Adsorptions of Plasma Proteins and Their Elutabilities from a

Anal. Chem. 2000, 72, 1523-1531

Adsorptions of Plasma Proteins and Their Elutabilities from a Polysiloxane Surface Studied by an On-Line Acoustic Wave Sensor Biljana A. Cavic and Michael Thompson*

Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada

Gold electrodes of thickness-shear mode acoustic wave sensors were modified with poly[(mercaptopropyl)methylsiloxane]. The flow-through adsorption of three major plasma proteins (human serum albumin, fibrinogen, and immunoglobulin G) was detected by acoustic network analysis. The elution of fibrinogen and albumin from coated and unmodified gold surfaces by sodium dodecyl sulfate was studied with respect to different adsorption times and protein concentrations. Both sequential and competitive adsorptions of the three proteins on polymermodified surfaces of sensors were examined as were simultaneous adsorptions from binary and ternary mixtures. The experimental results confirm that the competitive behaviors of proteins in terms of adsorptive processes are explained by factors other than displacement phenomena. Protein adsorption at the biomaterial-to-blood interface is a very complex process not yet fully understood. Studies involving interactions between specific materials and proteins performed with single-protein solutions are very useful. However, the consequences of mutual interactions among different protein molecules that could affect their adsorptive behaviors are often neglected. Among numerous investigations, the examination of competitive processes between various proteins has figured prominently. Such studies repeatedly tend to assume that a more realistic approach is represented by the partial simulation of physiological conditions, using blood or plasma as a complex medium, rather than mixtures of proteins in buffer solution. Nevertheless, uncertainty remains regarding phenomena related to protein behaviors at interfaces, especially since laboratory experiments do not necessarily reflect adsorptive processes in vivo. Since the pioneering work of Vroman,1,2 where the rapid loss in reactivity of fibrinogen to anti-fibrinogen upon adsorption was partially attributed to its possible displacement by other plasma proteins, many studies have examined the competitive adsorption behaviors of proteins. Among the means available to study such processes, the most frequently applied methods include radiochemical3,4 or fluorescent5 labeling of selected protein species that are added to the protein mixture or plasma. Another useful * Corresponding author. E-mail: [email protected]. (1) Vroman, L.; Adams, A. L. Surf. Sci. 1969, 16, 438-446. (2) Vroman, L.; Adams, A. L. J. Biomed. Mater. Res. 1969, 3, 43-67. 10.1021/ac9909094 CCC: $19.00 Published on Web 02/23/2000

© 2000 American Chemical Society

emerging method is total internal reflection fluorescence spectroscopy (TIRF) combined with ellipsometry, which can be employed to investigate the competitive adsorption of plasma proteins in situ and in real time.6 Acoustic wave sensors, particularly thickness-shear mode devices, have also been applied to the monitoring of protein adsorption processes.7-10 Interestingly, the study of related competitive phenomena has not been a subject that has attracted the attention of researchers in this field. Thickness-shear mode acoustic wave devices (TSMs) are composed of piezoelectric quartz crystals with metal electrodes in place for the application of an oscillating electric field. The generated transverse waves, when propagating into liquid, are attenuated by viscous forces. The resulting damping effect is associated with both the viscosity of the liquid medium and various interfacial processes. The most complete characterization of these devices in terms of electrical information connected with their operation in liquids is acquired by the application of the network analysis method. Values of the magnitude and phase of the impedance of the sensor are computed, followed by data-fitting to an equivalent electrical circuit. Series and parallel resonance frequencies, motional resistance, and interfacial capacitance are among the quantities that can be obtained from acoustic network analysis. These values provide information related not only to surface mass and the properties of the contacting liquid but also to interfacial properties such as interfacial free energy, liquid structure, and coating film properties.11,12 These parameters, in turn, influence acoustic coupling phenomena. Silicone polymers are among the most widely applied biomaterials. These substrates contain a siloxane (Si-O-Si) backbone together with various side functionalities. Their molecular struc(3) Wojciechowski, P.; ten Hove, P.; Brash, J. L. J. Colloid Interface Sci. 1986, 111, 455-465. (4) Horbett, T. A. J. Biomed. Mater. Res. 1981, 15, 673-695. (5) Lok, B. K.; Cheng, Y.-L.; Robertson, C. R. J. Colloid Interface Sci. 1983, 91, 104-116. (6) Lassen, B.; Malmsten, M. J. Colloid Interface Sci. 1997, 186, 9-16. (7) Yang, M.; Chung, F. L.; Thompson, M. Anal. Chem. 1993, 65, 3713-3716. (8) Rickert, J.; Brecht, A.; Go ¨pel, W. Anal. Chem. 1997, 69, 1441-1448. (9) Caruso, F.; Furlong, D. N.; Kingshott, P. J. Colloid Interface Sci. 1997, 186, 129-140. (10) Hook, F.; Rodahl, M.; Kasemo, B.; Brzezinski, P. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12271-12276. (11) Rickert, J.; Hayward, G. L.; Cavic, B. A.; Thompson, M.; Go ¨pel, W. In Sensors Update: Sensor TechnologysApplicationssMarkets; Baltes, H., Go ¨pel, W., Hesse, J., Eds.; Wiley-VCH: Weinheim, Germany, 1999; Vol. 5, pp 105139. (12) Yang, M.; Thompson, M. Anal. Chem. 1993, 65, 1158-1168.

