11822
J. Phys. Chem. C 2008, 112, 11822–11830
Studies of Adsorption and Viscoelastic Properties of Proteins onto Liquid Crystal Phthalocyanine Surface Using Quartz Crystal Microbalance with Dissipation Technique Sharmistha Paul,*,† Deepen Paul,‡ Tamara Basova,§ and Asim K. Ray† School of Engineering & Materials Science and Interdisciplinary Research Centre in Biomedical Materials, Queen Mary, UniVersity of London, Mile End Road, London E1 4NS, United Kingdom and NikolaeV Institute of Inorganic Chemistry, LaVrentieV pr., 3, NoVosibirsk 630090, Russia ReceiVed: February 1, 2008; ReVised Manuscript ReceiVed: April 30, 2008
This paper presents time-resolved adsorption behavior of lysozyme, bovine serum albumin (BSA), and immunogamma globulin (IgG) onto a liquid crystal phthalocyanine surface and concentrates on the kinetic, viscoelastic variation, interfacial hydration, and structural details obtained by quartz crystal microbalance dissipating monitoring (QCM-D) technique with the Voigt model. The rate of adsorption for lysozyme is faster than that of BSA and IgG. The Freundlich model can explain the adsorption isotherm of lysozyme, whereas an exponential growth model can describe that of BSA and IgG. Layer surface coverage has been found to increase for all three proteins with significant variation in surface packing density and viscoelastic parameters within the investigated concentration range. The adsorbed IgG and BSA form soft, water-rich multilayers with large energy dissipation. The layer viscosity and shear modulus have been found to decrease as the protein hydration increases with concentration in these cases. On the other hand, lysozyme forms a rigid, negligibly hydrated multilayer with higher values of viscosity, shear modulus. Among three proteins, IgG is found to be a good adsorbent for liquid crystal surface comparing their theoretical monolayer surface coverage. 1. Introduction Quartz crystal microbalance with dissipating monitoring (QCM-D) is a well-established technique for monitoring in situ adsorption and conformational changes of biomolecules.1–9 Biosensors based on this method are portable, simple, costeffective, label-free, and suitable for the real-time monitoring of biospecific interactions such as those between antigens and antibodies with high sensitivity and selectivity in flow-systems. Using the piezoelectric effect, QCM-D can simultaneously determine change in frequency and energy dissipation of a quartz crystal at nanoscale in real time. Adsorption of proteins on different scaffold materials—namely, metal, metal oxides, metal nano particles, quartz, self-assembled monolayer, and polymers—by the QCM method is reported in literatures.3–9 These processes include either chemical surface modification or physical alternations in the composition of the surface to improve the protein adhesion on synthetic materials while preserving protein activity and maximizing sensitivity, selectivity, reproducibility or even recyclibility for a specific application. The adsorption kinetics of different proteins by this method is often compared by several authors in literature with other techniques based on optics such as ellipsometry, surface plasmon resonance (SPR), and atomic force microscopy (AFM) both qualitatively and quantitatively. These optical techniques provide results that could be simply converted to adsorbed protein dry mass. In analogy, QCM-D provides valuable informations about the adsorbed mass of protein with hydro* Corresponding author. Tel. +44(0) 20 7882 5547, Fax. +44(0) 20 8981 9804, E-mail:
[email protected]. † School of Engineering & Materials Science, Queen Mary, University of London. ‡ Interdisciplinary Research Centre in Biomedical Materials, Queen Mary, University of London. § Nikolaev Institute of Inorganic Chemistry.
