of a glassy PS and a reactivePtBA block, respectively. The selective deprotection of the tert-butyl-ester groups in the PtBA skin layer by hydrolysisunder acidic conditions, the activation with N-hydroxysuccinimide and the subsequent derivatization with amino function-alized (bio)molecules were investigated. Based on contact angle, FTIR spectroscopy and XPS, fluorescence microscopyand AFM data, it was shown that the (bio)molecules were coupled covalently to the polymer films and that high molecularcoverages up to �2.4 poly(ethylene glycol) (PEG) molecules per nm2 (Mn = 500 g/mol) were obtained. Organic dyes, pol-yamidoamine dendrimers, polypeptides, proteins and amino end-functionalized DNA were efficiently and homogeneouslyimmobilized on the PS-PtBA platforms. Grafting of x-amino functionalized PEG afforded surfaces with substantiallyreduced non-specific adsorption of proteins and DNA. Owing to the glassy nature of PS and the covalent amide linkages,the derivatized films showed excellent stability under a broad range of processing conditions. Finally, the viability ofPS690-b-PtBA1210 platforms as versatile biointerfaces was demonstrated in DNA hybridization experiments, as well ascell-surface interaction studies using pancreatic cancer and K562 cells.� 2007 Elsevier Ltd. All rights reserved.
0014-3057/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.
A diversity of organic or polymeric surfaces hasbeen investigated for the fabrication of bio-func-tional interfacial architectures for potential use asaffinity coatings in genomics [1], proteomics [2],and as biosensors for biomedical purposes [3,4].Organic or polymeric surfaces can also be designedto serve as biointerfaces between the biological envi-ronment and man-made materials [6,5]. Biointer-faces are central to biology and medicine and arecrucial for implants, biosensors, drug delivery, andmany other fields [6]. In particular, biointerfacesplay an important role in cell engineering in the fieldof microfabrication, cell adhesion, and cell activityin terms of metabolism (Fig. 1a and b) [7].
Biomimetic films can be constructed as mono ormultilayers, or in the form of thin (polymeric) films.Because of their defined and flexible surface chemis-try and design, self-assembled monolayer (SAM)-based platforms, e.g. based on thiol–gold chemistry,have been intensively investigated in this context
Fig. 1. (a) Schematic of a biosensor based on organic or polymericimmobilized via a surface-attached antibody. The detection of the releamount of analyte bound to the sensor. (b) Schematic of cell interactichemical in nature, or both. (c) Schematic of PSn-b-PtBAm diblock copovia hydrolysis, activation and immobilization of amino end-functionalizfilms.
and found widespread application for biosensing,diagnostic assays, high throughput drug screening,and studies in cell biology [8].
In order to improve the long term stability, toenhance the versatility, as well as to addressessential requirements, such as combined robust(bio)functionalization with controlled molecularloading, topographical patterning with nanometerscale precision and control of modulus, polymeric
platforms have received increasing attention[9–21]. While the physisorption of macromolecularmaterials to various surfaces provides tailored sur-face properties in many cases, only the immobiliza-tion of tailored branched macromolecules withmultidendate/multivalent functionality [16] or thecovalent grafting of functional polymers yield therequired robust and stable surface properties[17,18,22]. Thick polymer films, in which the sur-face-attached macromolecules can be present inthe so-called brush regime [19,23], are most fre-quently synthesized via the so-called grafting-frommethodologies [20].
thin films. The analyte (A) displaces a label (L) that has beenased label, e.g. by optical techniques, allows one to quantify theng with a structured surface. The structure may be topographic,lymer and the corresponding film structure and functionalization
ed (bio)molecules on the PtBA skin layer of the diblock copolymer
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In addition to the tailored surface chemicalcomposition, truly functional biointerfaces callfor advanced design and preparation in order tomatch the sophisticated recognition ability of bio-logical systems, which includes the control ofchemical composition on multiple length scales
spanning the 100 lm to the sub-100 nm regime.Specifically, combined topographic and chemicalpatterns on surfaces are required in order tomatch typical spacings of proteins and proteinclusters on the 5–100 nm length scale and entirecells and cell aggregates on the 10–100 lm lengthscale [24–27].
