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Preventing Protein Adsorption from a Range of Surfaces Using an Aqueous Fish Protein Extract Saju Pillai,† Ayyoob Arpanaei,†,¶ Rikke L. Meyer,†,‡ Victoria Birkedal,† Lone Gram,§ Flemming Besenbacher,† and Peter Kingshott*,† Interdisciplinary Nanoscience Center (iNANO) and Department of Biological Sciences, Aarhus University, Ny Munkegade, 8000 Aarhus C, Denmark, and National Institute of Aquatic Resources, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark Received May 25, 2009; Revised Manuscript Received August 14, 2009
We utilize an aqueous extract of fish proteins (FPs) as a coating for minimizing the adsorption of fibrinogen (Fg) and human serum albumin (HSA). The surfaces include stainless steel (SS), gold (Au), silicon dioxide (SiO2), and poly(styrene) (PS). The adsorption processes (kinetics and adsorbed mass) are followed by quartz crystal microbalance with dissipation (QCM-D). Complementary surface information is provided by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). QCM-D shows no mass increases to any of the FPcoated surfaces upon treating with Fg or HSA. Also, when Fg- or HSA-coated surfaces are exposed to the FPs, a significant increase in adsorbed mass occurs because the FPs are highly surface-active displacing Fg. Additionally, fluorescence microscopy confirms that very little Fg adsorbs to the FP-coated surfaces. We propose that FP coatings prevent protein adsorption by steric stabilization and could be an alternative method for preventing unwanted bioadhesion on medical materials.
Introduction Preventing the nonspecific adsorption of proteins to surfaces is a major challenge in biomaterials research due to the cascade of adverse responses that subsequently occur, leading to implant rejection.1-3 Depending on the type of implant, different responses and outcomes can occur, including blood clotting,4 inflammation,2,3,5 immune responses,6 and bacterial adhesion leading to implant-centered infection.7,8 Surface modification of biomaterials is generally recognized as the best strategy for preventing protein adsorption, and there are clear indications that lack of protein at an interface often implies that cells will also be inhibited from attachment and growth. To date, the best strategies have employed the use of neutrally charged polymer chains that extend from the surface and provide a steric barrier against proteins interacting with the substrate. In this regard, poly(ethylene oxides) (PEOs) have proved most effective.9-12 However, the use of other synthetic polymers, such as poly(Nisopropyl acylamide)13 or polymers based on phosphorylcholine,14-16 has proved quite successful. In addition, natural polymers, such as polysaccharides (e.g., dextran,17 pullalan,18 and hyaluronic acid19), have been shown to reduce both protein adsorption and cell attachment. Recently, it has been demonstrated that a protein mixture extracted from fish can reduce bacterial adhesion significantly when adsorbed to a surface.20 The antifouling effect was demonstrated for a number of material surfaces including tissue culturing polystyrene (TCPS), stainless steel, and glass. In this study, we aim to extend these findings to determine whether the same fish protein (FP) coating has the potential to minimize * To whom correspondence should be addressed. E-mail: peter.kingshott@ inano.dk. † iNANO, Aarhus University. ‡ Department of Biological Sciences, Aarhus University. § Technical University of Denmark. ¶ Present address: Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, Iran.
the adsorption of proteins relevant to biomaterial surfaces and, therefore, be used as protein and cell-repellant surfaces. In particular, fibrinogen (Fg) and human serum albumin (HSA) repellency is interesting, as these proteins are implicated in triggering adverse cell responses to biomaterial surfaces. HSA has a molecular weight of 66 kDa and acts as a multifunctional transporter protein in the circulatory system.21 It is the most abundant protein in human blood plasma. Fg is a 46 nm long glycoprotein with a mass of 340 kDa and is abundant in the circulatory system at a concentration of 2.6 mg mL-1.22 Fg is involved in blood clotting and inflammation. We utilize quartz crystal microbalance with dissipation (QCM-D) to follow the adsorption processes of both the FPs and the Fg and HSA to the FP-coated surfaces. In addition, X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) are employed as complementary techniques to follow changes to the surface chemistry and topology after exposure to FPs. Finally, in order to confirm the protein-resistant capabilities of the fish protein layer, adsorption studies with fluorescently labeled Fg were employed.
