Immobilization of Biomolecules to Plasma Polymerized

Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany, and Instituto Químico Sarriá-Universitat Ramon Llull, Via Augusta ...
0 downloads 0 Views 1MB Size
2818

Biomacromolecules 2010, 11, 2818–2823

Immobilization of Biomolecules to Plasma Polymerized Pentafluorophenyl Methacrylate Luis Duque,†,‡ Bernhard Menges,† Salvador Borros,‡ and Renate Fo¨rch*,† Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany, and Instituto Quı´mico Sarria´-Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain Received August 6, 2010; Revised Manuscript Received August 20, 2010

Thin films of plasma polymerized pentafluorophenyl methacrylate (pp-PFM) offer highly reactive ester groups throughout the structure of the film that allow for subsequent reactions with different aminated reagents and biological molecules. The present paper follows on from previous work on the plasma deposition of pentafluorophenyl methacrylate (PFM) for optimum functional group retention (Francesch, L.; Borros, S.; Knoll, W.; Foerch, R. Langmuir 2007, 23, 3927) and reactivity in aqueous solution (Duque, L.; Queralto, N.; Francesch, L.; Bumbu, G. G.; Borros, S.; Berger, R.; Fo¨rch, R. Plasma Process. Polym. 2010, accepted for publication) to investigate the binding of a biologically active peptide known to induce cellular adhesion (IKVAV) and of biochemically active proteins such as BSA and fibrinogen. Analyses of the films and of the immobilization of the biomolecules were carried out using infrared reflection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The attachment of the biomolecules on pulsed plasma polymerized pentafluorophenyl methacrylate was monitored using surface plasmon resonance spectroscopy (SPR). SPR analysis confirmed the presence of immobilized biomolecules on the plasma polymer and was used to determine the mass coverage of the peptide and proteins adsorbed onto the films. The combined analysis of the surfaces suggests the covalent binding of the peptide and proteins to the surface of the pp-PFM.

Introduction The use of plasma polymerization techniques to deposit highly reactive films that show potential for biomaterial and biomedical applications is well described in the literature.3-7 Already since the 1970s the literature describes the deposition of polymers using plasma assisted processes and points out how functional group control can be achieved by simple control over process parameters. The density of a particular functional group and the degree of cross-linking within the polymer network can be largely tailored by careful control over the energy input during the deposition. The energy input can be varied either by minimizing the peak power (Ppeak),8 moving substrates into the afterglow zone,8,9 or by pulsing the applied power.10-14 It has previously been shown that the ratio of the plasma on-phase to the plasma off-phase during pulsed plasma deposition (i.e., the duty cycle ) ton/ton + toff) largely determines which reaction mechanisms predominate to form a plasma polymer film. The types of films that have been found to be particularly suitable for many biomaterial and biomedical applications are those containing reactive groups such as carbonyls,15 amines,16-19 carboxylic acids,20-23 and anhydrides.24-26 Much less attention has been given to precursors containing active esters, generally because of the difficulties encountered to retain the active functional groups during the plasma deposition. In previous work by the authors, the plasma assisted deposition of thin films from pentafluorophenyl methacrylate (PFM) and some of their properties have been discussed.1,2,27,28 It was previously shown that, while PFM can be deposited under a number of conditions, there is only a very narrow range of process conditions that allow for a high retention of the functional ester group. * To whom correspondence should be addressed. Tel.: +49-(0)6131379-766. Fax: +49-(0)6131-379-100. E-mail: [email protected]. † Max Planck Institute for Polymer Research. ‡ Instituto Quı´mico Sarria´-Universitat Ramon Llull.

It is generally understood that the adsorption of biomolecules follows a pathway in which initially physical adsorption via electrostatic or van der Waals forces predominate. If the availability of surface chemical groups is sufficient then chemisorption of the biomolecule may follow. Studies and discussion of covalent attachment of bioactive proteins and peptides to different materials have already been discussed by other authors.29-32 Surface plasmon resonance spectroscopy has been demonstrated to be a powerful technique to study the behavior of plasma polymerized films in aqueous solution and has previously been used for real time studies of protein attachment on plasma polymers.33-35 The work presented here carries on from the authors’ previous work on the reactivity of pp-PFM in solution and toward simple amines and is extended toward studying the immobilization of adhesion promoting peptides and proteins to these highly functional surfaces.