dynamically coupled water and the mechanical (viscoelastic)/ structural information of the adsorbed layer after analyzing the experimentally obtained data of energy dissipation in relation with the frequency shift. Metallophthalocyanines [Pc] with liquid crystalline property are widely used for several purposes such as nonlinear optical application, light absorption, electric conduction, photoconduction, energy conversion, photodynamic therapy, chemical sensor, and catalysis.10–17 Since the discovery of mesogenity, a variety of Pcs with supramolecular organization have been prepared.12,13,15 The Pcs offer three important advantages; high thermal and chemical stability, a rich substitution chemistry leading to a vast amount of compounds with modulated properties, and a very good processibility by several deposition methods. The structures and liquid crystalline property of copper octakishexylthiophthalocyanine [CuPcR8] have been studied in an earlier publication.18 Spin cast films of CuPcR8 are known to form a columnar hexagonal mesophase at room temperature. Adsorption behavior of bovine serum albumin (BSA) protein on CuPcR8 surface was characterized before by AFM, SPR, and UV-vis absorption techniques. Here, the effect of heat treatment of CuPcR8 film upon BSA adsorption has been discussed.19 In the present article we discuss in depth the surface-protein interaction considering different proteins based on molecular weight (MW), net charge, and conformational stability onto liquid crystal phthalocyanine surface with thickness in the order of several nanometer. Here, in situ adsorption kinetics of three proteins onto a CuPcR8 film surface with various concentrations is being presented for the first time by the QCM-D method. The obtained data was applied in the Voigt model to get viscoelastic information of the adsorbed layer.20,21 Using modeling, physical properties (mass, thickness, and density) and mechanical properties (viscosity and shear modulus) of the adsorbed layer have been discussed
10.1021/jp800975t CCC: $40.75 2008 American Chemical Society Published on Web 07/15/2008
Protein Adsorption onto CuPcR8 Surface
J. Phys. Chem. C, Vol. 112, No. 31, 2008 11823
Figure 1. (a) Molecular structure of octakishexylthio-substituted Cu(II) phthalocyanine derivative [CuPcR8], (b) FESEM image of spin cast CuPcR8 film.
considering the role of hydrodynamically coupled water as well as structural changes upon binding on the phthalocyanine surface. 2. Experimental Section 2.1. Materials. The molecular structure of CuPcR8 (where R) n-hexyl, C6H13) was given in Figure 1a. Synthesis and characterization of CuPcR8 was described in a previous publication.18,19 BSA, lysozyme, and IgG were purchased from Sigma Chemical Co. and were used without further purification. H2SO4, H2O2 (30% w/v), and CHCl3 were purchased from Aldrich and were used as received. Disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), and sodium chloride (NaCl) were purchased from BDH, UK. 2.2. Preparation of Phosphate Buffer Saline (PBS) Solution. Phosphate buffered saline (PBS, 0.5 mM NaCl, 0.02 mM NaH2PO4, and 0.08 mM Na2HPO4) aqueous solution was prepared at pH 7.4. The buffer solution was stored at room temperature and was used throughout the experiment. 2.3. Film Preparation. AT-cut quartz crystal coated with gold (fundamental frequency of 4.95 MHz) was obtained from Q-Sense AB (Sweden). The gold-coated quartz crystal was first cleaned by immersing in 20 mL of Pirnha solution for 2 min (3:1 mixture of conc. H2SO4 and 30% H2O2) and washed in plenty of Milli-Q water and dried in nitrogen gas (N2). A spin coating unit G3P-8 (Specialty Coating Systems, UK) used at 2000 rpm for 5 min allowed the formation of thin uniform films of CuPcR8 on cleaned substrates from a 0.2 mL spreading solution in chloroform of concentration 2 mg/mL. 2.4. Proteins Adsorption. The interactions between the CuPcR8-adsorbed quartz crystal surface and different proteins were studied using a commercial QCM-D apparatus (Q-Sense AB) with a Q-Sense D300 electronic unit. A polypropylene pipet tip connecting to the temperature-controlled chamber was initially filled with PBS buffer of pH 7.4. By opening the valve, buffer solution was filled in the QCM-D chamber via gravitational flow. After a stable baseline was established, different proteins in the same buffer were separately exposed to CuPcR8modified crystal surfaces. After obtaining an equilibrium signal in each concentration, the next higher concentration of protein solutions was applied without washing with buffer. The concentrations of lysozyme, BSA, and IgG proteins were varied form 0.015 mM to 0.3 mM, 0.015 mM to 1 mM, and 0.01 mM to 0.1 mM, respectively. Finally, the cell was washed in PBS buffer solution to remove loosely attached proteins. The adsorption was monitored, as a function of time, by simultaneously recording the shifts in the frequency (∆F) and in the