The currently available platforms do not yet pro-vide sufficiently sophisticated control on multiplelength scales from the 100 lm down to the molecu-lar size range, together with precise control of(bio)chemistry, ligand density, spacing and orienta-tion. Recent developments to fabricate miniaturizedpatterns in a massively parallel manner rely on‘‘top–down’’ techniques, such as photolithography[28] and soft lithography [29–31]. Microcontactprinting (lCP) and selective molecular assemblypatterning (SMAP) [32] are among the promisingapproaches for producing (bio)chemical patternson various solid substrates. As demonstrated, thesetechnologies are compatible with the mentionedpolymeric platforms [32,33]. In the future self-orga-nized block copolymer thin films [34] may contrib-ute to the development of new generations ofbiointerfaces with improved control over biofunc-tionality on the required length scales [35]. Theself-assembly of block copolymers into microphaseseparated morphologies allow one to access the15–100 nm size regime and together with advancedsynthetic methods a broad range of reactive blocksis in principle available. To enable further progressin the mentioned and other areas the developmentof novel and versatile approaches, as well as plat-forms, to obtain robust patterned bio-platforms istargeted.
As shown in this paper, block copolymer-basedreactive thin film platforms were developed for thefabrication of tailored biointerfaces. The surfacechemistry of the poly(tert-butyl acrylate) (PtBA)skin layer in polystyrene-block-poly(tert-butyl acry-late) (PS690-b-PtBA1210) diblock copolymer thinfilms [36] was systematically investigated to provideoptimized surface functionalization. The viability ofthe developed platforms as versatile biointerfaceswas shown in DNA hybridization experiments andcell-surface interaction studies.
2. Experimental
2.1. Materials
PS690-b-PtBA1210 diblock copolymers (Mw =202.4 kg/mol, polydispersity index (PDI) 1.03)were purchased from Polymer Source Com-pany (Dorval, Canada) and were used as received.Amino functionalized-labeled PEG (denoted asPEGn–NH2) was purchased from Nektar UK Com-pany (Mn = 500, 2000 or 5000 g/mol, PDI = 1.1).Fluoresceinamine was acquired from MolecularProbes Inc., The Netherlands. Fifth-generation(G5) amine-terminated PAMAM dendrimers wereobtained from Aldrich as a 5% wt methanolic solu-tion. DNA samples (probe DNA terminated withprimary amino group on the 5 0-terminus: 25 mer5 0-GGA ATG TGC CAT ACC GAA TCC GTGT-3 0; Cy5-labeled target DNA: 5 0-CAC GGA TTCGGC ATG-3 0-Cy5, Cy5-labeled mismatch DNA:5 0-TGT GCC TAA GCC ATA-3 0-Cy5, MWG BIO-TEC AG, Ebersberg, Germany) were used asreceived (HPLC-purified, purity >98%). The DNAsamples were stored at �4 �C until use. AlexaFluor�594 labeled isothiocyanate and bovine serumalbumin (BSA) with Alexa Fluor�594 conjugatewere bought from Molecular Probes Inc. and wereused as received. Lissamine rhodamine B sulfonylchloride was bought from Molecular Probes Inc.Poly(L)lysine (Product code P1274; Mw (viscosity) =84 kg/mol) was bought from Sigma and stored at�20 �C. Fibronectin (purity >95% pure determinedby SDS-PAGE) was purchased from Roche Diag-nostics GmbH (Penzberg, Germany). Fibronectinwas stored at 4 �C until use.
Cell culture media and trypsin solution for celldissociation were obtained from Cambrex Biosci-ence (Verviers, Belgium). Cell culture sera werebought from PAA Laboratories GmbH (Colbe,Germany). All other reagents for cell culture workand cell fixation were from Sigma Aldrich GmbH(Taufkirchen, Germany) and the cell culture plasticmaterial from Greiner (Germany).
K562 cells were a kind gift of the Tumor Immu-nology Laboratory of the Nijmegen Center forMolecular Life Sciences, the Netherlands. K562were transfected to express either the transmem-brane protein K-SIGN or its fluorescent mutantK-SIGN-YFP. The cells were kept in an incubator(37 �C, 5% CO2) and transferred to fresh mediumtwice a week (0.1/0.2 mL of old cell culture into5 mL fresh medium). Iscove’s 1 mg/mL neomycin,
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10% fetal calf serum, glutamine and antibioticantimyotic solution (pH 7.4) were used as amedium.
The investigated human pancreatic ductal adeno-carcinoma cell line PaTu 8988T were obtained fromGerman Collection of Microorganisms and CellCultures (DSMZ, Braunschweig, Germany).
Silicon (111) wafers (Okmetric N/Phosphorous(100) wafers, thickness 381 ± 15 lm), glass coverslides (Menzel-Glaser, Braunschweig, Germany),or optical glass for cell work (Menzel-Glaser,Braunschweig, Germany) were used as substrates.These substrates were cleaned prior to use by anoxygen plasma treatment (Pressure of O2: 0.5 bar;Current: 30 mA) using an Elektrotech twin systemPF 340 apparatus or a SPI Plasma PrepTM PlasmaCleaner (Structure Probe Inc, West Chester,USA). Alternatively, the substrates were cleanedin piranha solution (solution of 1:3 (v/v) 30%H2O2 and concentrated H2SO4) for 15 min andthen rinsed with copious amounts of high-puritywater (Millipore Milli-Q water) and ethanol. Cau-
tion: Piranha solution should be handled withextreme caution; it has been reported to detonateunexpectedly.