Experimental Section Materials. The aqueous extract of protein mixture was prepared from Atlantic cod fillets as described elsewhere. In brief, fillets of cod (Gadus morhua) were diced, 500 mL of tap water was added per kg of meat, and the mixture was boiled for 5 min. The aqueous extract was separated from the solid substance in a strainer. The extract was boiled for 5 min, left for 5 min at ambient temperature, and filtered through standard coffee filters; 0.10 M phosphate buffer, 0.056 M H2KPO4 (Merck 48711000), and 0.044 M HK2PO4 (Merck 51041000) were added, and pH was adjusted to 6.60 with NaOH/HCl. The extract was heated to 100 °C for 30 min. A concentration of 1 mg mL-1 was used throughout the experiment. Two proteins were chosen for protein adsorption studies: Fg, a sticky protein implicated in implant failure, and HSA, the most abundant serum protein. Human Fg (lyophilized powder) and HSA (lyophilized powder) were obtained from Kordia Life Sciences (The
10.1021/bm900589r CCC: $40.75 2009 American Chemical Society Published on Web 09/18/2009
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Netherlands) and Sigma Inc., respectively, and used as received. Stock solutions of Fg in aliquots were prepared as follows. Fg was hydrated to the original volume with 37 °C distilled buffer. The hydration should take place in 37 °C water bath to ensure that all protein solubilizes. After hydration, aliquots were frozen at -50 °C. Fg was used in concentrations of 60 µg mL-1 and 1 mg mL-1, and HSA in a concentration of 1 mg mL-1 for protein adsorption experiments. As proteins are usually more soluble in dilute salt solutions than in pure water, we used phosphate buffered saline (PBS) to prepare solutions. PBS with pH 7.4 at 22 °C was prepared freshly from PBS tablet (Sigma) and used for preparing protein solutions. QCM-D. QCM-D measurements were performed on Q-Sense E4 system (Q-Sense AB, Sweden). The FP solution was injected into each QCM chamber and allowed to adsorb on test surfaces before injection of the test protein solution (Fg or HSA). The reverse experiments were also performed to check the ability of FPs to replace other adsorbed proteins. PBS was used to obtain a baseline signal before injection of protein solutions. The measurements were performed in continuous flow mode (100 µL min-1) and under ambient temperature (22 °C). Four types of QCM sensor surfaces (Q-Sense AB, Sweden) were tested: gold (Au), stainless steel (SS), silicon dioxide (SiO2), and polystyrene (PS). All crystals were washed in 2% SDS for 5 h, rinsed with Milli-Q water several times, and UV/ozone treated for 40 min (except PS sensor surface) before use. The shifts in both frequency (∆F) and dissipation (∆D) were monitored simultaneously. The frequency drop was recorded as ∆F and measures the change in total coupled mass including the water layer. In addition, ∆D provides information about the viscoelasticity of the layers. The adsorbed protein mass (∆m) per unit area (Sauerbrey mass) was quantified using Q-tools (Q-Sense AB, Sweden) data analysis software. All data presented are from the seventh overtone. XPS. We used XPS to characterize QCM crystal surfaces before and after FP adsorption using a Kratos Axis UltraDLD instrument equipped with a monochromated Al KR X-ray source (hν ) 1486.6 eV) operating at 10 kV and 15 mA (150 W). A hybrid lens mode was employed during analysis (electrostatic and magnetic), with an analysis area of approximately 300 µm × 700 µm. For each sample, a takeoff angle (TOA) of 0° (with respect to normal) was used allowing a maximum probe depth (10 nm). Wide energy survey scans (WESS) were obtained over the range of 0-1400 eV binding energy (BE) at a pass energy of 160 eV and used to determine the surface elemental composition. High-resolution spectra were recorded for C 1s and N 1s at a detector pass energy of 20 eV. The Kratos charge neutralizer system was used on all samples with a filament current between 1.8 and 2.1 A and a charge balance of 3.6 V. Sample charging effects on the measured BE positions were corrected by setting the lowest BE component of the C 1s spectral envelope to 285.0 eV, corresponding to the C-C/ C-H species. Deconvolution of the high-resolution spectral regions was performed by subtraction of a linear background and application of a mixed Gaussian-Lorentzian synthetic peak. Full width at halfmaximum (fwhm) values for each component were often set to values that gave the best fits and made the most physical sense in relation to the resolving power of the instrument and experimental parameters used. The generated data were converted into VAMAS format and processed using CasaXPS software. Fluorescence Microscopy. Fluorescence microscopy was used to directly visualize adsorption of fluorescently labeled Fg (Alexa Fluor 488 conjugate of human Fg; Invitrogen A/S). Samples were prepared on thin glass coverslips previously cleaned with dilute HF solution and imaged with a widefield fluorescence microscope in epi geometry (Zeiss, Germany). Samples were excited at 488 nm with an argon laser (BeamLok 2060, Spectra Physics). The excitation power was around 0.17 kW/cm2 with a spot size around 15 µm in diameter. Fluorescence was passed through a dichroic mirror and several emission filters and detected with a sensitive peltier cooled back illuminated CCD (MicroMax 512BFT, Roper Scientific). The setup is able to detect single molecule fluorescence.
