Properties of Adsorbed Bovine Serum Albumin and Fibrinogen on Self

Article pubs.acs.org/JPCC

Properties of Adsorbed Bovine Serum Albumin and Fibrinogen on Self-Assembled Monolayers Lalit M. Pandey,†,‡ Sudip K. Pattanayek,*,† and Didier Delabouglise‡ †

Department of Chemical Engineering, Indian Institute of Technology, Delhi, New Delhi, 110016 Laboratory of Materials Physics and Engineering (LMGP), Grenoble Institute of Technology and CNRS, 3 parvis Louis Néel, BP 257, 38016 Grenoble cedex 1, France

‡

S Supporting Information *

ABSTRACT: We have studied kinetics of adsorption and properties of adsorbed bovine serum albumin (BSA) and fibrinogen (Fb) on a hydrophobic octyl surface, a hydrophilic amine surface, and a mixture of octyl and amine selfassembled monolayer (SAM) and newly synthesized hybrid SAM by using quartz crystal microbalance (QCM). In addition, we have proposed a combined kinetic and mass transfer constrained protein adsorption model. The model is fitted to a change in resonance frequency, ΔFn/n versus time data obtained from QCM to get the kinetic rate constants, mass transfer coefficient, and spreading of adsorbed proteins. Initial rate of adsorption increases with a decrease in surface energy of the substrate. The equilibrium adsorbed amount of BSA on the hybrid surface is less than that on the mixed surface and lies in between that on octyl and amine surfaces and that of Fb is the least on hybrid surface. The analysis of variation of the dissipation factor, ΔD, with ΔFn/ n indicates that BSA is more flexible than Fb and the adsorbed layer of both proteins is softest on the hybrid surface. The relaxation times of adsorbed proteins are the slowest on the octyl surface, while those on the hybrid surface are the fastest. The analysis of secondary structures of proteins using ATR-FTIR suggests secondary structures of the proteins change during adsorption. The content of α-helix of the proteins increases due to adsorption on the amine surface, while that decreases on all other surfaces. The total content of α-helix and β-sheet strongly depends on the adsorbed mass of the proteins and is weakly dependent related to elasticity and viscosity of the adsorbed proteins, respectively. atomic force microscopy (AFM),13 and quartz crystal microbalance (QCM).14,15 Various proteins such as albumin, fibrinogen, immunoglobulin, serum proteins, and so forth have been used in the above studies. Mixed SAMs of moderate wettability,8,11,12 which corresponds to contact angles of water in the range of 60°−80°, have been found to show considerable albumin adsorption and little fibrinogen adsorption. It is reported11,16,17 that the mixed SAM modified surfaces with noncompatible hydrophilic and hydrophobic groups produce phase-separated structures due to strong attraction between like molecules and strong repulsion between dissimilar molecules. To overcome the phase separation, we propose to make SAMs with hydrophobic and hydrophilic groups on the same molecule. We call this type of SAM a hybrid SAM. We have shown that adsorption of bovine serum albumin (BSA) follows the Langmuir adsorption isotherm at a low BSA concentration on the above surfaces.18 Further works are necessary to establish its effectiveness as a surface modifier of biomaterials. In our present study, we have explored the effect of chemical structure of substrates on kinetics of adsorption, change in conformations, and properties of adsorbed proteins.

1. INTRODUCTION Protein−surface interaction is the first step when a foreign material comes in contact with biological fluid. This is ubiquitous in various biomedical applications such as medical implants, biosensors, drug delivery, and so forth.1 The protein− surface−fluid interactions lead to the change in conformation of the protein. The change in conformation of the protein may be reversible or irreversible. Due to the irreversible change in native conformation of the protein, biofouling and failure of the implants2 are observed. Reversible adsorption of protein at the surface is essential to resist the fouling. The reversible adsorption of protein is expected on a modified surface of an implanted body with polymer film, grafted polyethylene oxide, and so forth.3−6 As polymer coated surfaces do not give controllable surface structure and chemical environment, researchers have been using silica and gold substrates coated with self-assembled monolayers (SAMs)7 for the last few decades. Various mono-SAMs such as CH3, NH2, COOH, and so forth and mixed SAMs of CH3/OH, CH3/ COOH, CH3/NH2, and so forth have been tested for cell adhesion and protein adsorption.8−12 Cells adhere more effectively on equimolar CH3/NH2 mixed SAMs than on CH3/COOH and CH3/OH mixed SAMs.11 Adsorbed masses of proteins on the above SAMs have been determined by radiolabeling, Fourier transform infrared reflection absorption spectroscopy (IRAS),12 surface plasmon resonance (SPR),11,13 © 2013 American Chemical Society