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ture consists of a polymer chain with an exceptional freedom of rotation and low intermolecular forces. One of the consequences of such a molecular arrangement is the low surface energy of the polymer, which is manifested as surface hydrophobicity.13 The investigation of competitive protein adsorption phenomena on these materials is undoubtedly of major importance for the evaluation of blood and tissue compatibility of silicone-based polymers. The adsorption of different plasma proteins on siloxane compounds coated on TSMs and detected by acoustic network analysis was the subject of a previous study.14 The experimental findings in this work have clearly shown that the sensor response is governed not only by the amount of adsorbed protein but also by the boundary conditions present at the protein-liquid interface. Device responses upon protein adsorption typically consist of a characteristic decrease in series resonance frequency and an increase in motional resistance, implying the introduction of a viscoelastic film to the system or perturbation of acoustic coupling to the liquid. In the present paper, we extend our earlier work related to single-protein adsorption on the gold electrodes of a piezoelectric sensor, and those modified with siloxane compounds, to the investigation of competitive adsorption behavior involving solutions of fibrinogen, immunoglobulin, and albumin. We consider the simultaneous and consecutive adsorptions of these three major blood proteins on gold surfaces modified with a polymer containing a siloxane backbone, together with their removal by rinsing processes. EXPERIMENTAL SECTION Reagents. The proteins fibrinogen (Fg) from human plasma (fraction I, type I, 69% w/w protein, 91% of protein clottable), immunoglobulin G (IgG) from human plasma (purified, reagent grade, 97.3% w/w protein), and albumin (HSA) from human serum (fraction V, powder, 96-99% w/w protein) were purchased from Sigma (St. Louis, MO) and used without further purification. A 0.01 M phosphate-buffered saline (PBS) solution at pH 7.4 (Sigma, Catalog No. P-4417) was used throughout the experiments. Concentrations of protein solutions in PBS were determined as protein weight. Sodium dodecyl sulfate (SDS) was obtained from ICN Biomedicals (Costa Mesa, CA). Poly[(mercaptopropyl)methylsiloxane] (specific gravity 1.06; viscosity 75150 cSt) PMPMS, [SH(CH2)3SiO(CH3)]n, was purchased from United Chemical Technologies, Bristol, PA (Catalog No. PS 927). The solvents used in the work were of analytical reagent grade. Apparatus. AT-cut 9 MHz piezoelectric quartz crystals with gold electrodes (International Crystal Manufacturing, Oklahoma City, OK) were incorporated into the flow-through system described previously in detail.14 Only one side of the sensor mounted in the cell was exposed to the liquid flow. A network analyzer (HP 4195A Network/Spectrum Analyzer, Hewlett-Packard, Colorado Springs, CO) was used to measure the impedance. X-ray photoelectron spectra were recorded on a Leybold MAX200 X-ray photoelectron spectrometer using an unmonochromatized Mg KR source run at 15 kV and 20 mA. The energy scale of the spectrometer was calibrated to the Ag 3d5/2 and Cu 2p3/2 peaks at 368.3 and 932.7 eV, respectively. Survey runs were performed (13) Cavic, B. A.; Thompson, M.; Smith, D. C. Analyst 1996, 121, 53R-63R. (14) Cavic, B. A.; Thompson, M. Analyst 1998, 123, 2191-2196.