Experimental Section Materials and Substrates. Pentafluorophenyl methacrylate (PFM) was purchased from Sigma-Aldrich, Germany. To degas the liquid monomer, it was freeze-thawed three times but not purified further. A 25 mL round-bottom flask containing fresh PFM was attached directly to the plasma reactor and the gas flow was controlled by a needle valve. The substrates used in the course of this work were Si-wafers for XPS analysis, BK7 glass slides coated with 2 nm chromium and 80 nm gold thermally evaporated onto the glass slides for Infrared reflection absorption spectroscopy (IRRAS). For surface plasmon resonance spectroscopy (SPR) substrates were LaSFN9 glass slides (Hellma Optik, Jena, Germany) coated with approximately 2 nm of chromium and 50 nm of gold. SPR reference measurements were carried out using each of the blank glass/Cr/Au substrates prior to deposition. To improve the adhesion of the plasma polymer on gold-coated substrate, a monolayer of 1-octadecanethiol (5 mM in ethanol, 10 min immersion) was self-assembled on the gold. The LaSFN9 glass/Cr/Au/SAM

10.1021/bm100910q  2010 American Chemical Society Published on Web 09/10/2010

Immobilization of Biomolecules to pp-PFM

Biomacromolecules, Vol. 11, No. 10, 2010

2819

substrates were dried after the SAM formation and placed into the plasma chamber. Sample Preparation and Plasma Polymerization. Plasma polymerization was carried out in a home-built 30 cm long cylindrical Pyrex reactor using an excitation frequency of 13.56 MHz, which was fed to the reactor via two concentric rings wound around the outside of the chamber separated by a distance of 13 cm. The substrates were placed on a glass platform half way between the electrodes in the center of the plasma glow. Before introduction of the substrate, the chamber was cleaned in a continuous wave O2/Ar (at a relative gas ratio of 1:4) plasma for approximately 1 h at a power of 100 W. The optimum deposition conditions for plasma polymerized PFM (pp-PFM), that gave films showing the highest -CF2 content, were found to be a pulsed plasma process using a duty cycle (DC ) ton/(ton + toff)) of 2/52 at an input power (Pinput) of 50 W, giving an equivalent power (Peq ) DC · Pinput) of 1.92 W. The process pressure during deposition was 0.2 mbar with a gas flow rate of 0.87 sccm. Plasma polymer film thickness was typically 50 ( 4 nm measured on Si-wafers subjected to the same sample preparation process. Chemical and Morphological Analysis. A Tencor Surface Alphastep Profiler 200 was used to determine film thickness. Contact angles were measured using the sessile drop method. A 0.8 µL drop of Milli-Q water was placed on the sample, the syringe needle was removed from the drop and the angle between the surface of the drop and the sample surface was measured. This was repeated 5 times and the values averaged. The measurements were used as a routine control for checking reproducibility of the films produced in this work. Only samples showing the previously determined value of 105.1° ( 3° were used in subsequent experiments. Analysis of film chemical structure and changes within the film upon immersion in different solutions was carried out by Infra Red Reflection Absorption Spectroscopy (IRRAS) using a Nicolet 850 spectrometer with an angle of incidence of 5°. For some selected samples X-ray Photoelectron Spectroscopy (XPS) was performed approximately 24 h after treatment using a Perkin-Elmer PSI 5000 series instrument equipped with a monochromator and an Al KR (1486.6 eV) X-ray source. The pass energy used was 17.9 eV giving a resolution of 0.6 eV for Ag (3d 5/2). The samples were stored under dry Ar until analysis. An electron flood gun was used to compensate surface charging. Analysis of core level peak shapes was performed using CASA XPS analysis software. Spectra were corrected for C 1s at 285 eV. For fitting of the C 1s spectra a 70/30 Lorentian/Gaussian peak shape was assumed and the minimum number of peaks possible was fitted to the experimental peak profiles. During the fitting routine the peak fwhm was fixed constant for all peak components (1.6 eV) and the iteration was allowed to vary peak position and height. The observed peak positions were correlated to values from the literature. Scanning force microscopy (SFM) measurements were made under ambient conditions using noncontact mode (Dimension 3100 CL, Veeco, Santa Barbara, U.S.A.). All measurements were performed with silicon cantilevers (Olympus OMCL-AC160TS, Japan) having a nominal spring constant of K ) 42 N/m and a resonance frequency of 300 kHz. All images have been flattened by using a first order plane. Topograghy and phase contrast were measured in a lateral scale of 5 × 5 µm. The root-mean-square roughness (rms) values were then calculated for all images.

