- Open Access
Adsorption of Fibronectin, Fibrinogen, and Albumin on TiO2: Time-Resolved Kinetics, Structural Changes, and Competition Study
© The Author(s) 2012
- Received: 19 June 2012
- Accepted: 18 July 2012
- Published: 9 August 2012
An understanding of protein adsorption process is crucial for designing biomaterial surfaces. In this work, with the use of a quartz-crystal microbalance with dissipation monitoring, we researched the following: (a) the kinetics of adsorption on TiO2 surfaces of three extensively described proteins that are relevant for metallic implant integration [i.e., albumin (BSA), fibrinogen (Fbg), and fibronectin (Fn)]; and (b) the competition of those proteins for adsorbing on TiO2 in a two-step experiment consisted of sequentially exposing the surfaces to different monoprotein solutions. Each protein showed a different process of adsorption and properties of the adlayer—calculated using the Voigt model. The competition experiments showed that BSA displaced larger proteins such as Fn and Fbg when BSA was introduced as the second protein in the system, whereas the larger proteins laid on top of BSA forming an adsorbed protein bi-layer when those were introduced secondly in the system.
- TiO2 Surface
- Protein Layer
- Shear Elastic Modulus
- Surface Mass Density
- Adsorbed Protein Layer
The first event taking place at the biomaterial-tissue interface is the interaction of water molecules and salt ions with the surface of the implant. Shortly after the formation of the hydration layer, blood proteins start to crowd the biomaterial surface [1, 2]. Eventually, those proteins form a layer on top of the implanted material. When cells reach the implant surface, they scan the layer of proteins looking for activation factors to attach to that surface and react accordingly. Thus, protein adsorption on biomaterial surfaces plays a crucial role in the integration of an implant in the human body.
Ideally, the surface properties of the implantable material will trigger appropriate responses for specific applications by controlling the type, amount, and conformation of the proteins that will adsorb on the implant. Understanding the process of protein adsorption is then crucial to the surface design of biomaterials.
Proteins are a special, highly complex, and important case of particles adsorbing at surfaces. Protein adsorption is a dynamic process involving non-covalent interactions such as hydrophobic interactions, electrostatic forces, hydrogen bonding and van der Waals’ forces . On the one side, the non-covalent interactions are controlled by multiple protein parameters, such as protein size, pI, secondary and tertiary structure [4, 5]. On the other side, surface properties such as surface energy, roughness and chemistry have been also identified as key factors influencing the process of protein adsorption [4, 6–8].
Protein adsorption to surfaces is the first step in many fundamental biological processes such as blood coagulation cascade and trans-membrane signalling . There are multiple proteins in human blood plasma. Among them, fibrinogen (Fbg), fibronectin (Fn) and albumin (Alb) which constitute a set of well-known, thoroughly characterized, extensively described proteins . Several studies have suggested that platelet adhesion and activation might be particularly affected by adsorbed fibrinogen, a mediator of platelet activation via its direct interaction with platelet receptors [10–12]. Fibronectin is a key component of the extracellular matrix (ECM), is a large dimeric glycoprotein triggering cell adhesion , undergoes cell-driven assembly in supramolecular fibrils, and provides specific binding sites for various ECM biopolymers . Albumin is the most abundant component of many biofluids, serving as transport media for various metabolites and as regulator of osmotic pressure [14, 15]. Moreover, albumin is a widespread protein used to block ‘non-specific’ cell adhesion , but some controversy exists given that albumin has been found to promote platelet and macrophage adhesion [17, 18].
Metal surfaces are, with few exceptions, covered with a metallic oxide layer of a few to several nanometers in thickness. As a consequence, interactions between metal implants, proteins, and cells are governed by the physical–chemical properties of their corresponding metallic oxides. The excellent chemical inertness, corrosion resistance, repassivation ability, and biocompatibility of titanium are thought to result from the chemical stability (high corrosion resistance) a thermodynamically stable state and structure of the titanium oxide film mainly composed of TiO2 at physiological pH values [19–22]. The following crystal structures have been recognized for TiO2: rutile (tetragonal), anatase (tetragonal), and brookite (orthorhombic). TiO2 also exists in an amorphous state . Regarding the surface oxide layer on c.p. Ti dental implant systems, spectroscopic studies suggest that the oxide is amorphous and its thickness is approximately 2–17 nm [22–24].
In vitro studies have shown that material surface properties, including the negatively charged and hydrophilic TiO2 layer, are of importance for protein adsorption . One reported issue is that in vitro adsorption of the same protein on the same type of surface may vary considerably between different studies as a consequence of the different experimental conditions. However, some general characteristics of protein interaction with titanium surfaces can be found. Proteins in solution accumulate spontaneously at materials interfaces and, in the case of titanium surfaces, most of them adsorb irreversibly since the process is highly energetically favorable . However, some adsorbed proteins can detach due to protein competition and displacement processes . The replacement of initially adsorbed proteins with high mobility by new proteins with lower mobility but higher affinity for the surface is the so-called Vroman effect . Different adsorption isotherms reported in the literature [1, 27, 28] showed high affinity of fibronectin to titanium surfaces.