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at a pass energy of 192 eV and from 0 to 1000 eV on the binding energy scale. Samples were analyzed at a 90° angle relative to the electron detector, using a 1 mm analysis spot size. Satellite subtraction and normalization were performed with software obtained from the manufacturer, and data analyses were performed using ESCA Tools software. Procedures. Film Deposition. Gold electrodes, cleaned with acetone, methanol, and water, were coated with PMPMS (5% w/v, dissolved in methyl ethyl ketone) by following the procedure described earlier.14 The siloxane-coated electrode surfaces exhibited higher hydrophobicity than untreated gold electrodes, as evidenced by the measurement of advancing contact angles with water (95° compared to 60°, respectively). The surfaces were characterized by X-ray photoelectron spectroscopy (XPS). Figure 1 shows XPS survey spectra for an unmodified gold electrode surface (Figure 1a), and for one coated with polysiloxane (Figure 1b). The latter confirms that complete coverage of the gold electrode surface with the polymer was achieved. Elution of Adsorbed Proteins. The system was flushed with PBS for 10 min, and single-protein solutions at concentrations of 1 or 0.1 mg/mL were introduced for monitoring adsorption for 3 or 120 min. After the system was flushed for 2 min (shorter protein residence time) or 10 min (longer protein residence time) with the buffer, 1% (w/v) SDS solution in PBS was introduced to interact with the protein layer for 45 min. Finally, the system was again flushed with buffer. Sequential Protein Adsorptions. The system was flushed with PBS continuously for 10 min before the introduction of 0.5 mL of each of the three proteins at concentrations of 1 mg/mL, in different sequences. The 1 h adsorption of each protein was followed by at least 10 min of rinsing with buffer. Simultaneous Protein Adsorptions. After 10 min of initial buffer flow, a 0.5 mL portion of a mixture of two or all three proteins present at concentrations of 1 mg/mL each was introduced to the system and adsorption was monitored for 1 h. Finally, the system was flushed with PBS for another 20 min. The same experiments were performed with three proteins present in the mixture at concentrations corresponding to their physiological ratio: HSA:IgG:fibrinogen ) 16:5:1 (4 mg/mL:1.25 mg/mL:250 µg/mL). The adsorptions for single-protein solutions of the above concentrations (10 times diluted compared to their physiological concentrations in plasma) were monitored for 1 h as well. All the experiments involving the described procedures (elution; sequential and simultaneous adsorptions) were performed at room temperature and repeated at least three times with different individual sensors. Acoustic Network Analysis. The network analyzer, calibrated for a frequency of 9 MHz, was used to calculate the values for the elements of equivalent circuits internally. The characteristic parameters were collected continuously over the scanned frequency range. A baseline drift for the series resonance frequency signal ranged from 0 to (10 Hz. RESULTS AND DISCUSSION Elutabilities of Adsorbed Proteins. Experiments involving elution (removal of surface-adsorbed protein molecules induced by surfactant solutions) result in valuable information with regard to the interactions between such macromolecules and the surface of a material, principally the binding strengths of particular

Figure 1. XP survey spectra of (a) an unmodified TSM gold electrode and (b) a TSM electrode coated with PMPMS.

molecules. Consequently, the study of elution processes is essential for obtaining a better understanding of the competitive behaviors of adsorbed proteins. It is generally assumed that a surface population of such species is heterogeneous in terms of the different modes of contact between the macromolecules and the substrate surface. This produces variations in the orientation or conformation of the adsorbed molecules. The various factors that lead to the existence of multiple states of adsorbed protein have been described.15 Different states of orientation and immediate and slow structural changes of adsorbed individual molecules lead to differences in the strengths of their binding to a substrate, resulting in variable degrees of elutability. In our experiments, we have examined the elution of fibrinogen and albumin from PMPMS-coated and unmodified gold surfaces by SDS, where the residence times of the adsorbed proteins on the surface and the concentrations of protein solutions were varied. Figure 2a illustrates the sensor signals (fs and Rm) obtained for exposure of a polysiloxane-coated TSM to both SDS (1% in PBS) and buffer in the absence of an adsorbed protein layer. A frequency change (∆fs) of less than 30 Hz is observed when the surfactant is introduced, which is accompanied by insignificant energy dissipation, as reflected in the change of motional (15) Horbett, T. A.; Brash, J. L. In Proteins at Interfaces. Physicochemical and Biochemical Studies; Brash, J. L., Horbett, T. A., Eds.; American Chemical Society: Washington, DC, 1987; pp 1-33.