Pillai et al. AFM. AFM imaging was performed under dry conditions at room temperature (23 ( 2 °C) using a MultiMode scanning force microscope equipped with Nanoscope IIIA controller (Veeco Instruments, Santa Barbara, CA). Si cantilevers (NSG 01, NT-MDT) with a typical curvature radius of approximately 10 nm were used. The force constants of the probes were in the range of 2.5-10 N/m and with a resonance frequency range of 120-180 kHz. The scan rate used was 0.5-1 Hz. Raw data were processed offline using Veeco image processing software. Surface roughness was reported in root-mean-square (rms) values. Contact Angle Measurements. The experiments were performed in a Kru¨ss DSA100 contact angle measuring system under ambient conditions. Measurements were made on QCM sensor surfaces precleaned by UV-ozone treatment for 40 min (except for PS surface). At least 3-5 measurements were made at different locations on each sample, and the average values were reported.
Results and Discussion Adsorption of Aqueous Extracts of FPs. Four QCM sensor surfaces were used in this study: a metal (Au), a metal oxide (SS), an inorganic oxide (SiO2), and a polymer (PS). AFM height images of bare QCM sensor surfaces (Figure 1, a1-d1) showed a granular morphology with almost identical surface roughness for all surfaces, whereas PS had an ultrasmooth topography with roughness of 0.4 nm (Table 1). The contact angle values for metal/inorganic and metal oxide surfaces were in the hydrophilic range, while PS was hydrophobic (Table 1). Adsorption of proteins from the FP solution resulted in a slight change in surface morphology, most likely due to protein layer build up (Figure 1, a2-d2), supported by a slightly higher surface roughness value for the surfaces. The reasons why the proteins are hardly visible in AFM could be (1) the soft nature of the protein layer being easily distorted by the AFM tip. The QCM data (below) suggest that the layers are quite flexible (from dissipation changes). (2) The proteins are not globular in nature and adopt a flat conformation on the surface when in air, thus the area per protein molecule is sufficiently larger than the AFM tip contact area, resulting in the generation of smooth images. Adsorption monitored in real time by QCM-D showed a shift in the QCM frequency for all four surfaces after exposure to the FP solution (1 mg mL-1), inferring that FPs were adsorbed (Figure 2). However, the adsorption behavior appears to be surface-type-dependent. Both Au and PS surfaces exhibited a rapid FP adsorption compared to SS and SiO2. Interestingly, the hydrophobic PS surface exhibited a lower shift in frequency than the hydrophilic oxide surfaces, although the adsorption occurred more rapidly. The difference in the amount of adsorbed FPs on the different surfaces may indicate a higher binding affinity for FPs to hydrophilic versus hydrophobic surfaces.23 SiO2 and SS surfaces showed slightly different adsorption behavior with an initial rapid adsorption followed by a second slower adsorption process before gradually approaching the plateau region after 1 min (Figure 2), indicating that slow protein conformational changes may occur or exchange FPs until a stable layer is obtained. The lines in the ∆D versus ∆F plots in Figure 4 for these two surfaces have a different slope, also suggesting two different rates of FP adsorption. It is interesting that protein adsorption from complex solutions is generally more pronounced on hydrophobic surfaces such as PS, yet this is not the case here. We believe that the FP extract contains proteins with more hydrophilic polar amino acid residues, and less unfolding takes place. Therefore, electrostatic interactions could be mediating the higher protein adsorption to the oxide surfaces.