energy dissipation (∆D) at the fundamental resonant frequency by periodically switching off the driving circuit and recording the decay of the damped oscillation along with the third, fifth, and seventh overtones, until a steady state of adsorption was reached. The stability of the frequency and energy dissipation were 1 Hz and 0.1 × 10-6 respectively, which were negligible when compared with the shifts due to adsorption. Q-Sense software was used to determine the resonance frequency (f0) and the decay time (τ0) of the exponentially damped sinusoidal voltage signal over the crystal. Energy dissipation has become a popular measure that indicates energy loss from the viscous layer against total vibration energy of the QCM plate. The dissipation factor (D), the inverse of the quality factor, can be obtained from eq 1,
D)
1 2 ) πf0τ0 ωτ0
(1)
where ω is the width-at-half-height of the resonance peak. D is related to the softness (viscoelasticity) of the film. A soft film will not fully couple to the crystal oscillation, which usually dampens the oscillation of crystals. For a rigid, thin, uniform film where change in dissipation factor ∆D is less than 1 × 10-6 for 10 Hz frequency change, adsorbed mass was calculated using the Sauerbrey equation (eq 2),22
M ) -(C ⁄ n)∆F
(2)
where ∆F, M, and n represent frequency change, adsorbed mass per unit area, and overtone number, respectively. C is the mass sensitivity constant (17.7 ng/cm2 Hz-1). For a viscous, soft adsorbing layer where the change in the dissipation factor (∆D) is greater than 1 × 10-6, the Sauerbrey equation underestimates the adsorbed mass. For this case, normalized data obtained from different overtones were used in the Voigt model for getting four unknown parameters such as effective density, viscosity, shear elastic modulus, and thickness of adsorbed layers. 2.5. Modeling of the QCM-D Response. The Voigt element consists of a parallel combination of a spring and dashpot to represent the elastic (storage) and inelastic (damping) behavior of a material, respectively. In the Voigt model, it was assumed that the oscillatory quartz plate is covered by a viscoelastic film of uniform thickness and density that is in contact with a semiinfinite Newtonian liquid under no-slip conditions. Details of the modeling can be found in literature.21 To obtain unknown parameters of the adsorbed layer, the temporal change in frequency and the change in dissipation were fitted by Qtools software (QSense). For modeling, we vary different parameters, namely, (i) the density of the layer from 1000 to 1400 kg/m3 (which are bulk densities of water and protein), (ii) the layer
11824 J. Phys. Chem. C, Vol. 112, No. 31, 2008 viscosity between 0.001 and 0.1 kg/ms, (iii) the layer shear between 104 and 1010 Pa, and (iv) the layer thickness between 10-10 and 10-6 m. Finally, the best fit between the Voigt model and experimental data for all overtones was obtained considering minimum value of the error function (χ2). This gives actual density, thickness, viscosity, and shear modulus of adsorbed protein layer. 3. Results 3.1. CuPcR8 Surface Characterization. The average thickness of spin cast film of CuPcR8 was 30 nm, calculated using the Sauerbrey equation over 9 measurements from ∆F (considering density of the liquid crystal film to be approximately 1.29 g/cm2) from the third overtone on the gold surface of the QCM electrode. The film is rigid, as the ∆F value for all overtones were very close to each other. The surface roughness was approximately 3.8 nm, and the contact angle of the surface using water as probe liquid was 65° (by separate measurements). Complete coverage of the QCM plate by CuPcR8 film was verified by scanning electron microscopy (Figure 1b). In this image, it forms a nanoribbon-like structure with an average width of 100 nm and length of more than 10 µm with intercolumn spacing of more than 500 nm. 3.2. Adsorption Behavior of Proteins onto CuPcR8 Thin Film. Normalized data of the frequency shift (∆F) and the energy dissipation shift (∆D) from the third overtone associated with protein adsorption onto CuPcR8 at different concentrations are shown in Figure 2, panels a-c. Each experiment was conducted at pH 