Proteins systems are very complex and thus, measurements with well-defined model systems carried out under well-controlled conditions are essential to understand the underlying mechanisms of protein adsorption . Different techniques have been used for quantitatively characterize protein adsorption on solid surfaces. Most of them either (a) rely on labelling adsorbing molecules with a radioactive or fluorescent tag and comparing their signal on the surface before and after adsorption or (b) are based in changes in the optical properties of the surface while the layer of proteins is being adsorbed, such as ellipsometry and surface plasmon resonance. The former are laborious procedures that do not allow real-time monitoring of the studied process. The later provides us with real-time data of the formation of the protein layer; however, information of the structural/conformational changes of the protein layer is not obtained. We used a quartz-crystal microbalance with dissipation monitoring (QCM-D). With QCM-D the kinetics of both mass changes and structural/mechanical properties changes are obtained [30–32]. This is possible because the equipment simultaneously collects both the resonant frequency and the energy dissipation signals of a quartz crystal sensor coated with the material in question. The change in mass of the sensor while the proteins adsorb produces changes in the frequency of the sensor. Also, the structural and conformational effects at the protein layer when water incorporates into and interacts with the protein layer can be monitored by tracking changes in the energy dissipation of the system . However, the specific structural changes have to be further investigated with complementary experimental techniques.
Even though increasing number of studies have investigated the process of protein adsorption on biomaterials there is still lack of fundamental knowledge on how some specific combinations of relevant proteins interact with synthetic substrates of clinical interest. We report here on (a) the kinetics of the protein adsorption process, (i.e. evolution of mass and structure of the protein layer on sensors coated with TiO2 from three different monoprotein solutions, Fbg, Fn, and Alb) and (b) the competition of the proteins for adsorbing on the TiO2 surfaces in two-step experiments; (i.e. sequentially exposing the surfaces to two different monoprotein solutions). All those experiments were performed using QCM-D.
2.1.1 TiO2-Coated Sensors
Titanium QCM-D sensors (QSX 310) were purchased at Q-Sense (Sweden). The sensors consisted of gold-coated quartz crystals (14 mm in diameter) covered using vapour deposition with a 50-nm thick TiO2 layer. The fundamental mode of the sensors was at 4.95 MHz. The cleaning protocol used before starting each experiment was: (1) 10 min sonication with ethanol (96 %, Panreac); (2) 10 min sonication with acetone (99.5 %, Panreac); (3) 10 min sonication with MilliQ® ultrapure water; and (4) 10 min UV/ozone chamber (BioForce Nanosciences, Ames, USA). During the cleaning process the sensors were held on a Teflon® Q-Sense Sensor Holder.
2.1.2 Protein Solutions
Human fibronectin (Fn, ≥95 %, CAS Number: 86088-83-7), human fibrinogen (Fbg, 50–70 %, CAS Number: 9001-32-5) and bovine serum albumin (BSA, ≥96 %, CAS Number: 9048-46-8) were purchased at Sigma Aldrich (Sant Louis, USA). The protein concentration of each solution was different depending on the type of protein and the specific test (see below).
2.2.1 Surface Characterization of TiO2-Coated Sensors
Roughness and wettability of TiO2-coated sensors (Q-Sense, Sweden) was measured.
Roughness measurements were performed with a white-light interferometer (Wyko NT1100, Veeco, USA). Images were taken using a 10× objective. A Gaussian filter was used to separate waviness and form from roughness of the surface by applying a cut-off value (λc) of 0.25 mm according to ISO 11562:1996 standard .
The static contact angle (SCA) of the TiO2-coated sensors was assessed using the sessile drop method. Ultrapure distilled water (Millipore) 3–6 μl drops were generated with a micrometric syringe and deposited on the substrate surface. Syringe and sample were placed inside a customized PMMA environmental chamber with two optical glass windows to saturate humidity during the experiments. The wettability studies were performed with a contact angle video based system (Contact Angle System OCA15plus, Dataphysics, Germany) and analysed with the SCA20 software (Dataphysics, Germany).
The chemical analysis of the TiO2 surfaces was performed with a X-ray photoelectron spectrometer (XPS) XPS experiments were performed in a PHI 5500 Multitechnique System (Physical Electronics) with a monochromatic X-ray source (Aluminium Kα line of 1,486.6 eV energy and 350 W), placed perpendicular to the analyzer axis (takeoff angle of 45°). The analyzed area was a circle of 0.8 mm diameter, and selected resolution for the fitted spectra was 23.5 eV of Pass Energy ands 0.1 eV/step. The analysis of the spectra obtained was made with the software Multipak. All the binding energies were referenced to the C1 s peak at 284.8 eV.