Received: September 24, 2012 Revised: February 18, 2013 Published: February 27, 2013 6151

dx.doi.org/10.1021/jp309483p | J. Phys. Chem. C 2013, 117, 6151−6160

The Journal of Physical Chemistry C

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solution (0.02 mg/mL) on octyl, amine, mixed, and hybrid SAMs was studied using quartz crystal microbalance (QCMz500). The surface of silica coated quartz crystal was modified with the required modifier within the QCM chamber. The modified crystal was initially washed thoroughly with toluene and subsequently with methanol. To confirm the modification, we took out the modified crystal in many cases and dried under nitrogen atmosphere. We have measured the contact angle of water on the modified crystal. The contact angle of the modified crystal and that of the silicon wafer with same SAMs are equal. This confirms the modification. After surface functionalization, 10 mL PBS of pH 7.4 was passed through the QCM chamber with the modified crystal. The crystal was ready for the protein adsorption. Then 10 mL of protein solution was passed through chamber. The chamber was kept with the protein solution for about 30 min. Adsorption data reached to equilibrium in this duration. After about 30 min, PBS solution was passed through the chamber to remove loosely bound proteins within the adsorbed protein on the quartz crystal. We obtained changes in resonance frequency of different overtones (ΔFn) (n = 3, 5, 7, 9, and 11) and dissipation factor (ΔDn) of the quartz crystal in real time due to adsorption of the proteins. QCM theory based on assumption of uniform rigid over layer, ΔFn/n, is independent of n and directly proportional to the adsorbed mass, Δm, and ΔDn is zero.14,15,20,21 The relation between ΔFn/n and Δm is called the Sauerbrey equation, given by21

We have studied the adsorption and change in secondary structures of BSA and fibrinogen (Fb) on octyl, amine, mixture of amine and octyl, and hybrid SAMs using quartz crystal microbalance (QCM) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, respectively. We have proposed a kinetic model to fit the resonance frequency change during adsorption of proteins on the above surfaces, to determine the kinetic rate constants and total coverage on the surfaces. We have determined viscoelastic properties of the adsorbed proteins on various substrates.

2. MATERIALS AND METHODS All chemicals used are purchased from Sigma-Aldrich if not mentioned otherwise. 3-Aminopropyl- trimethoxysilane (APTMS) and octyltrimethoxysilane (OTMS) were used for the formation of mixed SAMs. Dibulyltin dilaurate (95%), ptolyl isocynate (99%), and anhydrous toluene (99.8%) were used for synthesis of hybrid SAMs. Methanol, acetone (HPLC grade), sodium chloride, potassium chloride, disodium hydrogen phosphate anhydrous, and potassium dihydrogen orthophosphate were purchased from Merck. Single-sided polished silicon wafers (p-type, orientation 100) were used as substrate for contact angle measurements. We have done the following experimental steps: (i) Modification of surface of silicon wafers/quartz crystals and characterization of the modified surfaces, (ii) studies of adsorption of BSA and Fb from solution at various adsorbing surfaces using QCM, and (iii) studies of secondary structures of the adsorbed proteins using FTIR. 2.1. Surface Modification and Characterization. Silicon wafers were cleaned with “piranha” solution. The following SAMs were formed on the cleaned silica surfaces: (a) octyl, (b) amine, (c) 1:1 mixture of amine and octyl, and (d) hybrid. The characterization of surfaces were done by using (i) FTIR for detection of functional groups, (ii) AFM for measurement of surface heterogeneity, and (iii) a goniometer for measurement of contact angle against low molecular weight liquids. The procedures of surface modification and characterization of the modified surfaces have already been discussed in refs 18 and 19. Briefly, these are described below. The cleaned wafers were dipped individually into a 1% solution of OTMS, APTMS, and 1:1 v/v mixture of APTMS and OTMS in anhydrous toluene for 24 h at room temperature under nitrogen atmosphere to make octyl, amine, and mixed SAMs, respectively. In a separate set of reactions, a surface with amine headgroups was dipped into a 1% v/v solution of p-tolyl isocynate in anhydrous toluene. The catalyst dibulyltin dilaurate (2−3 drops) was added into the reaction mixture at room temperature (30 °C) under nitrogen atmosphere. The reaction time of 10 min is sufficient for the completion of the reaction.18 However, we did the reaction for about 2 h to ensure the completion of the reaction between amine group present on the surface and p-tolyl isocynate. The amine (−NH2) and isocynate (−NCO) groups reacted to form urea (NH−CO− NH) linkage. The formed monolayer had a hydrophilic urea group and hydrophobic toluene group. After the formation of SAMs, the wafers were washed consecutively in three different solvents: toluene, mixture of toluene and methanol (v/v = 1:1), and methanol for 2 min in each step. Finally, the wafers were dried under vacuum to get octyl, amine, mixed, and hybrid surfaces. 2.2. Study of Protein Adsorption. The adsorption behavior of BSA and Fb in phosphate buffer saline (PBS)