resistance (Rm), which is less than 1 Ω. Subsequent exposure of the system to buffer returns the signals close to the baseline. These signals can be attributed mainly to changes in bulk liquid properties, such as viscosity, density, and conductivity, which were caused by the introduction of the surfactant. In the presence of an adsorbed protein layer, an analogous experiment yields contrasting behavior (Figure 2b). Introduction of a 0.1 mg/mL solution of fibrinogen results in an initial frequency drop of approximately 300 Hz, while subsequent flushing with buffer after 2 h of adsorption initiates a slight frequency increase, indicating that partial removal of reversibly adsorbed protein has occurred. The exposure of the fibrinogen layer that is adsorbed on the polysiloxane surface to SDS solution results in a marked frequency drop (almost 200 Hz) followed by a frequency increase associated with the wash-off of eluted protein. The initial increase in motional resistance of 5 Ω associated with the introduction of a viscoelastic protein layer, is followed by further, substantially more pronounced energy dissipation (the change of motional resistance is approximately 30 Ω), which indicates significant energy losses associated with the presence of surfactant molecules at the surface. A final flushing with buffer produces a decrease in the signal, pointing to a decrease of energy losses connected with elution of the protein layer. In further studies, we examined the effect of the time of the adsorption on the elutability of protein layers. Figure 3 shows that, Analytical Chemistry, Vol. 72, No. 7, April 1, 2000

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Figure 2. Changes in series resonance frequency (fs), and motional resistance (Rm) for TSMs coated with PMPMS on exposure to SDS (1% in PBS) and PBS (pH 7.4) (a) without the presence of a preadsorbed protein layer and (b) after the adsorption of fibrinogen (Fg) from a 0.1 mg/mL solution for 120 min. The arrows indicate where different solutions were introduced into the system. The same procedure was used in all experiments involving elution.

Figure 3. Changes in series resonance frequency following the adsorption of fibrinogen from a 1 mg/mL solution on TSMs coated with PMPMS for (a) 3 min and (b) 120 min and subsequent exposure to SDS (1% in PBS) and PBS (pH 7.4).

in accord with what has been stated in other studies involving different systems,16,17 the elution of a preadsorbed fibrinogen layer

decreases with the increase of protein surface residence time (86% after 3 min (Figure 3a), compared to 57% after 2 h (Figure 3 b)).

(16) Slack, S. M.; Horbett, T. A. J. Colloid Interface Sci. 1989, 133, 148-165.

(17) Rapoza, R. J.; Horbett, T. A. J. Colloid Interface Sci. 1990, 136, 480-493.