SPR measurements were carried out with a home-built setup, which was based on the Kretschmann configuration, already well described in the literature.36,39 In this scheme, monochromatic, linearly p-polarized light is coupled through a prism mounted onto the backside of the glass/ Cr/Au substrate in contact with the dielectric medium under investigation. By choosing the appropriate inner angle of incidence (θSPR), resonant coupling between photons and surface plasmons can be obtained, resulting in a sharp minimum in the intensity of the reflected light. If the refractive index of the dielectric medium changes, for example, as a result of the adsorption of molecules at the surface, a proportional change in the angle (∆θSPR) can be observed. For quantitative treatment of the data, an already well-established procedure34,36,40-43 based on the Fresnel equations and transfer matrix algorithm was used to calculate the optical thickness of a general multilayer assembly. The SPR spectrum, shown as a % reflectivity versus angle curve, can be used to obtain the optical thickness (∆dAnA) of the dielectric medium, where dA is the adlayer thickness and nA is its refractive index. In the present work, the position (or value) of the resonance angle, θSPR, was followed as a function of time to obtain a plot of θSPR versus time. Reference SPR measurements using glass/ Cr/Au SPR samples were taken in air, in solvent, and in the protein solution. To measure protein adsorption in real time, the pp-PFM coated SPR sample was attached to a Teflon flow cell after plasma polymerization and the flow cell was filled with deionized water (DI H2O). After about 10 min the film was found to be stable in water (no further change in θSPR was observed). Following this, the peptide or protein solution was injected into the cell. The peptide binding kinetics was obtained by monitoring the change in the minimum angle over time. After the reaction was completed and equilibrium was reached, DI H2O was again injected to rinse the surface and remove any remaining physisorbed biomolecules. A final SPR measurement was recorded to obtain the optical thickness (dAnA) of the adsorbed layer. To calculate the thickness of the adsorbed layer from these experimental values (dAnA) the refractive index nA for the IKVAV peptide and the proteins (BSA and fibrinogen) were taken as 1.51, 1.45, and 1.39, respectively.34,35,44 In the present work, these values were chosen, as it can be assumed that the smaller peptide IKVAV will form a more compact layer with a higher density and refractive index than the large protein such as fibrinogen.44 The absolute value of n for these proteins is unfortunately not known and the literature report values ranging from the dried protein (n ) 1.53) to slightly above n of the solvent (1.33) for the proteins chosen in this work.15,44,45 To overcome this spurious source of errors in calculating the thickness of an adsorbed layer, the present work also investigated the application of Feijter’s equation to the experimental results from the SPR experiments to calculate the mass coverage. The results of the different interpretation procedures for the SPR data are discussed below. Feijter’s equation relates the measured optical thickness (dAnA) to the mass coverage and states:46

Surface Plasmon Resonance Studies for Peptide and Protein Immobilization. Surface plasmon resonance spectroscopy (SPRS) is a highly sensitive optical method for probing changes in the optical thickness (∆dAnA) of ultrathin adlayers on a metallic surface. The fundamentals of SPR are well established and have been summarized in a number of excellent reviews.36-38 Surface plasmon spectroscopy (SPS) is based on the electromagnetic mode which propagates along a metal surface in contact with a dielectric that is associated with an evanescent wave with a decay length normal to the surface of 150-200 nm. Changes occurring at the surface can be observed by a change in the resonance conditions of the propagating wave.