2.2. Film preparation
Thin films were prepared by spin coating poly-mer solutions in toluene (conc. 10 mg/mL) ontooxygen plasma-cleaned silicon wafers or glass sub-strates. The samples were spun at 3000 rpm for30 s using a P6700 spin coater (Specialty CoatingSystems Inc). All spin-coated samples were annealedat 135 �C for 24 h in vacuum before analysis. A filmthickness of 90 ± 5 nm was determined by ellipsom-etry (see below).
2.3. Hydrolysis
The thin polymer films were hydrolyzed atroom temperature in 3 M aqueous HCl, neat tri-fluoroacetic acid (TFA) or in a HCl atmospherefor specified intervals. For the gas phase hydroly-sis, the samples were exposed to vapors of HCl atroom temperature by placing them 10 mm abovethe liquid surface in a closed beaker filled with6 M HCl. After specified time intervals, sampleswere removed from the beaker. All hydrolyzedfilms were rinsed three times thoroughly usingMilli-Q water and were finally dried in a streamof nitrogen.
2.4. Activation of hydrolyzed polymer films
The hydrolyzed polymer films were activated byimmersion in an aqueous solution of 1-ethyl-3-(di-methylamino)-propylcarbodiimide (EDC, 1 M) andN-hydroxysuccinimide (NHS, 0.2 M) for 30 min.The samples were then rinsed with Milli-Q water,dried in a stream of nitrogen, and used immediatelythereafter.
2.5. Immobilization of (bio)molecules on activated
polymer films
PEGn–NH2 was coupled from phosphate buffer(PB) solution as described previously (100 lMPEG solution in PB, pH = 7.4) [37]. After the reac-tion the samples were taken out of the correspond-ing solution and thoroughly rinsed with Milli-Qwater. Fluoresceinamine and G5 PAMAM werecoupled from 2.0 · 10�4 M PB solution (pH = 7.4)and 5.0 · 10�4 M solution in methanol, respectively.BSA, fibronectin, and PLL were coupled to the acti-vated films using the corresponding solutions(100 lM in PB, pH = 7.4). DNA solutions in PBwere prepared with a concentration of 1.0 ·10�6 M (pH = 7.4). For covalent coupling to sur-face-bound reactive ester groups, the polymerfilms were immersed into the probe DNA solution.After a reaction time of 60 min, the samples weretaken out and rinsed with PB (pH = 7.4). Forhybridization, the films were placed in the targetDNA solution in PB (100 · 10�9 M, T = 25 �C,pH = 7.4), and after 30 min the films were takenout followed by a thorough rinse with PB. Allexperiments were carried out at T = 25 ± 2�C.
2.6. Labeling of adsorbates
Labeling of surface-immobilized fibronectin andPAMAM dendrimers was carried out by reactionwith lissamine rhodamine B sulfonyl chloride(100 lM in PB, pH = 7.4) for 30 min, PLL waslabeled with Alexa Fluor�594 labeled isothiocya-nate. Afterwards the samples were rinsed thor-oughly with PB, followed by Milli-Q water andfinally dried in a stream of nitrogen.
2.7. Cell preparation
Pancreas tumor cells (PaTu 8988T) were culturedin DMEM supplemented with 5% FCS, 5% horseserum and 2 mM L-glutamine at 5% CO2. For anal-
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ysis of adherence cells were trypsinized, seeded subconfluent on manufactured substrates, cultured for24 h and fixated with 1% glutaraldehyde in PBS[38]. After PBS washing cell adherence was studiedusing light microscopy and AFM.
Samples containing K562 cells were prepared bytaking a given volume of cell containing solutionout of the incubator followed by cell counting. Cellwere centrifuged, the medium removed and phos-phate buffered saline (PBS, pH 7.4) was added tothe cells. This setup was performed because themedium contains proteins which can adhere to thePLL functionalized surface reducing the attachmentof cells to the PLL. A (0.6 mL) droplet of cell-PBSsolution (approximately 90 · 104 cells/mL was thendeposited on the surface and cells were allowed tosettle/attach for 30–45 min (at 37 �C)). Finally, thesample was rinsed with PBS to remove loose cells.Microscopic experiments were performed in PBS.
Transmission mode FTIR spectra (spectral reso-lution of 4 cm�1, 1024 scans) were obtained usinga BIO-RAD model FTS575C FTIR spectro-meter equipped with a liquid nitrogen-cooled cryo-genic mercury cadmium telluride (MCT) detector.Background spectra were obtained using oxygenplasma-cleaned silicon wafers.