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These findings suggest an increase in the strength of attachment between adsorbed protein and the polymer, reflecting the attainment of more tightly bound conformational states of the protein following prolonged contact and unfolding of fibrinogen on hydrophobic PMPMS. Despite the fact that the results of a study involving elution of radiolabeled fibrinogen and albumin from polyethylene and polystyrene, with various surfactants including SDS at concentrations similar to those applied here, indicated that proteins adsorbed from more concentrated solutions are eluted more efficiently,17 our data do not support such findings. Comparison of the elutabilities of fibrinogen adsorbed from a 0.1 mg/mL solution (Figure 2b) and from a 1 mg/mL solution (Figure 3b) reveals no significant difference for this range of concentrations. In both situations, approximately 60% of the preadsorbed fibrinogen is eluted 2 h after adsorption. The nature of the surfactant-protein interaction that is involved in the elution process is undoubtedly complex but likely involves the formation of a surfactant-protein complex with a micellar structure, which is eluted from the surface.16 Frequency response curves shown in Figures 2b, 3a, and 3b generally exhibit a rapid, immediate decrease upon the introduction of the surfactant, followed by a slower frequency increase associated with the formation of micellar complexes. Significant increases occur for Rm (Figure 2b) after the introduction of the surfactant. This indicates the generation of a structurally new, viscoelastic complex layer and/or a change in acoustic coupling at the interface. The energy dissipation of the oscillating system into the liquid medium, influenced by the viscosity of the surrounding liquid environment, is also determined by shear coupling that depends on the slip conditions of the surface.12 The increased energy losses related to the presence of the surfactant indicate a decrease in the hydrophobicity of the adsorbed layer due to the formation of a micellar structure at the interface. An analogous study of the elutability of adsorbed human serum albumin failed to produce a definitive conclusion. As shown in Figure 4a, a full recovery of the frequency signal, indicating the apparent total reversibility of the adsorption of HSA on polysiloxane, is often found. However, in many experiments, full reversibility with respect to HSA adsorption is not observed (Table 1). Indeed, considerable irreproducibility was found with respect to the elution of preadsorbed albumin, implying a less uniform adsorption pattern for this protein. It has been suggested that the adsorption of fibrinogen is completely reversible, for elution in a similar surfactant system, even when it is adsorbed on the hydrophobic surface of acousticplate-mode devices modified with methyl-terminated (1-dodecanethiol) self-assembled monolayers.18 Our results show that the elution of fibrinogen from PMPMS surfaces ranges from approximately 80% to 85% following a short residence time and from approximately 60% to 65% after a longer residence time (Table 2). However, in the case of untreated gold surfaces, the elution of fibrinogen ranges from approximately 60% to 70% after a short residence time and from approximately 30% to 40% following a long residence time (Table 2). Evidently, gold surfaces tend to retain more adsorbed protein than those composed of polysiloxane (18) Seigel, R. R.; Harder, P.; Dahint, R.; Grunze, M.; Josse, F.; Mrksich, M.; Whitesides, G. M. Anal. Chem. 1997, 69, 3321-3328.

Figure 4. Changes in series resonance frequency after the adsorption for 2 h of (a) HSA (1 mg/mL) on PMPMS and (b) fibrinogen (1 mg/mL) on gold and subsequent exposure to SDS (1% in PBS) and PBS (pH 7.4). Table 1. Experimental Elutabilities of HSA Adsorbed on TSMs Coated with PMPMS concn of HSA in the soln (mg/mL)

adsorption time (min)

recoveries of the signals obtained in different expts (%)

0.1 0.1 1 1

3 120 3 120

70, 79, 100, 100 40, 54, 66, 100 70, 85, 100, 100 44, 53, 60, 100

polymer. Figure 4b illustrates the elution of 31% of fibrinogen adsorbed from a 1 mg/mL solution on an unmodified gold surface after 2 h of adsorption. It was shown previously that the high surface activity of fibrinogen favors its adsorption on hydrophobic surfaces,14 where entropically favorable hydrophobic interactions with smaller contributions from van der Waals attractions play a predominant role in the adsorption mechanism. However, the presence of disulfide bridges between cysteine residues in the fibrinogen molecule is responsible for the interaction with gold Analytical Chemistry, Vol. 72, No. 7, April 1, 2000

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Figure 5. Changes in series resonance frequency during the consecutive adsorptions of proteins (HSA, fibrinogen, and IgG) from 1 mg/mL solutions on TSMs coated with PMPMS.

Table 2. Experimental Elutabilities of Fibrinogen Adsorbed on Unmodified Gold TSM Electrodes and TSMs Coated with PMPMS

concn of fibrinogen in the soln (mg/mL)

adsorption time (min)

0.1 0.1 1 1

3 120 3 120

recoveries of the signals obtained in different expts (%) gold PMPMS 62, 64, 70, 71 29, 30, 36, 40 60, 61, 70, 72 31, 32, 35, 41

78, 83, 85, 86 57, 57, 64, 66 79, 83, 86, 87 57, 58, 60, 65

surfaces through the formation of surface thiolates,14,19 which are less susceptible to dissociation by the mechanisms connected with 1528


the process of elution. Stronger retention of other proteins (βcasein and β-lactoglobulin) on the gold electrode surfaces of TSMs, compared to hydrophobic gold, following elution has also been reported.20 Sequential Adsorptions of Proteins. Results of experiments involving the consecutive adsorptions of albumin, fibrinogen, and IgG on PMPMS-coated gold electrodes of TSMs are shown in Figure 5. The initial decrease in series resonance frequency was instigated by the introduction of the first protein: HSA (Figure (19) Nuzzo, R. G.; Zegarski, B. R.; Dubois, L. H. J. Am. Chem. Soc. 1987, 109, 733-740. (20) Murray, B. S.; Cros, L. Colloids Surf., B 1998, 10, 227-241.