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Figure 1. Tapping mode AFM, height images of bare QCM sensor surfaces (a1-d1) and FPs treated (a2-d2) Au, SS, SiO2, and PS surfaces, respectively. The scan size is 2 × 2 µm2, and the height scale is 20 nm. Scale bar is 0.25 nm. Table 1. Water Contact Angle and Surface Roughness of QCM Sensor Surfaces before and after Adsorption of FPs
surfaces
water contact angle before FP treatment (degrees)
rms roughness before FPs treatment (nm)
rms roughness after FPs treatment (nm)
Au SS SiO2 PS
45 47 43 90
1.04 ( 0.03 1.02 ( 0.07 1.40 ( 0.13 0.40 ( 0.04
1.06 ( 0.09 1.12 ( 0.05 1.59 ( 0.24 0.58 ( 0.07
In fact, the ζ potentials for planar SiO2 and 316L SS surfaces have been determined to be -7024 and -14 mV,25 respectively, confirming that they both have negative surface charges. However, the higher adsorbed amount of FP occurs on the SS not the SiO2 surface, which is more negatively charged, indicating that other factors are also contributing to the FP adsorption. For the Au surface, strong Au-S bond formation between the Au and the cysteine residues in the FPs cannot be ruled out as a contributing factor toward the rapid, irreversible adsorption to that substrate surface. It has been demonstrated that surface roughness affects protein adsorption, and more protein appears to adsorb to rough
Figure 2. QCM-D adsorption profile of FPs (1 mg mL-1) to Au, SS, SiO2, and PS surfaces. The drop in frequency refers to mass loaded onto the surfaces.
compared to smooth surfaces.21 As PS had a lower roughness than the other surfaces, this could be a possible explanation for the lower FP adsorption. However, the XPS (which measures only the dry mass) results (Figure 3) indicate that the adsorbed mass on hydrophilic and hydrophobic surfaces was comparable, indicating that the degree of FP hydration depends heavily on the adsorbate surface, as observed in QCM-D experiments. Due to the presence of the oxide layer on the hydrophilic surfaces, it can trap liquids (especially water molecules) in surface cavities and can hydrate protein molecules, resulting in large QCM frequency shifts as observed for all metal/oxide surfaces. Hydrophobic surfaces, however, do not wet easily, and proteins adsorbed to these most often form a rigid layer. The smaller shift in frequency and dissipation for PS surfaces may thus be explained by adsorption of more compact proteins with less hydration in the final adsorbed protein layer26 or different types of proteins from solution with different hydration properties. We observed a slight decrease in mass (i.e., protein desorption) upon rinsing the surfaces with buffer, which demonstrates the presence of both strongly (for the case of PS) and weakly (for the case of metal/oxide surfaces) bound proteins (Figure 2). The irreversible adsorption is presumably due to strong proteinsurface interactions, and reversible binding is due to less stable protein-protein interactions. On the basis of the QCM-D and XPS data, we believe that hydrophilic surfaces (SiO2, SS, and Au) have more hydrated proteins, which are less strongly bound to these surfaces, thus are easier to desorb upon rinsing. The advantage of using QCM-D compared to other protein adsorption monitoring techniques is the ability to provide information about the viscoelastic behavior of adsorbed layer from dissipation values (∆D). Higher ∆D/∆F values during adsorption indicate a more viscous and hydrated adlayer (Table 2). On plotting ∆D/∆F, we observed higher values for hydrophilic surfaces, indicating the existence of more hydrated protein layer on these surfaces compared to hydrophobic PS. Further, the estimated Sauerbrey mass calculation confirms this by showing that the mass of the adsorbed protein layer followed the trend Au > SS > SiO2 > PS. However, protein quantification using high-resolution XPS based on N 1s scans showed equal amounts of protein on all surfaces except for Au (Figure 3). It is known that XPS measures the dry mass where as QCM-D measures wet mass (mass coupled with water). Therefore, the