2.9. Ellipsometry
The measurements were performed with a Plas-mos SD2002 instrument (rotating analyzer method)with wavelength k = 632.8 nm and incidence angleequal to 70�. Film thicknesses were determinedbefore sample processing and during sample pro-cessing on (un)treated sample surfaces to serve ascontrol experiments. Furthermore samples wereplaced in a vacuum oven for 2 h at room tempera-ture before experiments were performed (to over-come height differences due to water uptake ofPEG from the air). Layer thicknesses were deter-mined using a 2-layer model. The first layer consistsof the substrate, the second layer consists of PS-b-PtBA or, after treatment, PS-b-PtBA with PEG.Depending on the top layer the refractive index ofPS-b-PtBA or PEG was used (PS-b-PtBA: 1.513,PEG: 1.4638 [39]; DNA: 1.375 [40]). For the Si sub-strate layer a refractive index of 3.865 and anabsorption coefficient of k = �0.019 was used. Amean squared error (MSE) method was used to
quantify the difference between experimental andcalculated model data. The Marquardt-Levenbergalgorithm was used to determine the best fit. Mea-surements were performed at ten different spots onthe surface (for PEG and polymer). Average layerthickness and errors were determined over these 10data points.
2.10. X-ray photoelectron spectroscopy (XPS)
XPS spectra were recorded on a PHI Quantum2000 Scanning ESCA microprobe using a mono-chromated X-ray beam (Al-anode; 100 lm diame-ter/25 W X-ray beam) scanned over 700 lm ·300 lm area at a fixed take-off angle of 45� (sam-pling depth �8 nm). Charging of all samples wascorrected by correcting the peak positions relativeto the position of neutral carbon at 285.0 eV.Atomic concentrations were determined by numeri-cal integration of the relative peak areas using theMultipak software with supplied sensitivity factors(C1s: 0.314; O1s: 0.733; N1s: 0.499) [41].
2.11. AFM
The AFM measurements were carried out with aNanoScope IIIa multimode AFM using a 10 lmscanner, as well as on a PicoForce unit operatedwith a NanoScope IVa controller (Digital Instru-ments/Veeco, Santa Barbara, CA). Tapping modeAFM scans were performed with silicon cantile-vers/tips (Nanosensors, Wetzlar, Germany) in air.The instrument was operated at frequencies slightlylower than the natural resonance frequency of thecantilever in air, the free amplitude was kept con-stant. The amplitude-damping (setpoint) ratio wasadjusted to �0.9.
2.12. Contact angles measurements
The contact angles were measured on a contactangle microscope (Data Physis, OCA 15Plus) withMilli-Q water as the probe liquid. Advancing andreceding contact angles were measured at roomtemperature.
2.13. Fluorescence microscopy
Fluorescence microscopy images of dry sampleson glass cover slips were recorded at room temper-ature on a Zeiss LSM 510 confocal laser scanningmicroscope using a Plan-Apochromat� 63·/1.4
Fig. 2. Static contact angles measured for PS-b-PtBA films onoxidized silicon under different reaction conditions vs. reactiontime.
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NA oil-immersion objective. For excitation a633 nm HeNe laser and the 488 nm line of an Ar+
laser were used. The fluorescence emission of thesedyes was recorded with photomultiplier tubes(Hamamatsu R6357) after spectral filtering. A500–550 nm bandpass filter was selected for experi-ments with fluoresceinamine (emission wavelength:k = 520 nm) and a 650 nm longpass filter wasselected for the labeled target Cy5-labeled DNA(emission wavelength: k = 670 nm) and BSA-AlexaFluor�594 (emission wavelength: k = 630 nm),respectively. Fluorescence micrographs were alsotaken using a Olympus IX 70 fluorescence micro-scope equipped with a U-MWG-2 fluorescent filterand a BA590 filterblock. Optical microscopy wasperformed using a Olympus BX 60 (standard setup).
3. Results and discussion
Thin spin-coated films of PS690-b-PtBA1210 on sil-icon or glass expose an approximately 8 nm thick skinlayer of PtBA [36] that can be hydrolyzed and furtherfunctionalized following activation with NHS(Fig. 1c). The resulting film architecture is very robustowing to the presence of water-insoluble, thermallystable PS cylinders in the interior of the film and thecovalent nature of the attachment of the (bio)mole-cules. As shown below, a wide range of moleculescan be grafted to these films with high loading.
3.1. Investigation of the surface chemistry
of PS690-b-PtBA1210 films
The surface chemistry of thin spin-coated films ofPS690-b-PtBA1210 on silicon or glass was investi-gated first under different conditions in order toobtain optimized covalent coupling routes that yieldaccess to robust tailored biointerfaces. The previ-ously reported hydrolysis data of the acid labiletBA groups in the PtBA skin layer in 3 M aqueousHCl [36] was complemented by hydrolysis experi-ments in trifluoroacetic acid (TFA), as well as ingaseous HCl (Fig. 1c).