5a,b), fibrinogen (Figure 5c,d), and IgG (Figure 5e,f). After 1 h, the system was exposed to buffer to remove any small amount of reversibly adsorbed protein layer from the surface. This resulted in a slight increase in the frequency signal. The remaining two proteins were introduced into the system, in different sequences, and submitted to the same treatment. An initial examination of the results suggests that, under the conditions employed, none of the proteins are displaced or removed at any level of significance by subsequent treatment with other proteins in terms of competition for surface sites. In the case of the initial adsorption of fibrinogen, as can be seen in Figure 5c,d, the subsequent adsorptions of other proteins are almost undetectable. As mentioned above, this correlates with the strength of binding of fibrinogen to a hydrophobic surface. Additional factors that probably hinder subsequent adsorptions of other proteins are likely the fact that fibrinogen exhibits a tendency for strong lateral interaction, leading to the formation of closely packed films and, finally, multilayers.21,22 In contrast, the adsorption of fibrinogen (Figure 5a,b,e,f) and, to a lesser extent, that of IgG (Figure 5a), can be detected following the preadsorptions of other proteins. Subsequent adsorption of fibrinogen is detected after initial adsorptions of HSA (Figure 5a,b) and IgG (Figure 5e,f), while subsequent adsorption of IgG is detected only after preadsorption of HSA (Figure 5a). In no case does HSA adsorb following attachment of other proteins. With regard to subsequent adsorption, the attachment of additional protein to a surface may be associated with available surface and/ or displacement phenomena. One study, involving FT-IR/ATR spectroscopy of sequential adsorptions of albumin and fibrinogen on various substrates indicated that nonhomogeneous adsorption of the former protein, resulting in island formation, yields an available surface for further adsorption processes.23 On the other hand, other experiments have suggested that there is an order of effectiveness with respect to displacement among three major blood proteins, with fibrinogen being the highest on this scale.24 Our results show that fibrinogen is just as effective in the apparent subsequent adsorption following attachment of IgG. Furthermore, rinsing with buffer after the introduction of a second dispersion of fibrinogen initiates a significant increase in fs. This effect is pronounced for the experiment involving addition of fibrinogen to preadsorbed albumin (Figure 5b). This result stands in sharp contrast to the case where rinsing with buffer following initial adsorption of fibrinogen to the device surface cannot produce such an effect (Figure 5c,d). If this reversal of the signal implies loss of material from the surface, then the only explanations for this result lie in loss of weakly bound material from the protein adlayer or from the surface itself. One possibility in terms of the latter is that the second introduction of fibrinogen yields adsorption at lower energy sites and mainly in an “end-on” position, which leads to weak binding to the substrate, but this seems unlikely. A more plausible scenario is the removal of weakly bound fibrinogen from multilayer configurations and/or fibrinogen-albumin complexes. (21) Feng, L.; Andrade, D. In Proteins at Interfaces II; Horbett, T. A., Brash, J. L., Eds.; American Chemical Society: Washington, DC, 1995; pp 66-79. (22) Sevastianov, V. I. In High Performance Biomaterials; Szycher, M., Ed.; Technomic: Lancaster, PA, 1991; pp 313-341. (23) Pitt, W. G.; Park, K.; Cooper, S. L. J. Colloid Interface Sci. 1986, 111, 343362. (24) Bale, M. D.; Danielson, S. J.; Daiss, J. L.; Goppert, K. E.; Sutton, R. C. J. Colloid Interface Sci. 1989, 132, 176-187.