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Figure 3. Upper panel shows the XPS high-resolution N 1s scans of surfaces, before and after adsorption of FPs (1 mg mL-1) to Au, SS, SiO2, and PS surfaces. The amount of protein adsorbed was quantitatively estimated based on a N 1s scan and is shown in the lower panel. Table 2. QCM Shifts in Frequency (∆F), Dissipation (∆D), and ∆D/∆F Values (7th Overtone) after Adsorption of FPs
surfaces
-∆F7 (Hz)
Au SS SiO2 PS
80.55 ( 12.35 63.5 ( 8.27 52.47 ( 10.13 16.83 ( 4.42
∆D7 ∆D7/∆F7 (1 × 10-6) (1 × 10-8 Hz-1) 7.85 ( 3.2 6.81 ( 1.4 5.20 ( 1.7 0.93 ( 0.4
9.75 10.72 9.91 5.53
Sauerbrey mass (ng/cm2) 1375 1000 824 224
difference in FP adsorption observed by QCM-D may thus be largely dependent on the difference in the amount of water bound in the protein layer. Effect of the FPs on HSA Adsorption. The adsorption behavior of HSA was separately investigated before testing the ability of the FP layer to prevent its adsorption (Supporting Information Figure 1). In this experiment, HSA in high concentration (1 mg mL-1) was injected to all four surfaces followed by injection of the FP solution at the same concentra-
tion. Despite binding constants for HSA being reported as the same for both hydrophilic and hydrophobic surfaces,22 we observed that HSA adsorbed more rapidly to PS and Au compared to SS and SiO2. Moreover, adsorption to SS and SiO2 was less stable compared to PS and Au. The shift in dissipation (∆D) was less (compared to FP adsorption) for all surfaces, indicating the formation of a more compact protein layer (Supporting Information Figure 1). Because HSA is globular, adsorption of a monolayer of protein would result in nearly the same mass regardless of the orientation of the individual proteins.22 The ∆D versus ∆F plots (Supporting Information Figure 1) also indicate that the adsorption of HSA to both SS and SiO2 surfaces was slow. This is most likely due to higher initial electrostatic repulsion between the oxide surface and HSA molecules. After injection of the FPs to the HSA-coated surfaces, a significant drop in frequency occurs for all surfaces, especially the Au, SS, and SiO2 surfaces, indicating that the FPs were capable of coadsorbing with or displacing the HSA.
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Figure 4. Plot showing shift in frequency vs dissipation (∆D vs ∆F) upon injection of HSA (1 mg mL-1) to surfaces previously exposed to FPs (1 mg mL-1).
In order to check whether the FP layer was capable of preventing protein adsorption, HSA in high concentration (1 mg mL-1) was injected into the QCM-D chamber after exposing all four surfaces to FPs (Figure 4). Interestingly, both the ∆F and ∆D values remained constant or decreased slightly following the addition of HSA. This indicates that little or no additional proteins were adsorbed to the surface. Upon continuous liquid flow for 100 min, a slight drift in frequency and dissipation was seen. This is not unusual for experiments that run in continuous flow mode and is not indicative of a change in mass of the adsorbed layer. The ∆D versus ∆F plots showed a slight decrease in ∆D relative to ∆F for all surfaces after the buffer rinse following HSA injection, indicating that protein conformational changes may have occurred. Given the fact that HSA is a globular protein, there is least a possibility for conformational changes to occur. There is a subtle change to the ∆D versus ∆F plot after HSA adsorption to the FP-extract-coated PS, In addition, the ∆F versus time plot (Supporting Information Figure 1) shows a slight reduction in ∆F immediately after HSA injection. Both sets of data suggest that a portion of the FP layer on PS could be removed by HSA. For the other three surfaces, the results strongly indicate that the FP layer significantly resists further HSA adsorption to all surfaces. Effect of the FPs on Fg Adsorption. Experiments were performed to test whether the FPs not only prevent adsorption of other proteins but also displace proteins that are already adsorbed to a surface. Here, a more “sticky” protein, Fg, at 0.060 mg mL-1 concentration was injected into the QCM-D chamber followed by the FPs. Similarly, as we observed for HSA, little or no Fg adsorbed to the surfaces that were already coated with FPs (Figure 5, upper panel). The ∆D and ∆F values did not change upon Fg injection. This implies that the conformation of the adsorbed FP layer does not affect its protein-repelling abilities, indicating the effect is most likely due to steric repulsion and is therefore independent of the substrate to which the protein was adsorbed. Furthermore, increasing the Fg