Grazing incidence X-ray diffraction (GIXD) (D8 Advance, Bruker® Axs) was also carried out on TiO2-coated sensors. The diffractometer was operated at 40 kV and 10 mA using a Ni filter, Cu Kα radiation, and was equipped with a 0.6 mm slit. The incidence angle was fixed at 1° thus limiting the X-ray beam penetration depth. A counting time of 8 s per 0.01° step was set for each measurement. A range of 2θ angles of 10–80° were scanned.
2.2.2 Protein Adsorption Tests
184.108.40.206 Adsorption from Monoprotein Solutions
220.127.116.11 Two-Step Protein Adsorption
BSA–Fbg: 500 μg/ml for BSA and 50 μg/ml for Fbg
Fbg–BSA: 50 μg/ml for Fbg and 500 μg/ml for BSA
BSA–Fn: 2 mg/ml for BSA and 20 μg/ml for Fn
Fn–BSA: 50 μg/ml for Fn and 5 mg/ml for BSA
2.2.3 Quartz Crystal Microbalance with Dissipation Monitoring
Equation 1 holds if the adsorbed layer is rigid, if the added mass is small compared to the weight of the crystal, if there is no slip in the metal/layer interface, and if the layer is homogeneously distributed on the surface. The Sauerbrey relation concludes that the change in resonance frequency is proportional to the change in the adsorbed mass if the adsorbed layer is much smaller than the mass of the crystal .
2.2.4 QCM-D Data Analysis
A time-resolved analysis of f and D shifts for the different overtones is required to assess the model to be used. Under normal conditions, the fundamental overtone is disregarded since it is too sensitive; whereas the third, fifth and seventh overtone are studied. The data will require viscoelastic modelling if D values are not close to zero, or if D-shifts (in 1E−6) are higher than 5 % of f shifts (in Hz). In addition, if the are significant differences between overtones, viscoelastic modelling is suggested .
A viscoelastic adlayer in all surfaces reported here was assessed and consequently analyzed. For modelling the adsorbed layer of proteins we have used the so-called Voigt-based representation of a viscoelastic solid, in which the adsorbed film is represented by a (frequency dependant) complex shear modulus. The description of the model and details on its implementation using a QCM-D are reported elsewhere . The calculation of the relevant parameters from the obtained protein layer, i.e. viscosity, shear elastic modulus and thickness, according to the Voigt model was performed with the appropriate software (Qtools, Q-Sense AB, Sweden). To do so, we initially fixed two parameters (fluid density and fluid viscosity) to those corresponding to water. This is a good estimation since the system includes a highly hydrated protein film. The density of the protein layer, also fixed to fit the model, might vary between 1,000 kg/m3 (water) and 1,350 kg/m3 (protein) [32, 48–50] depending on protein coverage. 1,200 kg/m3 was chosen on the basis that a saturated protein coverage is slightly above 50 % [32, 48, 50]. The rest of the protein layer should be covered by water.
3.1 Surface Characterization of TiO2-Coated Sensors
The arithmetic mean value ± standard deviation of the roughness, Ra, for the TiO2 surfaces was 5.11 ± 0.31 nm; and the mean static water ± standard deviation contact angle was 51.25 ± 2.87°.
3.1.1 Adsorption from Monoprotein Solutions
3.2 Two-Step Protein Adsorption
3.2.1 Interactions Between BSA and Fbg on TiO2
First step: solution with BSA–Second step: solution with Fbg (BSA–Fbg).
Figure 5 a shows time-resolved frequency and dissipation shift graphs during the two-step BSA–Fbg protein adsorption process. BSA adsorption caused a final Δf = −12 Hz; then, the addition of Fbg increased the frequency shift of the studied system to a final value of −18 Hz. The ΔD versus t graph showed a continuous increase in energy dissipation during BSA adsorption followed by additional increase during the first 20 min of Fbg adsorption up to a steady value that indicated saturation of the layer of proteins.
Figure 5b shows the ∆D versus. ∆f plot for the experiment presented in Fig. 5a. The process of BSA adsorption showed a similar response than the one obtained for the one-step adsorption from monoprotein solutions (Fig. 3), where three different slopes/phases were detected. These phases had increasing ∆D/∆f slope for increasing ∆D. That indicated an ongoing decrease of the shear elastic modulus of the adlayer during the process of BSA adsorption on the TiO2-surfaces. Further Fbg adsorption on the BSA layer also showed the same type of response than in the case of the adsorption from a one-step Fbg solution (Fig. 3); i.e., two slopes/phases and final near zero ∆D/∆f slope values.
First step: solution with Fbg–Second step: solution with BSA (Fbg–BSA).