where M is the mass coverage, dA is the thickness of the adsorbed layer, and nA and nsol are the refractive indices of the adsorbed layer and cover media, respectively. As the solution was DI H2O, nsol was taken to be 1.33. dn/dc is the refractive index increment of proteins and it has been quoted in the literature to be typically 0.182 g/cm3/g.44,46 The SPR experiment thus offers the unique opportunity to experimentally measure the product dAnA with extremely high accuracy irrespective of errors or uncertainties in the refractive index nA or the adlayer thickness dA and allow for the calculation of mass coverage. Three adsorption curves were recorded for all biomolecules and experimental conditions. The data shown are representative of each of these.

M ) dA

nA - nsol dn/dc

(1)

2820

Biomacromolecules, Vol. 11, No. 10, 2010

Duque et al.

Figure 1. General schematic of reaction of the pp-PFM with biomolecules. Table 1. XPS Elemental Composition of the Surface of pp-PFM before and after Immobilization of IKVAV, BSA, and Fibrinogen in DI H2Oa sample pp-PFM pp-PFM+IKVAV/ DI H2O pp-PFM+BSA/ DI H2O pp-PFM+fibrinogen/ DI H2O a

Figure 2. IRRAS spectra of pp-PFM before and after immobilization of (a) IKVAV, (b) BSA, and (c) fibrinogen.

Results and Discussion It was found that pp-PFM reacts readily in PBS in a hydrolysis reaction that is believed to release the pentafluorophenyl group from the polymer network to form a polymeric material with residual carboxylic acid groups. In the present case, the active ester is assumed to react with the aminated end groups of the biomolecules (Figure 1). Surface Analysis. The formation of the amide groups could easily be monitored by observing the intensity changes of infrared bands at 1730 cm-1 for the ester group and that at 1650 cm-1 for the amide link. At the same time, as the bulky pentafluorophenyl group leaves the polymer network a decrease in the band at 1500 cm-1 could be observed. Figure 2 shows the IRRAS spectra for pp-PFM films (Peq ) 1.92) immersed in a 0.1 mg/mL deionized water solution of (a) IKVAV, (b) BSA, and (c) fibrinogen over a period of 15 min. As observed previously for the simple amines,2 the reaction of pp-PFM with the peptide and the proteins led to a decrease in the band at 1730 cm-1 and an increase in the band at 1650 cm-1, indicating the conversion of an ester to an amide. When comparing the relative intensities of these bands in Figure 2

C%

O%

F%

N%

S%

68.1 ((1) 8.2 ((1) 23.7 ((1) 70.4 ((1) 16.5 ((1) 1.3 ((1) 11.2 ((1) 0.6 ((1) 60.8 ((1) 23.3 ((1)

1.1 ((1) 11.2 ((1) 3.6 ((1)

69.5 ((1) 17.8 ((1)

2.5 ((1) 10.2 ((1) 0.3 ((1)

The reaction time for the experiments was 60 minutes.

for (a) IKVAV, (b) BSA, and (c) fibrinogen, it appears that the greatest adsorption occurs for the larger protein fibrinogen. The IRRAS spectra shown are the sum of the spectra of the plasma polymer plus the peptide or protein adlayer. IKVAV, being the smallest of the three, also shows the smallest change, indicating that the immobilized layer of peptide after 15 min is much thinner than the layer of the plasma polymer. For the immobilization of BSA, a larger change in the relative intensities of the three peaks was observed. In fact, after 15 min of reaction, the peak at 1730 cm-1 completely disappeared, which may suggest that the attachment to the modified surface is complete. For the larger fibrinogen, the effect is even more pronounced, and after 15 min of reaction, a very strong band at 1650 cm-1 representing amide groups within the immobilized protein and at the surface is dominant in the spectrum. The persistently high intensity at 1500 cm-1, indicative of the pentafluophenyl ring, suggests that the immobilization of IKVAV, BSA, and fibrinogen is specific on the interface. Ester groups further within the film structure appear not to have been hydrolyzed or aminated within the time frame of the experiments. A more quantitative consideration of the relative amounts of immobilized material is considered later in this paper. Contact angle goniometry showed that the sessile drop contact angle of the surface decreased from around 105.1° for the as deposited pp-PFM to values ranging from 34 to 40° upon reaction with the peptide and the two proteins. The corresponding XPS analysis showed an O/C ratio of 0.12 and a F/C ratio of 0.35 for the as deposited pp-PFM films polymerized using the given conditions. This is slightly lower than the theoretical values of 0.2 and 0.5 for O/C and F/C, respectively, suggesting some loss of functionality from the precursor molecule in this work. Table 1 shows the elemental composition of the surface before and after reactions of the pp-PFM with IKVAV, BSA, and fibrinogen using deionized water as solvent. It shows clearly a substantial loss of fluorine from the uppermost surface accompanied by a gain in nitrogen for all three reacting molecules. For surface immobilized BSA, the analysis also clearly showed the presence of sulfur after the reaction. For the surface immobilized IKVAV and fibrinogen sulfur could only barely be identified above spectral noise and can only be given within the error range in Table 1. Figure 3 gives the XPS C 1s spectra of the samples from Table 1. Pp-PFM deposited at low input powers showed the characteristic peaks of C-C, C-O, C-F, and O-CdO