concentration from 0.060 to 1 mg mL-1 did not substantially increase Fg adsorption (Supporting Information Figure 2). Although there was some shift in frequency after the injection of Fg to metal/oxide surfaces coated with FPs, this did not remain after flushing with buffer. Moreover, ∆D/∆F values (data not shown) were nearly identical with both Fg concentrations for all surfaces before and after treatment of Fg over FP-coated surfaces. When these tests were conducted in reverse, that is, Fg was injected into the QCM-D chambers and followed by FPs, we observed an increase in adsorption after injecting the FPs (Figure 5, lower panel). Although binding affinity and surface saturation values were similar for both HSA and Fg, Fg unfolding depends on surface properties.27 Here, the QCM-D results suggest that there is stronger interaction between Fg and hydrophobic PS surfaces, resulting in more Fg adsorption to PS compared to other surfaces (Figure 5, lower panel). Interestingly, ∆D/∆F values (data not shown) were lower for adsorption of Fg compared to FP, inferring a more rigid protein layer. However, after injecting FPs, the ∆D/∆F value increased substantially, indicating that the protein adlayer became more hydrated. As discussed earlier, the adlayer formed by the FPs was more viscous than the Fg adlayer, and the change in ∆D/ ∆F thus infers that desorption of Fg by the FPs occurs. For the metal and oxide surfaces, we observed a second slower adsorption process at longer adsorption times (ca. 2 h), indicating perhaps that the rearrangement of protein molecules had occurred. Finally, a representative ∆F versus time plot is shown in Figure 6 for the experiment, demonstrating that after injection of Fg to the FP-coated surfaces no sharp frequency shifts are observed for the SS, Au, and SiO2 surfaces, with only a minor shift for the FP-coated PS. This supports the data presented in Figure 5. The data in Figures 4 and 5 for both HSA and Fg adsorption to the FP layers on the different surfaces indicate that the FP layers more readily prevent Fg adsorption than HSA, although the former is still largely repelled. Most likely the differences
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Figure 5. Upper panel: Plot showing shift in frequency vs dissipation (∆D vs ∆F) upon injection of Fg (60 µg mL-1) to surfaces previously exposed to FPs (1 mg mL-1). Lower panel: Plot showing shift in frequency vs dissipation (∆D vs ∆F) of the FP (1 mg mL-1) adsorption after treating all four surfaces with Fg (60 µg mL-1).
are attributed to the amount of FPs on the surfaces in relation to the size of the proteins. Thus for surfaces with lower levels of FP (i.e., PS), the smaller HSA molecules can penetrate the FP layer and coadsorb between the gaps, and Fg would require less FP surface coverage to achieve the same effect. Fluorescence microscopy was used to confirm that Fg did not bind to FP-coated surfaces (Figure 7). Alexa Fluor 488 labeled Fg at two different concentrations, 60 µg mL-1 (Figure
6, 1a-c) and 1 mg mL-1 (Figure 7, 2a-c), was used in the experiment. Glass coverslips, aimed at mimicking the SiO2 used in QCM-D experiments, were used. In the top two images (Figure 7, 1a and 2a), surfaces were exposed to Fg for 30 min and then washed with PBS. The intense signal indicates a protein layer that fully covers the surface. In the middle two images (Figure 7, 1b and 2b), surfaces were exposed to labeled Fg for 30 min, washed with buffer, and incubated with FPs for 2 h.
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Figure 6. ∆F vs time plot for Fg adsorption to the various surfaces after preadsorption with FPs.