Figure 5 a reports on frequency and dissipation shift versus time during the two-step protein adsorption process of BSA–Fbg solutions in PBS. During adsorption of Fbg on TiO2-coated sensors a rapid and significant decrease of frequency of the sensor was coupled with a continuous increase of the dissipation shift. Interestingly, the further exposure of the Fbg-coated TiO2-surface to the BSA solution caused a continuous increase of Δf—decrease of mass—as well as a variation of ΔD with its maximum reached near after the initiation of the adsorption of BSA.
The first portion of the ∆D versus ∆f plot for the Fbg–BSA test on TiO2-coated sensors (Fig. 5d) showed a two slope/phase response that was mainly characterized by a significant decrease of the ∆D/∆f slope in the final part of Fbg adsorption. That was the same response than the one obtained during one-step monoprotein solution studies (Fig. 3). The Fbg-coated TiO2-surfaces were exposed to a BSA solution during the second step of the experiment resulting in a four-stage adsorption process that was ended in a phase of adsorption with steady near-zero ∆D/∆f slope.
18.104.22.168 Interactions Between BSA, Fn and TiO2
First step: BSA solution–Second step: Fn solution (BSA–Fn).
A rapid decrease in frequency shift was detected during adsorption of the first protein; i.e., BSA (Fig. 6a). During the intermediate step of washing with PBS 1× certain quantity of BSA desorbed from the adlayer as showed by the slight increase in frequency shift. The notable higher decrease in frequency shift in this BSA–Fn experiment than in Fbg–BSA and BSA–Fbg experiments can be attributed to the higher concentration of BSA in the solution for this experiment. D increased with time during BSA adsorption (Fig. 6a). Further adsorption of Fn resulted in both a total frequency shift variation of −1 Hz and an increasing value of the dissipation shift.
The ∆D versus ∆f plot (Fig. 6b) for the BSA–Fn test showed a single ∆D/∆f slope/phase process during the initial BSA adsorption. Then, after exposure of the BSA-coated TiO2 surfaces to the Fn-solution the adlayer showed an abrupt increase in its ∆D/∆f slope.
First step: Fn solution–Second step: BSA solution (Fn–BSA).
Figure 6c shows time-resolved frequency and dissipation shift graphs during the two-step Fn–BSA protein adsorption process. During the exposure of BSA to Fn-coated TiO2-surfaces, an increase of 2 Hz in frequency was assessed. The increase in frequency was due to a loss of mass from the protein-coated surface. Interestingly, this was coupled to a continuous slight decrease in dissipation shift.
Figure 6d shows ΔD versus Δf plots for the two-step Fn–BSA tests. A constant ΔD/Δf slope was recorded during the Fn adsorption as it was the case during the one-step monoprotein adsorption experiments (Fig. 3). The further adsorption of BSA slightly increased the ΔD/Δf slope.
Figure 7 shows the evolution of the shear elastic modulus comparing the change in rigidity of the resulting protein layer when Fbg (Fig. 7a) or Fn (Fig. 7b) were the first protein to be introduced in the system in the two-step competition experiments. When BSA was introduced in the system after Fn the resulting layer was increasingly more rigid; however, a sudden notable drop in rigidity was recorded when BSA interacted with the previously adsorbed Fbg layer.
4.1 Single-Protein Adsorption Studies
Protein concentration in solution and molecular weight of the molecule tested are two of the most influencing properties on mass and thickness of protein adlayers. Other properties, such as protein conformation when adsorbed on the surface and amount of trapped water into the adlayer may also have an important effect on the specific characteristics of the layer of proteins obtained. Our results confirmed previous findings by others : the higher the protein concentration in solution the higher the amount of adsorbed proteins on the surface (Fig. 2a, b).
As previously discussed, the Sauerbrey equation—Eq. 1—provides an accurate estimation of the adsorbed mass only if the adsorbed protein layer on the studied surface is rigid. To determine which model, Sauerbrey or Voigt, is the most appropriate for assessing properties of the protein layer, the frequency and the dissipation shift versus time of the different overtones should be plotted (data not shown). The data will require viscoelastic modelling if D values are not close to zero, or if D-shifts (in 1E−6) are higher than 5 % of f shifts (in Hz). In addition, if there are significant differences between overtones viscoelastic modelling is suggested . The significant ΔD recorded for all the performed tests here indicated that a viscoelastic layer was obtained and thus, the use of the Sauerbrey equation would underestimate the thickness of the protein adlayers on the TiO2 surfaces. Subsequently, the Voigt model was used to calculate, compare, and discuss the parameters of the protein layers obtained—thickness, viscosity and shear elastic modulus. In addition, surface mass density was calculated from the thickness values using the Voigt model as well.