Immobilization of Biomolecules to pp-PFM

Biomacromolecules, Vol. 11, No. 10, 2010

Figure 3. C 1s spectra of (a) as deposited pp-PFM and surface immobilized (b) IKVAV, (c) BSA, and (d) fibrinogen on pp-PFM. Table 2. Deconvolution of the C 1s Peaks and Assignment of Peaks C-C C-O pp-PFM

C-F N-CdO O-CdO nfπ*

position 285.0 286.5 287.6 % area 48.1 7.4 37.7 position 285.0 286.3

288.2

% area 46.1 30.2 position 285.0 286.3

23.7 288.3

% area 50.3 31.4 pp-PFM+fib. position 285.0 286.3 % area 51.7 30.5

18.4 288.3 17.8

pp-PFM + IKVAV pp-PFM + BSA

289.3 4.0

294.5 2.9

appearing at binding energies of 285, 286.5, 287.6, and 289.3 eV, respectively.1 In addition, a shake up satellite, representative of the fluorinated aromatic ring, was clearly observed at 294.5 eV. Identification and quantification of peaks as obtained in this work are shown in Table 2. The C1s peak profiles for pp-PFM after reaction with IKVAV, BSA and fibrinogen show a clear loss of the pentafluorophenyl groups (loss of the C-F peak component at 287.6 eV). The other very striking feature of these data is the shift of the peak component at 289.3 to 288.2 eV. This shift to lower binding energy is at least partially indicative of the conversion of an ester group (289.3 eV) adjacent to a

2821

fluorinated aromatic ring to an amide group (288.2 eV) that could be associated with the covalent link between the peptide/ proteins and the plasma polymer surface. At the same time, the elemental analysis shows the uptake of nitrogen, Table 1. The high relative intensity of the peak component at 288.2 eV originates from amide bonds within the attaching protein as well as any amide linkages forming at the interface. The most striking proof of reaction of the ester group is the loss of the πfπ shake up satellite observed at 294.5 eV in the C 1s spectrum of the as deposited pp-PFM. This satellite is characteristic of the fluorinated aromatic ring and is seen to completely disappear upon reaction with each of the biomolecules in this work. Finally the small shift of the peak component at 286.5 eV, indicative of C-O-C bonds in the unreacted ppPFM, to 286.3 eV in the protein derivatized plasma polymer film may suggest the presence of newly formed C-N bonds. Thus, the new peak at 286.3 eV is associated here with the sum of contributions from C-N and C-O groups. These observations are clear indicators that the reactive ester groups of ppPFM react under the given conditions and that immobilization of the protein via covalent linkages to the pp-PFM has taken place. Scanning force microscopy for analysis of surface roughness shows the as deposited films to have an rms roughness of 31 nm, typical for these films. After immobilization of the small peptide IKVAV, the roughness decreased considerably to approximately 6 nm. Immobilization of BSA and fibrinogen to the original pp-PFM surface showed a surface with a typical rms roughness ranging between 14-16 nm. Refractive Index Determination of pp-PFM. The refractive index of the pp-PFM films was measured with a combination of step profiler and surface plasmon resonance techniques to obtain values required for subsequent studies of protein immobilization. First, the geometrical thickness (d) of several dry films, deposited on a Si-wafer, was determined using the Alphastep profiler. SPR measurements were performed on SPR-samples coated with pp-PFM during the same plasma deposition processes, assuming a comparable film thickness over the samples. Fitting of the plasmon curves and applying the measured geometrical thickness (d) allowed for the calculation of the refractive index (nPFM) of the pp-PFM. In this way the refractive index for pp-PFM was calculated in this work to be 1.47 for the films in air and 1.46 for the films in aqueous solution. This falls into the expected range for a typical polymeric material. Protein and Peptide Adsorption. Protein adsorption on the pp-PFM films was measured in real time using SPR kinetic