7.4 at a constant temperature of 25 °C and were repeated three times. Lysozyme from chicken egg white (MW 14,000 Dalton, d1 ) 3.5 nm, d2 ) 4.5nm and pI ) 11) is used.23,24 Figure 2a displays the time-resolved ∆F and ∆D value for lysozyme adsorption on the CuPcR8 surface. After the injection of each concentration, the immediate sharp decrease in ∆F and a little increase in ∆D have been observed. The steady state is quickly reached (within 10 min) in each concentration. These sharp changes in ∆F and ∆D indicate the quick adsorption of lysozyme on the CuPcR8 surface. The theoretical monolayer coverage is approximately 207 ng/cm2 (side on) to 310 ng/cm2 (end on), causing an approximately 15 Hz frequency change as we obtained a ∆F value for the 0.015 mM concentration.25 Lysozyme adsorption on a bare gold QCM surface was reported earlier in literature.26,27 The amount of adsorbed protein was reported to be 290 ng/cm2 following exposure to 1 mg/mL lysozyme in PBS buffer, pH 7.4, at 25 °C.27 In similar conditions, the amount of adsorbed lysozyme (460 ng/cm2 from Sauerbrey, 540 ng/cm2 from Voigt model) on a CuPcR8 surface is larger than the reported value, indicating higher adsorption affinity toward this surface. The variation of the ∆D value with concentrations is smaller than the corresponding ∆F values, which indicates the adsorbed layer is nondissipated. BSA is an ellipsoid protein (MW 69,000 Dalton, 2.7 × 2.7 × 11 nm3, and pI 4.9). It contains 143 carboxyl groups and 101 amine groups and has a net negative charge at biological pH.28 Immediately after the injection of each BSA solutions as shown in Figure 2b, there was a gradual decrease in ∆F and an increase in ∆D, followed by much slower changes of ∆F and ∆D until steady states were reached with 1 h (i.e., the frequency shift was within 1 Hz). BSA adsorption on a bare gold QCM surface was reported earlier in the literature where BSA binds through hydrophobic interaction and formed a multilayer. The reported ∆F is higher than the ∆F value obtained on the CuPcR8 surface.29,30 This means that CuPcR8 limits the amount of BSA
Paul et al. adsorption and therefore controls the spreading (denaturation) of BSA to the surface. The high ∆D value (as compared with ∆D for lysozyme) with similar concentration leads to the formation of a dissipated layer for this case. IgG protein (MW ) 1,46,000 Dalton, dimension 14 × 10 × 4 nm3, and pI ) 5.8- 7.3) is slightly negative in charge at biological pH.31,32 As described in Figure 2c at an initial concentration of 0.01 mM, there was a sharp decrease in ∆F (90 Hz) and an increase in ∆D (6 × 10-6, high compared to others) values, which indicates the IgG layer is highly dissipative. From the nature of variation of ∆F and ∆D with time at different concentrations it was observed that ∆F has reached a stable value within 40 min, whereas the ∆D value is still increasing. This indicates the slow adsorption process for big proteins on the CuPcR8 surface, which includes initial attachment to the surface as well as configuration change. In addition, due to its Y-shaped structure, IgG molecules can easily bring extra energy loss to the system by trapping more water molecules than other proteins. However, the increase in solvent density and viscosity for higher concentration could affect the energy dissipation of the adsorbed layer. The reported frequency change and energy dissipation by the QCM method due to binding of polyclonal IgG on the bare gold surface was different from our case.33–35 In our case, the initial concentration of IgG is much higher than the above references. Therefore, it is not possible to quantitatively compare the adsorbed amount. But considering the large dissipation change compared to frequency change, we could expect the interaction of IgG with the CuPcR8 surface to be different from that of pure gold. 3.3. Viscoelastic Modeling. 3.3.1. Adsorbed Mass Calculation. The fitted data of ∆F and ∆D during protein adsorptions obtained after using the Voigt model (viscoelastic solid) are also shown in Figure 2 with experimentally observed data. The fit was good (χ2 ) 1.2- 9.4 × 10-22) for all cases when we did fitting separately for each concentration. Voigt mass (Mvoigt) is obtained after multiplying modeled values of density and thickness. The thickness values have increased with concentration for all three proteins, but density behaves differently. For lysozyme, it increases slowly; for BSA, it increases up to 0.45 mM and then remained constant; and for IgG, it is constant all throughout the concentration range, near