The significant reduction in signal intensity indicates displacement of the Fg, but some of the Fg remained on the surface at both Fg concentrations. The bottom two images (Figure 7, 1c and 2c) show exposure of the FPs (2 h adsorption, 1 mg mL-1)
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followed by exposure to labeled Fg for 30 min. The observed low signal intensity confirms that no significant Fg was adsorbed to FP-coated surfaces, which is in good agreement with the QCM-D results. By comparing the total fluorescence intensity of samples, we estimated that Fg adsorption to the FP-coated surfaces was less than 1% of the fully covered Fg surface at the low Fg concentration and less than 3% at the high Fg concentration. The protein repelling effect thus could be universal because it can also minimize the adsorption of large sticky proteins such as Fg. The composition of the FP solution is highly complex, and tropomyosin has been identified as one component adsorbing to the surface.28 In addition, our recent work (to be submitted) using gel electrophoresis and MALDI-MS/MS of trypsin-digested proteins, followed by more accurate database searching, has identified four proteins as common adsorbates to all surfaces including tropomyosin 3, muscle troponin, apolipoprotein, and myosin. It may well be the adsorption of a protein complex, as opposed to a single protein, to the surfaces that is demonstrating the protein repellency. However, there are most likely many large proteins or even protein complexes that adsorb to the surfaces. This hypothesis is related to the fact that it is well-known that there is a time dependence in the sequence of protein adsorption when surfaces are exposed to
Figure 7. Fluorescence micrographs of samples (1a-c) incubated with a lower Alexa Fluor 488-Fg concentration of 60 µg mL-1, and samples (2a-c) incubated with a higher Alexa Fluor 488-Fg concentration of 1 mg mL-1. The scale bar is 2 µm. Samples in images 1a and 1b were incubated with Fg. Image accumulation times were 0.5 and 0.33 ms, respectively. In images 1b and 2b, samples were incubated with Fg and subsequently with FPs. Images accumulation times were 2 and 0.33 ms, respectively. Samples in images 1c and 2c were incubated with FPs and subsequently with Fg. Images accumulation times were 50 ms. The six images above are on the same scale, and the intensity scale is shown at the bottom of the figure.
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complex solutions such as human plasma. Generally, small proteins adsorb first and are later displaced sequentially by larger proteins (the so-called Vroman effect). Since preventing protein adsorption is a prerequisite for minimizing longer term adverse responses caused by cell attachment and proliferation, the approach presented here may prove to be a cheaper alternative to the synthetic approaches often employed to prevent bioadhesion. However, we are aware that immune responses to foreign materials will need to be determined, and work is underway in that area.
Conclusion We demonstrate that an aqueous protein extract from fish can significantly reduce the nonspecific adsorption of fibrinogen and albumin when adsorbed to different surfaces, including hydrophilic metal and oxide surfaces and hydrophobic PS. The FPs were shown to adsorb rapidly to both PS and Au, resulting in a more strongly bound protein layer on these surfaces compared to metal-oxide surfaces. Quantification of nitrogen by XPS confirmed that the amount of FP adsorption was in the order Au > SS > SiO2 > PS, which is in good agreement with QCM-D observations. Higher ∆D/∆F values for all metal and oxide surfaces indicate a more hydrated protein layer on these surfaces compared to hydrophobic PS. This revealed that the FPs bind quite differently to each surface type and undergo conformational changes during adsorption. However, protein adsorption preventing the ability of FPs is the same regardless of surface type and amount of proteins adsorbed, indicating that this may be a universal effect and could be applied to different types of biomaterial surfaces. The protein-repelling ability of FPs may be due to steric repulsion. Fluorescence micrographs support QCM-D results, in that the FPs can prevent adsorption of a more sticky protein such as Fg and even replaces Fg. Protein adsorption preventing the ability of the FPs may infer inflammatory cell-repelling characteristics under real biological environments. Acknowledgment. This work has been financed by the Danish Ministry, Department of Research and Development DFFE (3304-05-66). Supporting Information Available. Plot showing QCM-D adsorption profile (∆F vs time) of HSA (1 mg mL-1) after treating all four surfaces with FPs (1 mg mL-1). Plot showing shift in frequency vs dissipation (∆D vs ∆F) of the FP (1 mg mL-1) adsorption after treating all four surfaces with HSA (1 mg mL-1). Plot showing QCM-D adsorption profile (∆F vs time) of FPs (1 mg mL-1) after treating all four surfaces with (A) Fg (60 µg mL-1) and (B) HSA (1 mg mL-1). Plot showing shift in frequency vs dissipation (∆D vs ∆F) upon injection of Fg (1 mg mL-1) to surfaces previously exposed to FPs (1 mg
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mL-1). This material is available free of charge via the Internet at http://pubs.acs.org.
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