Figure 4. Scanning force micrographs and rms values obtained for pp-PFM before and after IKVAV, BSA, and fibrinogen immobilization.

2822

Biomacromolecules, Vol. 11, No. 10, 2010

Duque et al.

The combined information leads us to believe that in the present work it has been possible to prove the immobilization of at least one monolayer of biomolecules on the pp-PFM surfaces studied.

Conclusions

Figure 5. SPR kinetic of surface immobilization of IKVAV, BSA, and fibrinogen on pp-PFM, IRRAS spectra of pp-PFM before and after immobilization of (a) IKVAV, (b) BSA, and (c) fibrinogen. Table 3. Refractive Indices, Geometrical Thickness, Optical Thickness, and Mass Coverage Values Calculated Using Feijters Equation sample measured

nA

dA (nm)

dAnA (nm)

M (ng/mm2)

(a) ppPFM in air (b) ppPFM in DI H2O (c) -IKVAV (d) -BSA (e) -fibrinogen

1.471 1.467 1.510 1.450 1.390

56.6 54.2 11.5 14.5 21.1

16.6 21.0 29.2

10.7 93.2 65.8

minimum mode tracking, where the resonance angle (θSPR) was followed over time. The kinetics of IKVAV, BSA, and fibrinogen adsorption onto pp-PFM films are shown in Figure 5. The adsorption of IKVAV on bare Au is also given as a reference measurement in Figure 5. An initial physisorption of the peptides on the gold surface is clearly observed by the change in the resonance angle. However, subsequent rinsing of the surface after 30 min completely removed this adlayer as is seen by the sharp drop in the resonance angle at t ) 30 min. Figure 5 also shows the kinetics of the immobilization of IKVAV, BSA, and fibrinogen on the stabilized pp-PFM surface. When rinsing the surface with DI H2O a drop in the resonance angle was observed that can be related to a loss of physisorbed peptides and proteins from the surface. The change in the resonance angle for each of the biomolecules was around 1°. Calculating a reliable adlayer thickness from these values requires precise knowledge of the refractive index of the peptide and proteins. Using the values of nA from the literature, as discussed above, leads to adlayer thickness values (d), as provided in Table 3. The thickness (d) of the protein on the surface was calculated assuming a general multilayer assembly and applying Fresnel theory. A thickness of around 11 nm was calculated for the immobilized IKVAV assuming a refractive index of 1.51. For BSA, a thickness of 14 nm was obtained, and for fibrinogen, a thickness of 21 nm was calculated. Using eq 1, however, it is also possible to estimate a mass coverage much more accurately since this removes any discrepancies in refractive index values for the individual layers in the multilayer system. For the IKVAV peptide (smallest molecule), a mass coverage of 10.7 ng/mm2 could be estimated, while for the larger protein molecules, a mass coverage of 93 and 66 ng/mm2 was determined for BSA and fibrinogen, respectively. This data, of course, does not provide any insights into the orientation of the proteins on the surface or their tertiary structure once immobilized.