water density. Figure 3 gives the mass difference for different proteins calculated from the Sauerbrey and Voigt models. Although the calculated mass using the Sauerbrey equation is an underestimate, due to the high energy dissipation value, the equation remains useful because it gives qualitative information about mass adsorption. So Sauerbrey mass (Msauerbrey) was calculated directly from frequency change considering the density of the bulk protein. Adsorbed mass calculated from both processes was compared for initial concentration (0.01 mM for IgG and 0.015 mM for BSA and lysozyme, as these concentrations are very close to each other), which is high for IgG compared to lysozyme and BSA. BSA and lysozyme have more-or-less the same value at this concentration, but later, mass difference calculated between these two processes increases with concentration for the cases of IgG and BSA, whereas it decreases for lysozyme (Figure 3). 3.3.2. Mechanical Properties of Adsorbed Film. The obtained QCM-D response (∆F, ∆D) from different normalized overtones did not coincide with each other for our experiment, which suggests a strong contribution from viscoelastic variation during the adsorption of protein layers. Figure 4 shows viscosity and shear modulus values obtained after applying the Voigt model. Viscosity and shear modulus are increased with concentration for lysozyme, whereas the opposite behavior is seen
Protein Adsorption onto CuPcR8 Surface
J. Phys. Chem. C, Vol. 112, No. 31, 2008 11825
Figure 2. Real-time experimental and fitted data by the Voigt model of frequency (left axis) and energy dissipation (right axis) shifts for the third overtone of QCM resonator for (a) lysozyme, (b) BSA, and (c) IgG adsorptions on CuPcR8 surface. The first arrow indicates the addition of proteins in PBS buffer, and the second arrow indicates addition of the next higher concentration of protein without washing.
in the cases of BSA and IgG. Viscosity of the adsorbed layer increases from 0.001 to 0.011 kg/ms for lysozyme, whereas it decreased from 0.004 to 0.0015 kg/ms for IgG and BSA. Silimarly, shear elastic modulus increases from 105 to 2 × 106 kg/ms2 for lysozyme but decreases from 4 × 105 to 104 kg/ms2 for the case of BSA and IgG. The increased viscosity and shear value probably indicates formation of a protein multilayer expelling water outside the lysozyme. Because of the increased hydration level (high ∆D value) in the other two proteins films, viscosity as well as elasticity decreased with increase in protein bulk concentration in solution. For this case, the surface packing density of protein multilayer was fixed even though total
thickness of the adsorbed layer increased due to hydrodynamically coupled water mass, resulting in IgG and BSA layers with more dynamic and fluid-like structure on CuPcR8. We will discuss this in detail later. 3.4. Surface Coverage. Surface coverage (Γ) can be obtained by dividing the sensed mass by the molecular mass and by the active area of the QCM resonator. Figure 5 gives calculated Γ values of different proteins based on Voigt mass against concentrations. The behavior of Γ with concentration is different for all three proteins. Surface coverage of different proteins was fitted to a linearized form of the Freundlich model, expressed as eq 3,
11826 J. Phys. Chem. C, Vol. 112, No. 31, 2008
log Γ ) log k + 1 ⁄ nlog C
Paul et al.
(3)
Where C is the concentration of the protein solution, n is the exponential parameter representing the affinity of adsorption, and k is the maximum capacity of adsorption.36,37 Freundlich model coefficients together with correlation coefficients (R2) of different adsorbed proteins is shown in Table 1. The adsorption isotherm is better explained by the Freundlich model (R2 ) 0.998) than the Langmuir model (R2 ) 0.986) for lysozyme. Here lysozyme adsorption isotherm proves the formation of multilayer or aggregation of protein molecules, which has higher affinity constant (n). The adsorption isotherm for the other two proteins is best fitted by exponential growth (R2 ) 0.998 for IgG and 0.984 for BSA) than by the Freundich model. BSA and IgG formed protein multilayer or aggregation on the CuPcR8 surface, which never reached a saturation plateau within the concentration range investigated in this paper. Nonlinear surface coverage behavior of IgG in higher concentrations reported by
Figure 4. Variation of film (a) viscosity and (b) shear modulus with concentration for lysozyme (black 9), BSA (red b), and IgG (green 2) adsorbed on a CuPcR8 surface are shown.