Fn is a relatively big protein with a molecular weight of 450–500 kDa which is higher than the one of Fbg, 340 kDa. One would expect that the surface mass density for the adsorbed layers from Fn solutions would be higher than those obtained for Fbg layers. This is not the case as shown in Fig. 4b. The higher concentration of Fbg in solution, 100 μg/m, than the one for the Fn solution, 40 μg/ml, may explain the higher thickness of the Fbg layer. However, we obtained Fbg layers with lower viscosity and shear elastic modulus than the Fn layers (Fig. 4), which is an indication of a less compact Fbg layer in comparison with the Fn layer. The later might be associated to the effect of a protein rearrangement in the layer or to an increased amount of trapped water until the layer is saturated and the change in dissipation is almost constant (Fig. 3). This was further confirmed by the fact that the shear elastic modulus of the layer of Fbg adsorbed from the lower concentration solution, 80 μg/ml, was higher than the one from the higher concentration solution, 100 μg/ml (data not shown).
A complex relationship between all these properties could be speculated as further analysis of BSA adsorption was performed. During BSA adsorption, water was continuously being incorporated and trapped in the layer of proteins, as seen in the increasing ΔD in Fig. 3 and demonstrated by low values of shear elastic modulus (Fig. 4d). However, the thickness and surface mass density obtained for the BSA-adsorbed layer were significantly lower than for both Fn- and Fbg-adsorbed layers. From those results when comparing with the other types of proteins tested a prevalent influence of the molecular weight (66 kDa), size, and globular-like shape of albumin is suggested. Roach et al.  concluded that BSA adsorbs on hydrophilic and hydrophobic surfaces with minimal conformational changes. Thus, the three slopes/phases obtained for BSA adsorption (Fig. 3) might be due to a process of multilayer adsorption, as once demonstrated by Höök et al.  studying haemoglobin adsorption on gold-coated sensors.
4.2 Two-Step Protein Adsorption Studies
For all the sequential protein adsorption experiments, the adsorption response of the proteins for the first solution introduced in the system was similar to that obtained for single-protein adsorption studies on TiO2-surfaces. If differences were found, those were mainly attributed to the different protein concentrations in solution used for the single-protein and two-step studies. Macromolecules that are being adsorbed on a surface can use several adsorption sites depending on their structure and molecular mass. At low concentrations, adsorbed proteins are in an unfolded state with more located binding sites to adsorb . Protein adsorption, such as Fn and human serum albumin [27, 54] are related to structural rearrangements in the molecules that enable them to overcome the unfavourable conditions offered by an electrostatic repelling surface. At higher protein concentrations the adsorption layer becomes more compressed and molecules with different degrees of unfolding will coexist at the interface , which makes more difficult for some molecules to overcome the unfavourable conditions to reach adsorption. This fact can explain the higher rigidity of the protein layer obtained for the lowest Fn and BSA concentrations since protein molecules could be more tightly bound to the surface. The opposite occurred when testing at a higher protein concentration, as the 2 mg/ml BSA concentration used for two-step studies (BSA–Fn). A more viscoelastic protein layer was obtained in comparison with 500 μg/ml (BSA–Fbg) and 100 μg/ml (monoprotein solution studies) BSA concentrations (results not shown).
4.2.1 Interactions Between BSA and Fbg on TiO2
After the initial adsorption of BSA on TiO2-coated sensors Fbg adsorbed on the TiO2-BSA surface as indicated by the continuous decrease in frequency. The frequency shift following Fbg adsorption was aprox. −10 Hz lower than during the monoprotein solution test (Fig. 2). BSA had a high affinity for the TiO2-crystal surfaces which resulted in adsorption of Fbg either on top of BSA-layer or on the few BSA-free available spaces on those crystals. In any case, that resulted in reduced Fbg adsorption on top of the BSA-coated surfaces in comparison with the monoprotein solution test where Fbg interacted directly on top of the TiO2 surface. Additionally, this sequence of protein interactions did not change the structural and/or water entrapped in the BSA and Fbg adlayers in comparison with the same protein layers from separated monoprotein solution experiments, as shown in ∆D–∆f plots (Figs. 5b, 3, respectively). This suggested that a multilayered coating with minimal BSA–Fbg interaction was obtained, i.e. Fbg did not displace, but rather layered on top of the BSA layer.