Static water contact angles (CA) measured on thefilms after hydrolysis under different conditions areplotted in Fig. 2 as a function of hydrolysis time.It can be observed that the CA decreased very fastafter treatment with TFA, while it decreased slowerwhen the hydrolysis was carried out in HCl vapor.The hydrolysis in 3 M aqueous HCl proceededmuch slower. In full agreement with FTIR spectro-scopic data (see Supplementary material Figure S-1)
it was found that the hydrolysis of the polymer filmssurface did not reach completion in 3 M HCl com-pared to the other two reaction conditions. Thisobservation can be attributed to differences in reac-tant penetration into the film and mass transportlimitations. These results also suggest that a morerapid and likely efficient hydrolysis of PS690-b-PtBA1210 films can be achieved in TFA.
Tapping mode atomic force microscopy (TM-AFM) revealed that the films stay intact even afterhydrolysis of the entire PtBA in the films and repeatedthorough rinsing and drying. However, an apparentroughening of the film surface was observed afterhydrolysis (Figure S-2, Supplementary material).The mean roughness (Ra) [42] estimated from1.0 lm2 topographical images increased from 0.9nm before hydrolysis to 1.4 nm (3 M HCl), 5.1 nm(HCl, gas) and 5.3 nm (TFA), respectively, afterhydrolysis and drying in a vacuum oven. The morepronounced roughening for the completely hydro-lyzed films (after HCl (g) or TFA treatment) is inparts attributed to a restructuring of the film (as aresult of the decrease in overall film thickness) andpossibly swelling due to water uptake. From a com-parison of the surface roughness values, we can con-clude that the surface area of the films after hydrolysisin HCl gas and TFA will be larger compared to filmshydrolyzed in 3 M HCl solution by �15%.
3.2. Covalent coupling of PEGn–NH2 to activated
PS690-b-PtBA1210 films
The carboxylic acid groups generated in thehydrolysis reaction of the PS690-b-PtBA1210 films
Fig. 3. Thickness changes of PS-b-PtBA films after graftingPEGn–NH2 (n = 500, 2000, 5000) layer for various times asdetermined by ex situ ellipsometry.
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can be activated with NHS ester moieties using arecipe as described in reference [37]. The subsequentcovalent coupling reaction of amino groups termi-nated PEG500–NH2 with the activated NHS estergroups at and near the surface of PS690-b-PtBA1210
films from buffered aqueous solution was investi-gated by ellipsometry, XPS, and FTIR spectros-copy. Grafted PEG layers render these thin filmplatforms antibiofouling [22].
Evidence for covalent attachment of the PEGthrough amide linkage formation was found in theFTIR spectra of grafted films (Supplementary mate-rial, Figure S-3). Two new bands at 1645 cm�1 and1544 cm�1 (attributed to amide I and amide IIvibrations, respectively) were observed in the spec-tra. By contrast, the typical PEG C–O ether stretchvibration was not clearly resolved due to spectraloverlap with C–O vibrations in the PtBA andPAA, respectively.
Additional evidence for the successful surfacemodification reaction of PEG500–NH2, as well asthe high coverage, was obtained in XPS measure-ments using a take-off angle of 45� (sampling depth8 nm; see Supplementary material, Figure S-4) [36].For neat PS690-b-PtBA1210 films, only the C1s peaksat 284.3 eV and O1s at 532.0 eV [43] were observed.After coupling of PEG–NH2 to the polymer filmsurface, not only the C1s and the O1s peaks, but alsoa N1s peak at 401.5 eV, were observed. The C/Nratio of PEG500–NH2 functionalized films wasabout 7. The surface composition in the sampleddepth hence corresponds to a polymer film in which�10% has been reacted with PEG500–NH2. A graft-ing density of PEG of �2.9 PEG molecules per nm2
was thus calculated, which is in good agreementwith the ellipsometry results shown below.
The kinetics of immobilization of PEGn–NH2, aswell as the grafted thickness, were determined byellipsometry. Fig. 3 shows the thickness of thegrafted PEG layer (deduced from the increase infilm thickness after the coupling reaction) for thethree molar masses investigated for different reac-tion times. The maximum thickness of PEGincreased from �1.9 nm, �2.5 nm to �4.0 nm forPEG500–NH2, PEG2000–NH2 and PEG5000–NH2,respectively. Corresponding grafting densities of�2.4, 0.8, and 0.5 PEG molecules per nm2 were thuscalculated [44].