Figure 5. Surface coverage (Γ) of lysozyme (black 9), BSA (red b), and IgG (green 2) calculated from Voigt mass onto a CuPcR8 surface have been fitted to the Freundlich model.
TABLE 1: Freundlich Model Parameters with Correlation Coefficient
Figure 3. Calculated mass based on Sauerbrey (red 9) and Voigt (black 0) model with concentration for adsorbed (a) lysozyme, (b) BSA, and (c) IgG on a CuPcR8 surface is shown.
protein
log k
1/n
R2
lysozyme BSA IgG
1.654 57( 0.008 98 1.836 75( 0.074 47 2.544 08( 0.239 76
0.269 37( 0.007 63 0.901 51( 0.090 16 0.913 62( 0.164 58
0.9984 0.9433 0.8604
Zhou et al. has supported our observation.35 The surface coverage (Γ) can be explained by surface packing density that depends on orientation of protein molecules. The surface packing densities of BSA and IgG are different. Surface packing density of BSA gradually increases up to certain concentration after which no further increase occurs, whereas for lysozyme it increases throughout the concentration range.
Protein Adsorption onto CuPcR8 Surface
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Figure 6. Variation of (a) film hydration level and (b) swelling coefficient of water inside the film with concentration of lysozyme (black 9), BSA (red b), and IgG (green 2) adsorbed on a CuPcR8 surface are shown.
3.5. Hydration Level of Adsorbed Film. Viscoelastic and hydration properties of biomolecules have generated much interest, because these properties result in the structure and function of biomolecules such as stabilities, folding, structural changes, molecular recognition, and enzymatic activities. According to the Voigt model, a homogeneous viscoelastic film is composed on the sensor surface from aqueous solution. Therefore, water and salt molecules are trapped within adsorbed biomolecules and are probed hydrodynamically. Trapped water dissipates energy due to nonlaminar motion of the liquid. So the mass of adsorbed biomolecules calculated based on this model are composed of the mass of itself together with trapped water.38–40 Here we can have a rough estimate of the hydration level of the film, calculated from the mass difference between the Sauerbrey and Voigt models shown in Figure 6a. We considered adsorbed mass Msauerbrey as dry protein mass because the ∆D value was below 1 × 10-6 at low concentration for lysozyme. The hydration level of the film has reached up to 90% for the case of BSA and IgG, whereas it decreased for lysozyme from 20 to 0% with concentration variation. The swelling coefficient of water (Q) inside the film can be calculated based on the following equation (eq 4),
Q)
Mwater Fprotein × Mprotein Fwater
(4)
where M is the mass, and F is the density. The mass of water (Mwater) can be estimated considering the mass difference between Msauerbrey and Mvoigt. Figure 6b shows rough estimated Q values of water in different adsorbed proteins layers with concentrations, considering Msauerbrey as adsorbed protein mass. Q has a higher value in the case of IgG compared to BSA, whereas lysozyme has a negligible Q value. This means IgG and BSA formed hydrogel films, whereas lysozyme formed a rigid layer that does not swell at all. 3.6. Conformation Discussion with the Help of ∆F-∆D Plot. ∆D-∆F plots are important because they provide information on the energy dissipation per unit mass added to the crystal. Large dissipation changes are commonly associated with the extent of water coupled and flexible conformation of the attached biomolecules, whereas small dissipation reflects dehydrated and compact layers.41,42 Figure 7a-c, shows ∆D-∆F plots of initial concentration 0.015 mM for lysozyme and BSA and 0.01 mM for IgG, eliminating time scale. The slope of the plots is defined as K (where K ) ∆D/∆F), which is indicative for kinetic and conformational change processes during adsorption.35,41,42 Small values of K indicate a rigid layer, whereas a high value indicates a soft and water-rich layer.