The work described in this paper carried on from previous work investigating the chemical and biomaterial properties of plasma polymerized pentafluorophenyl methacrylate. In the present work it could be shown that a typical adhesion motif such as IKVAV and two typical proteins such as BSA and fibrinogen could be covalently bonded to the active ester surface. This could be clearly shown using IRRAS, detailed XPS analysis, and surface plasmon resonance spectroscopy, which was used to calculate adlayer thickness as well as mass coverage of the peptide and protein on the pp-PFM surface. The data could be supported by roughness measurements using scanning force microscopy and by changes in wettability upon reaction. The combined data obtained demonstrate that reaction occurred at the pp-PFM surface and that at least a full monolayer of peptide and proteins could be immobilized. IRRAS and XPS analysis suggest that covalent attachment of the proteins to the surface takes place. Acknowledgment. The authors wish to thank Daniela Mo¨ssner of IMTEK, University of Freiburg, for the XPS analysis of the samples, we wish to thank Helma Burgfor the AFM analysis, Ru¨diger Berger for the useful discussions, and the DAAD-La Caixa action for their economic support.

References and Notes (1) Francesch, L.; Borros, S.; Knoll, W.; Foerch, R. Langmuir 2007, 23, 3927. (2) Duque, L.; Queralto, N.; Francesch, L.; Bumbu, G. G.; Borros, S.; Berger, R.; Fo¨rch, R. Plasma Process. Polym. 2010, accepted for publication. (3) Siow, K. S.; Britcher, L.; Kumar, S.; Griesser, H. J. Plasma Process. Polym. 2006, 3, 392. (4) Foerch, R.; Chifen, A. N.; Bousquet, A.; Khor, H. L.; Jungblut, M.; Chu, L. Q.; Zhang, Z.; Osey-Mensah, I.; Sinner, E. K.; Knoll, W. Chem. Vapor Deposition 2007, 13, 280. (5) Favia, P.; d’Agostino, R. Vide-Sci. Tech. Appl. 2002, 57, 40. (6) Oehr, C. Nucl. Instrum. Methods Phys. Res., Sect. B 2003, 208, 40. (7) Foerch, R.; Zhang, Z. H.; Knoll, W. Plasma Process. Polym. 2005, 2, 351. (8) Alexander, M. R.; Duc, T. M. J. Mater. Chem. 1998, 8, 937. (9) Fally, F.; Doneux, C.; Riga, J.; Verbist, J. J. J. Appl. Polym. Sci. 1995, 56, 597. (10) Yasuda, H.; Hsu, T. J. Polym. Sci., Part A: Polym. Chem. 1977, 15, 81. (11) Ryan, M. E.; Hynes, A. M.; Badyal, J. P. S. Chem. Mater. 1996, 8, 37. (12) Savage, C. R.; Timmons, R. B.; Lin, J. W. Chem. Mater. 1991, 3, 575. (13) Rinsch, C. L.; Chen, X.; Panchalingam, V.; Savage, C. R.; Wang, Y. H.; Eberhart, R. C.; Timmons, R. B. Abstr. Pap.-Am. Chem. Soc. 1995, 209, 141. (14) Llewellyn, I. P. S. G.; Heinecke, R. A. Proceedings of the SPIEsThe International Society for Optical Engineering, San Diego, California, July 11-13, 1990, SPIE: Bellingham, WA, 1990; Vol. 1148, p 84. (15) Benesch, J.; Tengvall, P. Biomaterials 2002, 23, 2561. (16) Scho¨nherr, H.; van Os, M. T.; Foerch, R.; Timmons, R. B.; Knoll, W.; Vancso, G. J. Chem. Mater. 2000, 12, 3689. (17) Kurosawa, S.; Hirokawa, T.; Kashima, K.; Aizawa, H.; Park, J. W.; Tozuka, M.; Yoshimi, Y.; Hirano, K. J. Photopolym. Sci. Technol. 2002, 15, 323. (18) Zhang, Z.; Knoll, W.; Foerch, R.; Holcomb, R.; Roitman, D. Macromolecules 2005, 38, 1271. (19) Choukourov, A.; Biederman, H.; Slavinska, D.; Hanley, L.; Grinevich, A.; Boldyryeva, H.; Mackova, A. J. Phys. Chem. B 2005, 109, 23086. (20) Otoole, L.; Beck, A. J.; Short, R. D. Macromolecules 1996, 29, 5172.