Δm = C

−ΔFn n

(1)

where the sensitivity constant C equals to 17.7 ng.·cm−2·Hz−1 using physical parameters of the quartz crystal and Δm is Sauerbrey mass. The QCM theory based on viscoelastic layer, the ΔFn/n and ΔDn, is dependent on viscoelastic properties of the adsorbed protein. The following relations have been derived22−24 for the quartz crystal loaded with a viscoelastic layer and immersed in a Newtonian liquid. ΔFn = −

⎡ ⎛ μ ⎞2 2h1μ1ωn 2 ⎤ 1 ⎢ μ2 ⎥ + h1ρ1ωn − ⎜ 2 ⎟ 2 2πρ0 h0 ⎢⎣ h2 ⎝ h2 ⎠ η1 + (ωnμ1)2 ⎥⎦ (2)

2 ⎡ 2h1η1ωn ⎤ 1 ⎢ μ2 ⎛ μ2 ⎞ ⎥ ΔDn = +⎜ ⎟ 2 ρ0 h0ωn ⎢⎣ h2 ⎝ h2 ⎠ η1 + (ωnμ1)2 ⎥⎦

(3)

Here, the thickness, density, viscosity, and elasticity are represented by h, ρ, μ, and η respectively; suffixes 0, 1, and 2 represent the quartz crystal, viscoelastic layer, and the Newtonian liquid, respectively. The angular frequency is related to frequency by the relation ωn = 2πf n. One can observe that eqs 2 and 3 can be combined to get the relation between ΔFn and ΔDn, given by ΔFn = −

⎡ μ ⎛ μ ⎞ 1 ⎢ h1ρ1ωn + 2 ⎜⎜1 + 1 ωn⎟⎟ η2 ⎝ η1 ⎠ 2πρ0 h0 ⎢⎣

⎤ ⎛μ ⎞ − ΔDn⎜⎜ 1 ⎟⎟ρ0 h0ωn 2 ⎥ ⎥⎦ ⎝ η1 ⎠ 6152

(4)



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Relaxation time, τr, is a measure of time required for the stored energy to dissipate. This is defined by the relation ⎛μ ⎞ τr = ⎜⎜ 1 ⎟⎟ ⎝ η1 ⎠

surfaces with SAM of amine headgroup show IR peaks at 3300−3500 and 1500−1600 cm−1 corresponding to N−H stretching and bending, respectively. Also, we found a peak at around 1642 cm−1 corresponding to the carbonyl group of urea linkage for the hybrid surface.18 Surface morphology was characterized by AFM. We analyzed surface roughness and average height (Ra) from AFM images. Surfaces with different SAMs resulted in nanoscale uniform surfaces, and Ra values for octyl, amine, mixed, and hybrid SAMs were 0.83 ± 0.1 nm, 0.63 ± 0.1 nm, 1.61 ± 0.2 nm and 0.67 ± 0.1 nm, respectively (see refs 18 and 30). The surface energies of the silica modified surfaces are determined from its contact angle against deionized water, ethylene glycol, and methylene diiodide. The contact angles were measured by using a contact angle goniometer (Kruss DSA-10). The characteristics of the surfaces are almost equal to the earlier reported data18,19 within the error limit of 10%. From the contact angle data, we found the surface energy (γs) of octyl, amine, mixed, and hybrid surfaces to be 31.67, 44.94, 37.95, and 39.94 mJ/m2 respectively. Details of the data can be found in ref 18. 3.2. Adsorption Kinetics of Proteins. To understand the physical phenomenon happening during adsorption of proteins, we have investigated variation of ΔFn/n with time by using following kinetic model. We assume that a solid surface is in contact with the solution (see Figure 1). There are two thin

(5)