The interaction processes between these two proteins were different when Fbg was first adsorbed on TiO2-surfaces followed by BSA adsorption (Fig. 5c, d) in comparison with the initial adsorption of BSA followed by Fbg adsorption. Most notably, an increase in frequency shift was recorded after the BSA solution was introduced in the system and interacted with the already formed Fbg-layer on TiO2-crystals. This indicated that BSA was able to displace some of the Fbg molecules that were previously adsorbed on the surfaces. In fact, some previously reported works have concluded that some proteins can displace pre-adsorbed molecules because they have a superior affinity and they are able to bind strongly with the analyzed surface . Although others have shown that BSA has not strong affinity for surfaces with contact angles comparable to those of the TiO2-sensors tested here , the small size of BSA can favour this molecule to reach some of the non-covered surface points as well as to compete with Fbg molecules for the already occupied positions on the crystals. If this is the case, BSA molecules showed a higher affinity for TiO2 surfaces than Fbg molecules. This conclusion is aligned with the results discussed in the previous paragraph; i.e., BSA formed a stable layer on the TiO2 surfaces that was not disturbed by the later presence of Fbg in solution. The transition stage resulting from the PBS washings after Fbg was adsorbed also indicated that a small percentage of the adsorbed Fbg molecules were removed—those that were only weakly adsorbed to the surface, as exemplified by an increase of the frequency shift and ΔD/Δf slope before BSA was further introduced in the system. Thus, the displacement of Fbg molecules by BSA molecules can be also favoured by a stronger affinity of the BSA protein to the TiO2-surfaces than Fbg for this system. During the subsequent BSA adsorption, the mechanical properties of the layer significantly dropped (Fig. 7) and some structural rearrangements might have occurred, as shown in Fig. 5d but those potential structural changes of the layer did not notably affect the shear elastic modulus of the overall protein coating (Fig. 7). That confirmed that the mechanical properties of the final coating where mainly determined by the properties of the adsorbed BSA layer and thus, strongly influenced by a significant displacement of the Fbg molecules.
4.2.2 Interactions Between BSA, Fn and TiO2
The two-step sequential protein adsorption during the BSA–Fn experiments showed a rapid and dramatic decrease in frequency when BSA is introduced in the system, mainly due to its high concentration in PBS. Note that the concentration is 4 times higher than the one used for the BSA–Fbg experiments. Since the solution had an elevated concentration many molecules were attached immediately on the surface. Ramsden  concluded that depending on the protein, surface, and solution conditions the occupied area per adsorbed molecules is inversely proportional to the rate the proteins reach the surface. Thus, the rapid BSA adsorption most-likely caused a nearly non-spread molecules layer onto the surface. This was further confirmed by the significant desorption of BSA molecules when the surfaces were washed with PBS that in turn, made the BSA adlayer more rigid, as shown by a decreased dissipation factor. This was an occurrence that did not happen when the BSA–Fbg system was tested. A slight decreased in the frequency shift was recorded during further interaction of Fn with the BSA-layer adsorbed on TiO2-crystals. Thus, Fn is being adsorbed on top of the surface but in a very small quantity. In fact, an almost negligible 1 %-increase in both thickness and surface mass density of the adlayer was calculated after adsorption of Fn. The adsorption of Fn on top of the BSA layer also resulted in a less rigid, less viscous layer with a similar ΔD/Δf plot than the one obtained during the monoprotein Fn solution test. This, as aforementioned for the BSA/Fbg experiments, indicated that minimal interaction between the two types of proteins resulted in a multilayered coating.
Again, as in the case of the interaction with Fbg, when BSA was introduced in the second solution during the Fn–BSA two-step sequential adsorption experiments, an increase of frequency shift was recorded, i.e. the coating decreased in total mass. In this case, though, a continuous decrease in dissipation shift during the adsorption of BSA was determined. That indicated that some of the Fn molecules were displaced by BSA molecules with the displacement interactions resulting in a reduced thickness and surface mass density, and an increased rigidity of the resulting adlayer (Fig. 7).
All the previously discussed results showed on the one hand the ability of BSA to compete for locations to be adsorbed and finally displace bigger proteins that were previously adsorbed on the TiO2-coated sensors and thus, demonstrated that BSA has a higher affinity for TiO2 surfaces than Fn and Fbg. On the other hand, BSA adsorption had different effects on the structure and mechanical properties of the resulting layer when Fn or Fbg were pre-adsorbed on the TiO2-surfaces as demonstrated by the significant differences found in the effects on the mechanical properties of the final adlayer.
The ‘Vroman effect’ relates to the competition between two or more proteins for the same adsorbent surface. The generalized Vroman effect  demonstrated that adsorption from blood plasma involves a complex series of adsorption and displacement steps in which low molecular weight, MW, proteins arriving first at a surface are displaced by relatively higher MW proteins arriving later. Literature has not consistently supported the Vroman effect theory, though. Brash and Lyman  proposed that in protein mixtures, such as blood, the proteins would simply adsorb in proportion to their concentrations in solution. Noh et al.  agreed with Brash and Lyman’s conclusion, except when mass balance mandates a discrimination against larger proteins adsorbing from a mixture of smaller-and-larger proteins.
Concerning to our results, the studied Fbg/BSA and Fn/BSA experiments showed that BSA displaced larger proteins such as Fn and Fbg whereas in BSA/Fbg and BSA/Fn the larger proteins laid on top of BSA forming an adsorbed protein bi-layer. Overall, we can conclude that in the system studied here (Fn, Fbg, BSA on TiO2) the Brash and Lyman effect prevailed since a lower molecular weight and more concentrated protein in solution adsorbed preferentially to TiO2 surfaces.