The maximum thickness for PEG500–NH2 is towithin the experimental error identical to the previ-ously reported PNHSMA polymer system, yetexceeds the coverage on a 2-dimensional SAM by
a factor of 3 [37]. Thus also for the block copolymerplatforms discussed here, high molecular loading ofthe activated films can be achieved in coupling reac-tions with PEG500–NH2.
A significant reduction of protein adsorption toPEG functionalized surfaces was observed in exper-iments with dye-labeled bovine serum albumin(BSA) (see Supplementary material, Figure S-5).Without any PEG coating, strong fluorescenceemission was observed, which indicates a significantcoverage of the corresponding fluorescent adsor-bate. With increasing thickness of the graftedPEG500–NH2 layers, a significant reduction ofadsorbate coverage was observed. The ratio of thefluorescence emission intensity on films with1.9 nm thick PEG layer vs. the background fluores-cence of neat PS690-b-PtBA1210 was about 2. There-fore, it can be concluded at this point that thegrafted PEG layers with 1.9 nm thickness effectivelyinhibit the immobilization of BSA [22,45,46].
3.3. Covalent coupling of biomolecules to activated
PS690-b-PtBA1210 films
The covalent grafting of amino functionalizedmolecules, as demonstrated above for PEGn–NH2,can be extended to other amino functionalized(bio)molecules. The grafting of probe DNA (25mer) on activated PS690-b-PtBA1210 films from buf-fer solution (100 nM) was studied by ellipsometry,as shown in Fig. 4. A maximum coupling thicknessof 4.0 nm was achieved, which is again similar to thedata obtained for PNHSMA films [37]. The maxi-mum grafting thickness of 25 mer DNA on SAMs
Fig. 4. Thickness of the grafted DNA layer on PS690-b-PtBA1210
film vs. reaction time. The thickness was determined ex situ byellipsometry.
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under comparable conditions was reported to beabout 2.2 nm [47], which is about 2 times lower thanthe value obtained on PS690-b-PtBA1210 films. Thisobservation suggests that the DNA has been cou-pled not only to NHS ester groups exposed at thevery film surface, but also within the topmost regionof the film.
To further investigate the spatial homogeneity ofthe coupling reactions of relevant dyes, hyper-branched polymers, polypeptides and proteins onhydrolyzed and subsequently activated PS690-b-PtBA1210 films, fluorescence microscopy was used.The immobilization of G5 PAMAM dendrimers(as hyperbranched bioplatform) [17,18,48], dye-labeled BSA, as well as fibronectin and PLL werestudied. The unlabeled adsorbates were subse-quently labeled by reaction with lissamine rhoda-mine B sulfonyl chloride and Alexa Fluor�594
Fig. 5. Fluorescence microscopy images and fluorescence emission histofilm grafted with PLL (Alexa Fluor�594 labeled), and (c) PS-b-PtBA fi
labeled isothiocyanate, respectively. As a referenceexperiment, a neat PS690-b-PtBA1210 film was mea-sured (Fig. 5 and Figure S-6, Supplementarymaterial).
No fluorescence emission was detected for theneat (Figure S-6a) and for the PEG5000 coveredfilms (Fig. 5a), while a homogeneous fluorescenceemission was observed for the other (labeled) adsor-bates (Fig. 5b and c and S-6). These data show thatproteins and polypeptides can be successfully immo-bilized and that homogeneous coverages areobtained.
To demonstrate the functionality of the fabri-cated biointerfaces, the hybridization of targetDNA was investigated using fluorescence micros-copy. The corresponding micrographs are shownin Fig. 6. Similar to immobilization of proteins onPS690-b-PtBA1210 films, amino group-functionalizedprobe DNA with 25-mer was first immobilized onthe NHS ester activated PS690-b-PtBA1210 filmsfrom solution. Then the dye-labeled complementarytarget DNA reacted with polymer films modifiedwith covalently attached probe DNA in solution.
Strong fluorescence emission was detected, asshown in Fig. 6a. The observed fluorescence emis-sion demonstrates that the probe DNA is accessibleto solution-borne complementary target DNA,hence activated PS690-b-PtBA1210 films can be usedin principle as a platform to detect the DNA. As ref-erence experiment, the total mismatch target DNAwas applied; after rinsing no fluorescence emissioncould be detected (Fig. 6b).
The successful immobilization of biomoleculeson PS690-b-PtBA1210 films suggests that this poly-mer system may be very useful in applications wheretailored biointerfaces are required. This platformmay thus be of use to prepare robust biosensors
grams of (a) PS-b-PtBA film grafted with PEG5000 (b) PS-b-PtBAlm grafted with fibronectin (labeled with lissamine rhodamine B).