The latter could involve protein-protein interactions which induce restructuring of protein in the preadsorbed layer. This perturbation will lead to less tightly bound states of the albumin molecule and, thus, ease of removal on rinsing with buffer. Finally, one additional factor cannot be discounted, and that is that signal reversal may be, at least in part, connected with perturbation of the acoustic signal by changes in adsorbed layers in terms of their interfacial viscosity or viscoelastic properties. Simultaneous Adsorptions of Proteins. Competitive adsorptions from binary and ternary mixtures of albumin, IgG, and fibrinogen on TSMs coated with polysiloxane are shown in Figure 6. All response curves show frequency decreases that follow the introduction of protein solutions. Following 2 h of adsorption, flushing the system with buffer yields increases in signals, indicating the removal of reversibly adsorbed protein. Figure 6a depicts a comparison of responses for adsorptions from singleprotein solutions and from a ternary mixture where the proteins are present at equal concentrations (1 mg/mL), while Figure 6b presents the sensor responses to the adsorptions of proteins from binary mixtures at the same concentrations. Figure 6c shows the signals for sensor exposure to adsorptions from single-protein solutions and a ternary mixture with concentrations comparable to their concentrations in plasma. Our results confirm some of the previously observed trends with respect to the competitive behaviors of proteins interacting with hydrophobic surfaces. The smaller size of the albumin molecule favors its faster diffusion to surfaces (D20,w ) 6.1 × 10-7 cm2/s).25 This results in a faster rate of adsorption of this molecule compared to the cases for fibrinogen (D20,w ) 1.97 × 10-7 cm2/s)25 and IgG (D20,w ) 4.0 × 10-7 cm2/ s).25 Therefore, we note the strong influence of the presence of albumin in all situations, particularly when it is present at concentrations corresponding to plasma conditions (Figure 6c). However, the high surface activity of fibrinogen is clearly evident in situations where proteins are adsorbed from binary mixtures at equal concentrations (Figure 6b). When fibrinogen is adsorbed from a ternary mixture at equal concentrations (Figure 6a) and from a binary mixture with HSA (Figure 6b), the signals show significant increases during both adsorption and washing with buffer. These results concur, to some degree, with work indicating that the amount of total adsorbed protein from an albumin/ fibrinogen binary mixture (at the physiological ratio of concentrations) decreased after approximately 1000 s, suggesting that HSA is effectively reducing the adsorptions of both fibrinogen and IgG.26 The appearance of the curve for the fibrinogen/IgG binary mixture (Figure 6b) does not suggest a similar influence of the presence of IgG on the adsorption of fibrinogen. When fibrinogen is present at a comparatively lower concentration, as in the ternary mixture at concentrations simulating plasma conditions, such behavior is not observed (Figure 6c). The dominant adsorption of albumin from such a mixture is expected in view of its concentration coupled with the high diffusivity value for this protein. A study using TIRF and ellipsometry to examine simultaneous adsorptions to a hydrophobic surface of the three plasma proteins at concentrations corresponding to their physiological ratio showed that IgG and albumin dominate adsorptions from the ternary protein mixture, while fibrinogen is present in the adsorbed layer only to (25) Schultze, H. E.; Heremans, J. F. Molecular Biology of Human Proteins; Elsevier: Amsterdam, 1966; Vol. 1, pp 176-181. (26) Lassen, B.; Malmsten, M. J. Colloid Interface Sci. 1996, 180, 339-349.


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Figure 6. Changes in series resonance frequency (a) during single-protein adsorptions of HSA, IgG, and fibrinogen from 1 mg/mL solutions and from a solution where three proteins (Fg/HSA/IgG) were present simultaneously in concentrations of 1 mg/mL, (b) during adsorptions from solutions where HSA/IgG, Fg/HSA, and Fg/IgG were present simultaneously in concentrations of 1 mg/mL, and (c) during single-protein adsorptions of HSA, IgG, and fibrinogen from the solutions where they were present in concentrations of 4 mg/mL (HSA), 1.25 mg/mL (IgG), and 250 µg/mL (Fg) (10 times diluted compared to concentrations in plasma) and from the solution where three proteins (Fg/HSA/IgG) were present simultaneously at the ratio of concentrations corresponding to physiological values: HSA:IgG:Fg ) 16:5:1. The adsorptions were monitored on surfaces of TSMs coated with PMPMS.