This work was supported by the CICYT- Interministerial Comission of Science and Technology of the Ministry of Science and Technology of Spain, Grant MAT2009-13547. The authors thank Dr. Elisabeth Engel and Dr. George Altankov for experimental help with protein solution preparation. MP would like to thank the Universitat Politècnica de Catalunya (UPC) for funding through a fellowship Grant to complete her PhD graduate studies.
- MacDonald DE, Deo N, Markovic B, Stranick M, Somasundaran P (2002) Biomaterials 23:1269–1279View ArticleGoogle Scholar
- Puleo DA, Nanci A (1999) Biomaterials 20:2311–2321View ArticleGoogle Scholar
- Andrade JD, Hlady V (1986) Adv Polym Sci 79:1–63View ArticleGoogle Scholar
- Rabe M, Verdes D, Seeger S (2011) Adv Colloid Interface Sci 162:87–106View ArticleGoogle Scholar
- Haynes CA, Norde W (1994) Colloids Surf B Biointerfaces 2:517–566View ArticleGoogle Scholar
- Haynes CA, Sliwinsky E, Norde W (1994) J Colloid Interface Sci 164:394–409View ArticleGoogle Scholar
- Deligianni DD, Katsala N, Ladas S, Sotiropoulou D, Amedee J, Missirlis YF (2001) Biomaterials 22:1241–1251View ArticleGoogle Scholar
- Michael K, Vernekar V, Keselowsky B, Meredith J, Latour R, García AJ (2003) Langmuir 19:8033–8040View ArticleGoogle Scholar
- Schmidt DR, Waldeck H, Kao WJ (2009) Protein adsorption to biomaterials. In: Puleo DA, Bizios R (eds) Biological interactions on materials surfaces. Springer, Berlin, pp 2–17Google Scholar
- Béguin S, Kumar R (1997) Thromb Haemost 78:590–594Google Scholar
- Kumar R, Béguin S, Hemker HC (1994) Thromb Haemost 72:713–721Google Scholar
- Tzoneva R, Heuchel M, Groth T, Altankov G, Albrecht W, Paul D (2002) J Biomater Sci Polym Ed 13:1033–1050View ArticleGoogle Scholar
- Scotchford CA, Ball M, Winkelmann M, Voros J, Csucs C, Brunette DM, Danuser G, Textor M (2003) Biomaterials 24:1147–1158View ArticleGoogle Scholar
- Renner L, Pompe T, Salchert K, Werner C (2005) Langmuir 21:4571–4577View ArticleGoogle Scholar
- Ramsden JJ (1994) Q Rev Biophys 27:41–105View ArticleGoogle Scholar
- Smith RA, Mosesson MW, Daniels AW, Kent T (2000) J Mater Sci Mater Med 11:279–285View ArticleGoogle Scholar
- Sivaraman B, Latour RA (2010) Biomaterials 31:1036–1044View ArticleGoogle Scholar
- Godek ML, Michel R, Chamberlain LM, Castner DG, Grainger DW (2009) J Biomed Mater Res Part A 88A:503–519View ArticleGoogle Scholar
- Williams DF (1976) Corrosion of implant materials. Annu Rev Mater Sci 6:237–266Google Scholar
- Liu XY, Chu PK, Ding CX (2004) Mater Sci Eng R Rep 47:49–121View ArticleGoogle Scholar
- Textor M, Sittig C, Frauchiger V, Tosatti S (2001) Properties and biological significance of natural oxide films on titanium and its alloys. In: Brunette DM, Tengvall P, Textor M, Thomsen P (eds) Titanium in medicine. Springer, Berlin, pp 172–224Google Scholar
- Lausmaa J (1996) J Electron Spectrosc Relat Phenom 81:343–361View ArticleGoogle Scholar
- Wälivaara B, Aronsson B, Rodahl M, Lausmaa J, Tengvall P (1994) Biomaterials 15:827–834View ArticleGoogle Scholar
- Paine M, Wong J (2003) Osteoblast response to pure titanium and titanium alloy. In: Ellingsen J, Lyngstadaas S (eds) Bio-implant interface. Improving biomaterials and tissue reactions. CRC Press, Boca RatonGoogle Scholar
- Holgers K-M, Esposito M, Kalltorp M, Thomsen P (2001) Titanium in soft tissues. In: Brunette DM, Tengvall P, Textor M, Thomsen P (eds) Titanium in Medicine. Springer, Berlin, pp 513–560View ArticleGoogle Scholar
- Tengvall P (2001) Proteins at titanium interfaces. In: Brunette DM, Tengvall P, Textor M, Thomsen P (eds) Titanium in Medicine. Springer, Berlin, pp 457–484View ArticleGoogle Scholar