Fig. 6. Fluorescence microscopy images and fluorescence emission histograms (insets) of (a) PS-b-PtBA film grafted with probe DNAfrom PB solution (100 nM, pH: 7.4), followed by hybridization with complementary DNA. (b) PS-b-PtBA film grafted with probe DNAfrom PB solution (100 nM, pH: 7.4), followed by hybridization with total mismatch DNA from PB solution (100 nM, pH: 7.4).
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and, as shown below, to address important aspectsof cell membrane organization.
3.4. Cell adhesion studies on PtBA-b-PS films
The feasibility to fabricate tailored biointerfacesusing the block copolymer platform introducedhere, was addressed in studies of the interaction ofnon-adherent (K562) [49] and adherent (pancreaticcancer) cells with functionalized PS690-b-PtBA1210
films.The wide field optical microscopy images shown
in Fig. 7 indicate that the interaction of K562 cells
Fig. 7. Representative wide field optical micrograph of (a) PLL covePEG500–NH2 covered PS690-b-PtBA1210 film (scale: 150 lm · 150 lm). (PLL) vs. polymer surface composition.
with the platform depends on the surface chemistryof the polymers films. While PLL leads to substan-tial surface coverages of these cells, a grafted layerof PEG500 effectively eliminates cell adsorption.
It is important to mention that a large number ofcell receptors (ICAMs, the integrin receptor LFA-1and the pathogen recognition receptor SIGN) canbe easily transfected in K562, making it a veryrobust cell line for immunology studies. The cluster-ing of these receptors on the cell membrane playsan important role in cell function [50,51] and assuch, the study of protein clustering in the presenceof modified substrates like the ones presented
red PS690-b-PtBA1210 films (scale: 150 lm · 150 lm) and (b) ofc) Histogram of cell-surface coverage (normalized to coverage on
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here can lead to unique information in the way mol-ecules interact with each other in order to build upfunctional clusters that trigger cell signaling andresponse.
Subsequently, the interactions of adherentPaTu8988T pancreas adenocarcinoma cells [52]with PLL, fibronectin and PEG functionalizedPS690-b-PtBA1210 films were investigated. Thesecells grow as monolayers and are characterized bya high proliferation rate and rapid migration. Rest-ing cells appear rounded and develop an average cellheight of 15 lm. Migrating cells appear flattenedwith a spread fibroblast like morphology. MigratingPaTu8988T cells show extended filopodia cell struc-tures involved in the formation of new focaladhesions.
In the experiments reported here the blockingability of PEG layers with different number averagemolar mass Mn was investigated. It is known thatthe chain length and grafting density of PEG mole-cules exert a large influence on the antifouling prop-erties [53]. For blocking of proteins an optimum Mn
of 2000 g/mol was reported [16c]. The wide field
Fig. 8. Representative wide field optical micrograph of (a) PLL covercovered PS690-b-PtBA1210 film (scale: 90 lm · 160 lm), (c) PS690-b-PtBAPS690-b-PtBA1210 film (scale: 90 lm · 160 lm). (e) Histogram of cell-surface composition.
optical microscopy images shown in Fig. 8 provideevidence for a marked dependence of the cell-sur-face interaction on surface chemistry as well as thedifferent blocking capability of the three types ofPEG-modified films. For full layers of PLL andfibronectin the cells not only adhered, but alsostretched out on the film (Fig. 8a and b). This indi-cates a full adhesive capacity of PaTu8988T cellsand the unrestricted formation of focal adhesions.
Surfaces grafted with different molar masses ofPEGn–NH2 showed markedly different behavior.The number of cells, as well as their morphology,was found to differ systematically (see also Supple-mentary material, Figure S-8). On PEG500 modifiedfilms, larger numbers of cells were observed, whileon PEG2000 and PEG5000 only very few isolatedround, and hence not adhering, cells were detected.The lowest number of cells per unit area wereobserved for grafted PEG2000 (Fig. 8e). Thus, inagreement with the literature, PEG with high Mn
was found to be much more efficient for blockingthe surface compared to low Mn PEG moleculesand an optimum was observed for PEG2000 [16c].
ed PS690-b-PtBA1210 films (scale: 90 lm · 160 lm) (b) fibronectin
1210 film (scale: 90 lm · 160 lm), and (d) PEG5000–NH2 coveredsurface coverage (normalized to coverage on PLL) vs. polymer
Fig. 9. TM-AFM height images of fixated cancer cells on (a), (c) fibronectin and (b), (d) PEG5000-functionalized PS-b-PtBA (dataacquired in air).
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The differences in number of cells on the differentPEG layers can be related to the differences in block-ing ability against integrin cell adhesion receptors.
The interaction of the pancreatic cancer cells withblock copolymer films with two widely different sur-face chemical compositions was finally interrogateddown to the nanometer scale by TM-AFM. Cellswere fixated using a procedure that leaves subcellu-lar structures as filopodia intact and preserves cellmorphology in liquid media.