Immobilization of Biomolecules to pp-PFM (21) Daw, R.; Candan, S.; Beck, A. J.; Devlin, A. J.; Brook, I. M.; MacNeil, S.; Dawson, R. A.; Short, R. D. Biomaterials 1998, 19, 1717. (22) Mattioli-Belmonte, M.; Lucarini, G.; Virgili, L.; Biagini, G.; Detomaso, L.; Favia, P.; D’Agostino, R.; Gristina, R.; Gigante, A.; Bevilacqua, C. J. Bioact. Compat. Polym. 2005, 20, 343. (23) Kelly, J. M.; Scutt, A. M.; Short, R. D.; Brook, I. M. J. Dental Res. 2000, 79, 1184. (24) Schiller, S.; Hu, J.; Jenkins, A. T. A.; Timmons, R. B.; SanchezEstrada, F. S.; Knoll, W.; Foerch, R. Chem. Mater. 2002, 14, 235. (25) Chifen, A. N.; Foerch, R.; Knoll, W.; Cameron, P. J.; Khor, H. L.; Williams, T. L.; Jenkins, A. T. A. Langmuir 2007, 23, 6294. (26) Jenkins, A. T. A.; Hu, J.; Wang, Y. Z.; Schiller, S.; Foerch, R.; Knoll, W. Langmuir 2000, 16, 6381. (27) Francesch, L.; Garreta, E.; Balcells, M.; Edelman, E. R.; Borros, S. Plasma Process. Polym. 2005, 2, 605. (28) Queralto, N.; Bumbu, G. G.; Francesch, L.; Knoll, W.; Borros, S.; Berger, R.; Fo¨rch, R. Plasma Process. Polym. 2007, 4, S790. (29) MacDonald, C.; Morrow, R.; Weiss, A. S.; Bilek, M. M. M. J. R. Soc. Interface 2008, 5, 663. (30) Salim, M.; O’Sullivan, B.; McArthur, S. L.; Wright, P. C. Lab Chip 2007, 7, 64. (31) Lassena, B.; Malmsten, M. J. Colloid Interface Sci. 1997, 186, 9.

Biomacromolecules, Vol. 11, No. 10, 2010

2823

(32) Matsuda, A.; Kobayashi, H.; Itoh, S.; Kataoka, K.; Tanaka, J. Biomaterials 2005, 26, 2273. (33) Zhang, Z.; Chen, Q.; Knoll, W.; Foerch, R. Surf. Coat. Technol. 2003, 174, 588. (34) Zhang, Z.; Menges, B.; Timmons, R. B.; Knoll, W.; Foerch, R. Langmuir 2003, 19, 4765. (35) Chu, L. Q.; Foerch, R.; Knoll, W. Langmuir 2006, 22, 2822. (36) Knoll, W. Annu. ReV. Phys. Chem. 1998, 49, 569. (37) Raether, H. Springer Tr. Mod. Phys. 1988, 111, 1. (38) Schasfoort, R. B. M.; Tudos, A. J. Handbook of Surface Plasmon Resonance; RSC Publishing: Cambridge, U.K., 2008. (39) Kretschmann, E.; Raether, H. Z. Naturforsch. 1968, 23, 2135. (40) Aust E.F., I. S.; Sawodny, M.; Knoll, W. Trends Polym. Sci. 1994, 2, 313. (41) Kambhampati, D.; Nielsen, P. E.; Knoll, W. Biosens. Bioelectron. 2001, 16, 1109. (42) Chu, L. Q.; Knoll, W.; Foerch, R. Chem. Mater. 2006, 18, 4840. (43) Vasilev, K.; Knoll, W.; Kreiter, M. J. Chem. Phys. 2004, 120, 3439. (44) Voros, J. Biophys. J. 2004, 87, 553. (45) Schaaf, P.; Dejardin, P. Colloids Surf. 1987, 24, 239. (46) Defeijter, J. A.; Benjamins, J.; Veer, F. A. Biopolymers 1978, 17, 1759.

BM100910Q