Lysozyme has only one K value, whereas BSA and IgG have two K values, as shown in Figure 7a-c. This indicates that the adsorption kinetic process is different for each case. For lysozyme, only one slope is associated with direct adhesion on the CuPcR8 surface. More than one slope indicates direct adhesion and orientation change associated with hydrodynamically coupled water. Table 2 for lysozyme, Table 3 for BSA, and Table 4 for IgG give all calculated K values during their kinetic process for all concentrations considered. Comparing the K value for three cases for initial concentration, it can be concluded that lysozyme adsorption is quick and forms a rigid layer (lower K value). BSA and IgG have a higher initial slope (K1 value) than lysozyme, indicating that formation of the layer is slow and water rich. Comparing K1 and K2 of BSA and IgG, it was found that K2 (2nd slope) for BSA and IgG was high than K1 (initial slope). This indicates aggregation and a more flexible layer. IgG and BSA adsorb on the surface, which is quick (K1 < K2). Then they change their orientation in order to get low free energy state in equilibrium. With the increasing concentration of proteins as shown in Tables 3 and 4, all K values have increased, which indicates that all adsorbed multilayer are finally soft and water rich. Figure 7d supports the above explanation considering high ∆D and ∆F values obtained at saturation for all concentrations of the three proteins. 4. Discussion The main phenomena leading to protein adsorption are hydrophobic, electrostatic, steric interactions, changes in the state of protein hydration, and rearrangement in the protein structure. It has been shown that the surface charge, the topography, and the surface chemistry can influence protein adsorption.43 In our study,thesurface-proteininteractionisweakerthanprotein-protein interaction, which largely depends on hydration level and conformational changes of proteins. Lysozyme. It is known that a lysozyme molecule contains 7-10 carboxyl groups and 17-19 amine groups and has a net positive charge at pH 7.4. Lysozyme is a small, robust protein. It was probably adsorbed by attaching the hydrophobic part of the protein to the aliphatic side chain of CuPcR8 molecules in a liquid crystal layer, with the hydrophilic part facing outward. For the initial concentration of 0.015 mM, the calculated surface coverage of 10.5 × 1012 proteins/cm2 is found to be within the theoretical monolayer surface coverage25 of (8.6-12.9) × 1012 proteins/cm2. For all higher concentrations, after completion of the monolayer by side on orientation, a higher mass is adsorbed on the initial layer, forming a dense layer that has gradually driven out water molecules. The surface packing density value
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Paul et al.
Figure 7. ∆D-∆F plots for (a) lysozyme at 0.015 mM, (b) BSA at 0.015 mM, and (c) IgG at 0.01 mM adsorbed on the CuPcR8 surface are shown. Lines on the figures are drawn only as a guide. Figure (d) shows the plot considering ∆D and ∆F values at saturation for each concentration of lysozyme (black 9), BSA (red b), and IgG (green 2) proteins.
TABLE 2: Slopes in ∆F-∆D Plot for Lysozyme in Figure 7a slope K1 (× 10-6) Hz-1
lysozyme concentration (mM) 0.015 0.075 0.15 0.3 0.45
0.021 0.05 0.053 0.11 0.095
TABLE 4: Slopes in ∆F-∆D Plot for IgG in Figure 7c IgG concentration (mM) 0.01 0.02 0.03 0.04 0.06 0.08
first slope K1 (× 10-6) Hz-1
2nd slope K2 (× 10-6) Hz-1
0.057 0.072 0.186 0.142 0.165 0.236
0.477 0.521 0.679 0.836 0.881 0.906
TABLE 3: Slopes in ∆F-∆D Plot for BSA in Figure 7b BSA concentration (mM) 0.015 0.075 0.15 0.45 0.6 0.75 0.9 1
first slope K1 (× 10-6) Hz-1
2nd slope K2 (× 10-6) Hz-1
0.045 0.025 0.043 0.049 0.024 0.057 0.060 0.553
0.076 0.097 0.065 0.790 0.673 0.819 0.712 1.714
from the Voigt model has not overestimated the theoretical surface packing density value because of the negligible viscoelastic variation (