- Sousa SR, Moradas-Ferreira P, Barbosa MA (2005) J Mater Sci Mater Med 16:1173–1178View ArticleGoogle Scholar
- MacDonald DE, Markovic B, Boskey AL, Somasundaran P (1998) Colloids Surf B Biointerfaces 11:131–139View ArticleGoogle Scholar
- Norde W, Lyklema J (1978) J Colloid Interface Sci 66:257–265View ArticleGoogle Scholar
- Voinova M, Rodahl M, Jonson M, Kasemo B (1999) Phys Scr 59:391–396View ArticleGoogle Scholar
- Hook F, Kasemo B, Nylander T, Fant C, Sott K, Elwing H (2001) Anal Chem 73:5796–5804View ArticleGoogle Scholar
- Hook F, Voros J, Rodahl M, Kurrat R, Boni P, Ramsden JJ, Textor M, Spencer ND, Tengvall P, Gold J, Kasemo B (2002) Colloids Surf B Biointerfaces 24:155–170View ArticleGoogle Scholar
- Hook F, Rodahl M, Brzezinski P, Kasemo B (1998) Langmuir 14:729–734View ArticleGoogle Scholar
- Geometrical Product Specifications (GPS)—Surface texture: Profile method—Metrological characteristics of phase correct filters. ISO 11562:1996. 1996. Ref Type: GenericGoogle Scholar
- Wassell DT, Embery G (1996) Biomaterials 17:859–864View ArticleGoogle Scholar
- MacDonald DE, Markovic B, Allen M, Somasundaran P, Boskey AL (1998) J Biomed Mater Res 41:120–130View ArticleGoogle Scholar
- Tamada Y, Ikada Y (1993) J Colloid Interface Sci 155:334–339View ArticleGoogle Scholar
- Bai Z, Filiaggi MJ, Dahn JR (2009) Surf Sci 603:839–846View ArticleGoogle Scholar
- Grunkemeier JM, Tsai WB, McFarland CD, Horbett TA (2000) Biomaterials 21:2243–2252View ArticleGoogle Scholar
- Oliva F, Avalle L, Cámara O, De Pauli C (2003) J Colloid Interface Sci 261:299–311View ArticleGoogle Scholar
- Andersson M, Andersson J, Sellborn A, Berglin M, Nilsson B, Elwing H (2005) Biosens Bioelectron 21:79–86View ArticleGoogle Scholar
- Thompson M, Arthur CL, Dhaliwal GK (1986) Anal Chem 58:1206–1209View ArticleGoogle Scholar
- Fredriksson C, Kihlman S, Rodahl M, Kasemo B (1998) Langmuir 14:248–251View ArticleGoogle Scholar
- Hemmersam AG, Foss M, Chevallier J, Besenbacher F (2005) Colloids Surf B Biointerfaces 43:208–215View ArticleGoogle Scholar
- Ward MD, Buttry DA (1990) Science 249:1000–1007View ArticleGoogle Scholar
- Rodahl M, Hook F, Krozer A, Brzezinski P, Kasemo B (1995) Rev Sci Instrum 66:3924–3930View ArticleGoogle Scholar
- Q-Sense. QCM-D. 2008. http://www.q-sense.com/. Accessed 21 February 2008
- Weber N, Pesnell A, Bolikal D, Zeltinger J, Kohn J (2007) Langmuir 23:3298–3304View ArticleGoogle Scholar
- Hovgaard M, Rechendorff K, Chevallier J, Foss M, Besenbacher F (2008) J Phys Chem B 112:8241–8249View ArticleGoogle Scholar
- Vörös J (2004) Biophys J 87:553–561View ArticleGoogle Scholar
- Roach P, Farrar D, Perry CC (2005) J Am Chem Soc 127:8168–8173View ArticleGoogle Scholar
- Hook F, Rodahl M, Kasemo B, Brzezinski P (1998) Proc Nat Acad Sci USA 95:12271–12276View ArticleGoogle Scholar
- Yang YZ, Cavin R, Ong JL (2003) J Biomed Mater Res Part A 67A:344–349View ArticleGoogle Scholar
- do Serro APVA, Fernandes AC, Saramago BDV, Norde W (1999) J Biomed Mater Res 46:376–381View ArticleGoogle Scholar
- Miller R, Treppo S, Voigt A, Zingg W, Neumann AW (1993) Colloids Surf 69:203–208View ArticleGoogle Scholar
- Jung S, Lim S, Albertorio F, Kim G, Gurau MC, Yang RD (2003) J Am Chem Soc 125:12782–12786View ArticleGoogle Scholar
- Vroman L, Adams AL (1969) Surf Sci 16:438–446View ArticleGoogle Scholar
- Brash JL, Lyman DJ (1969) J Biomed Mater Res Part A 3:175–189View ArticleGoogle Scholar
- Noh H, Vogler EA (2007) Biomaterials 28:405–422View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. Open AccessThis article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.