a smaller extent, suggesting that minor displacement between the adsorbed proteins takes place.27 Ellipsometric studies of competitive adsorptions of fibrinogen and albumin on surfaces displaying different degrees of hydrophobicity lead to the conclusion that albumin is involved in the removal of fibrinogen.28 However, an earlier study concerning the competitive behaviors of radiolabeled fibrinogen and albumin on various surfaces stressed that proteins do not adsorb from plasma in proportion to their concentrations, indicating fibrinogen enrichment at the surface with respect to albumin.29 Finally, it has been suggested that plasma proteins displace each other in the sequence albumin, IgG, fibrinogen, high molecular weight kininogen, and factor XII, which actually represents the order from most to least concentrated protein species in plasma.30 The displacement of a preadsorbed protein by other plasma proteins, because of their ability to adsorb more favorably at a surface, has become known generally as the Vroman effect. In (27) Lassen, B.; Malmsten, M. J. Colloid Interface Sci. 1996, 179, 470-477. (28) Warkentin, P. H.; Lundstro ¨m, I.; Tengvall, P. In Proteins at Interfaces II; Horbett, T. A., Brash, J. L., Eds.; American Chemical Society: Washington, DC, 1995; pp 163-180. (29) Brash, J. L.; Uniyal, S. J. Polym. Sci. 1979, C6, 377-389. (30) Vroman, L.; Adams, A. L. In Proteins at Interfaces. Physicochemical and Biochemical Studies; Brash, J. L., Horbett, T. A., Eds.; American Chemical Society: Washington, DC, 1987; pp 154-164.

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the case of hydrophobic surfaces, such as PMPMS, we can assume that attractive hydrophobic interactions will favor the restructuring of protein molecules to more extended states, inducing their molecular relaxation (spreading) and denaturation. Although globular albumin molecules may achieve a strong configurational entropy gain by spreading on a hydrophobic surface, it is more probable that proteins of larger size yielding more contact points with surfaces, particularly conformationally labile fibrinogen, can spread to a larger degree and adapt with facility to the interface.15,30 However, there are strong indications that the adsorption of a second protein can also take place without displacement of the preadsorbed species.5,31 It is possible that proteins form a proteinprotein complex (BSA and IgG on polystyrene)31 and form multilayers (fibrinogen in competition with albumin on silicone rubber).5 Experimental studies suggest that albumin mainly adsorbs in a “side-on” configuration, while IgG adsorbs mainly in an “end-on” position.26,32 The orientation of adsorbed fibrinogen molecules is described as random, depending on the overall adsorption conditions, with a pronounced reorientation to the “endon” position at higher surface coverages.26 Such assumptions, together with the supposition that albumin may adsorb in patches, (31) Elgersma, A. V.; Zsom, R. L. J.; Lyklema, J.; Norde, W. J. Colloid Interface Sci. 1992, 152, 410-428. (32) Soderquist, M. E.; Walton, A. G. J. Colloid Interface Sci. 1980, 75, 386-397.

lead to an explanation of our signals. The overall appearance of the response curves in this work implies that the adsorptions of both IgG and fibrinogen take place at surface sites which are not occupied by albumin and, therefore, do not displace it. Additionally, fibrinogen, because of its high surface activity, displays a clear tendency to form less tightly bound multilayers (Figure 6a,b). In situations where it adsorbs simultaneously with albumin, the shapes of the curves, Figure 6a (Fg/HSA/IgG) and Figure 6b (Fg/HSA), are distinctively different. The observed increases in the series frequency responses indicate possible alteration in surface free energy induced by the formation of multilayers. Pronounced wash-off of the adsorbed protein layer in the abovementioned situation suggests that such multilayer structures are partially reversible. CONCLUSIONS The findings described in this paper, though qualitative in character, offer a clear picture of the competitive behaviors of three major plasma proteins in terms of their adsorptions on hydrophobic polysiloxane surfaces. Obviously, it is not possible to distinguish particular protein species within an adsorbed layer. However, monitoring of the series resonance frequency signals of TSMs does provide a reliable, on-line method for the monitoring

of protein adsorption processes in situ, which avoids the use of labeled proteins. Previous postulates regarding the occurrence of protein displacement phenomena that are generally applied to explain the competitive behavior of proteins tend to present a much simpler picture involving only consideration of kinetic and surface affinity factors. Evidence for protein adsorption without displacement of adsorbed proteins is provided. Also, the sensor system described here offers further possibilities for investigating conformational restructuring within the adsorbed protein layer and consequent alteration in surface free energy through a detailed examination of impedance and equivalent-circuit analyses. ACKNOWLEDGMENT We are greatly indebted to Dow Corning, Midland, MI, and the Natural Sciences and Engineering Research Council of Canada for support of this work. We also thank Dr. Rana Sodhi of the Surface Science Unit, University of Toronto, for assistance with the acquisition of XPS data.

Received for review August 10, 1999. Accepted January 4, 2000. AC9909094


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Adsorptions of Plasma Proteins and Their Elutabilities from a

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