The widely different interaction of the cells withsurfaces functionalized with a cell-adhesive protein(fibronectin) and a protein-resistant PEG manifestthemselves in a different cell morphology (Fig. 9).On fibronectin the cells show a spread-out appear-ance, similar to the optical microscopy images (videsupra). Many very long filopodia can be recognizedthat possess a width very similar to the distancesgiven by the intrinsic microphase separated mor-phology of the underlying block copolymer film(�90–100 nm). By contrast, on PEG the cell-film
boundary appears to be sharp and the presence offilopodia was not observed.
In both cases the TM-AFM images reveal theunderlying microphase separated morphology ofthe block copolymer film. This morphology reflectslikely some minor differences in topography, as wellas different local energy dissipation due to the differ-ent underlying materials inside the film [54]. It mustbe emphasized that the surface of the films, which isin contact with the cancer cells, is homogeneously
functionalized. The functionalization extends intoin the top few nanometers of the PtBA skin layeras can be concluded from the thickness of this layer[36] and the high coverages observed for, e.g., cova-lently grafted PEGn–NH2 (vide supra).
By using appropriate procedures that lead to theexposition of both constituent blocks at the surface,the exploitation of the microphase separation ofblock copolymers may open avenues towards thecontrolled fabrication of self-organized nanoscalestructured and functionalized biointerfaces [35].
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The complete absence of filopodia on the PEG-grafted substrate, and the very long (>10 lm)filopodia on the fibronectin-functionalized surfaceindicate that PEG completely abolished regular celladhesion and the formation of focal adhesions. Bycontrast, the functionalization of surfaces and thebinding of cell-adhesive substrates, such as PLLand fibronectin, completely restored the expectedadhesion capacity of pancreas tumor cells towardsthese substrates.
The directed manipulation of cell binding capac-ities of selected surfaces offers the possibility of adetailed cell-substratum interaction manipulationrequired for better understanding of crucial cellularprocesses as cancer cell migration, substrate depen-dent proliferation and dedifferentiation.
These results on the surface derivatization of thePtBA skin layer show that the PtBA-b-PS filmscomprise a promising platform to study cell-surfaceinteractions. In conjunction with micro- and nano-patterning approaches described elsewhere [35,55]and facile topographic structuring also cell-surfaceinteractions with combined biochemically and topo-graphically patterned polymer platforms can benow investigated and promise to yield insight intoimportant aspects of cell behavior.
4. Conclusions
A robust and versatile platform for the fabrica-tion of functional biointerfaces based on thePS690-b-PtBA1210 diblock copolymer system withreactive tert-butyl acrylate ester groups has beensuccessfully introduced. Owing to the presence ofPS domains in the microphase separated films, theselayers are stable under a wide range of processingconditions. Spin-coated ultrathin PS690-b-PtBA1210
films can be hydrolyzed under different controlledconditions. Following an activation with NHS estergroups, a variety of amino functionalized (bio)mol-ecules can be covalently immobilized in laterallyhomogeneous, high coverages. The function of anti-fouling layers (based on grafted PEG), DNAhybridization, as well as protein immobilization,were demonstrated. Using this polymer system asa platform to engineer designed biointerfaces pro-vides, also in conjunction with novel patterningapproaches [55], new possibilities for fundamentalbiological research involving cell biology, as shownby the studies on cell-surface interactions. In addi-tion, PS690-b-PtBA1210 films and related systems
may comprise attractive platforms for the prepara-tion of high throughput screening arrays and(bio)affinity assays.
Acknowledgements
The authors would like to thank Dr. LourdesBasabe Desmonts and Dr. Henk-Jan van Manenfor their help with the fluorescence experiments.The support of the EU (NoE Nano2Life) is grate-fully acknowledged. This work has been financiallysupported by the MESA+ Institute for Nanotech-nology of the University of Twente, the Councilfor Chemical Sciences of the Netherlands Organiza-tion for Scientific Research (CW-NWO) in theframework of the vernieuwingsimpuls program(grant awarded to HS) and a NWO middelgrootgrant, and by the BMBF (grant to JS).
Appendix A. Supplementary material
Transmission FTIR spectra of hydrolyzed films,TM-AFM height and phase image, transmissionmode FTIR data of derivatized films, XPS surveyscans, various fluorescence microscopy controlexperiments, optical micrograph of cells on PEGn-functionalized surfaces. Supplementary data associ-ated with this article can be found, in the onlineversion, at doi:10.1016/j.eurpolymj.2007.03.021.
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[49] K562 cells are probably the most widely used experimentalmodel of chronic phase of chronic myelogenous leukemia