ΔFn/n versus time is plotted for various overtones 3, 5 ,7, 9, and 11. It is found that ΔFn/n of all the overtones lie in the range of 10% standard deviation. The deviations are due to the viscoelastic nature of the adsorbed layer. We have chosen average of normalized ΔFn/n for calculation of the Sauerbrey mass, given by relation 1. We have evaluated thickness, viscosity, and elasticity of the adsorbed layer (i.e., h1, μ1, η1) by fitting the obtained data ΔFn (n = 3, 5, and 7) and ΔDn at different times using QcmZBrowse software. In this software, we have fitted rigid film, viscoelastic film, and liquid (RF+VF +L) model using the relationship among complex shear modulus (G), viscosity, and elasticity given by G = η + jωμ. Wet mass, Δmw, is determined from the product of thickness, h1, and density, ρ1, of the adsorbed viscoelastic layer. The values of ρ1, ρ2, and μ2 are 1.20 g/cm3, 1.0089 g/cm3, and 0.890 mPa·s respectively. The value of ρ1 is taken from the refs 21, 25, and 26, and the values of ρ2 and μ2 are measured at room temperature. The density and thickness of the quartz are ρ0 = 2.65 g/cm3 and h0 = 333 μm, respectively. All QCM experiments were done at room temperature and were repeated at least twice. The obtained results were within 10% standard deviation of the presented average values. 2.3. FTIR of Proteins and Adsorbed Proteins. We have used a VERTEX V70 FTIR spectrophotometer to get the characteristics peaks of amide-I band of adsorbed Fb and BSA on the surfaces. The amide-I band is due to carbonyl stretching vibrations in wavenumber 1600−1700 cm−1 range. The FTIR spectra are analyzed to determine the various secondary structures such as α-helix, β-sheet, β-turn, random, and side chain. Peak positions were determined from the second derivative of obtained FTIR spectra using the OPUS 6.5 software. Peaks were fitted with Gaussian shape curves. The peak area changes due to change in secondary structure adsorption at different surfaces. The changes were analyzed to see the effect of surfaces on the change in secondary structure of proteins. The solution of Fb and BSA were prepared at 0.02 mg/mL concentration in PBS buffer solutions. D2O was used instead of water to prepare buffer. The proteins were allowed to adsorb on amine, octyl, mixed, and hybrid SAMs at room temperature for 60 min individually. After adsorption, the samples were washed with D2O and dried using a nitrogen gun. FTIR spectra of adsorbed proteins were recorded against silicon wafer with only corresponding SAMs.

Figure 1. Schematic representation of model of adsorption of proteins (A) layers near solid surface, (B) arrangement of proteins on the substrate, and (C) projection of proteins.

layers of liquid near the surface in between the solid surface and the bulk solution. Near the bulk solution, the layer is termed as boundary layer, whose thickness is about 20 μm. The resistance due to mass transfer lies within this layer. A very thin layer of liquid, which is denoted as surface layer, is located at the other side this boundary layer. The thickness of the surface layer is equal to the longest molecular size at the contact point. Protein molecule present in this layer jumps to the surface according to its attractions toward surface. Protein molecule may be in bound or in unbound state in this layer. A similar concept has been used for adsorption of polymer from solution on solid surfaces.31 The concentration of proteins in the unbound state at surface layer at any time is[P1(*,t)], while at bulk it is [P1]0. The unbound protein molecule adsorbs on surface through two consecutive steps. The first step is the reversible adsorption of appropriately orientated protein P1 to give (P1S)I and second step is rearrangement of the (P1S)I to (P1S)II as shown in Figure 1B and C. The surface area of substrates in the (P1S)I state depends on the attraction of surface for a protein and orientation of the protein near the surface. The approach of

3. RESULTS AND DISCUSSION 3.1. Characterization of Substrates. We detected functional groups of the modified surfaces by FTIR. We took the FTIR spectra of the modified surfaces of silicon wafer in the transmission mode on a Nicolet 6700 FTIR spectrophotometer. The processing of the spectra was done using OMNIC 7.3 software. FTIR spectra can be found in ref 18. In all the spectra of the modified surfaces, peaks appeared around 2965, 2920, 2880, and 2855 cm−1, which are due to CH3 asymmetric (νaCH3), CH2 asymmetric (νa-CH2), CH3 symmetric (νs-CH3), and CH2 symmetric (νs-CH2) vibrations, respectively.27−29 These correspond to alkyl chains of the silane modifier. The 6153



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proteins is expected to be in most of the cases through end on orientation, which is the highest resistance offering surface32 at the low velocity of a particle. The Brownian force may make the particle to be away from surface, but the solid makes the protein to come at an appropriate orientation. The two steps described above can be written as (6)

k2

k3

(P1S)I + cS ↔ (P1S)II

(7)

k4

where ki (i = 1−4) are various rate constants and (P1S)I and (P1S)II are the two forms of adsorbed proteins. The (P1S)I form occupies S area per molecule, and the (P1S)II form occupies (1 + c)S area per molecule. The rate equations of the eqs 6 and 7 in terms of the surface concentration of species can be written as d[P1S]I = k1[P1(*, t )][S] − k 2[P1S]I + k4[P1S]II dt − k 3[P1S]I [S]

(8)

d[P1S]II = k 3[P1S]I [S] − k4[P1S]II dt

(9)

where [P1S]I and [P1S]II are the concentrations of the surface protein complexes (P1S)I and (P1S)II, respectively, and [S] is the area fraction of surface available for adsorption. Using material balance of protein at the surface layer and resistance at the boundary layer, we can write the variation in concentration of protein in the unbound state at the surface layer as δ

d[P1(*, t )] = k h([P1]0 − [P1(*, t )]) dt − (k1[P1(*, t )][S] − k 2[P1S]I )

(10)

where δ is the thickness of the surface layer and kh is the mass transfer resistance of the boundary layer. The initial conditions for eqs 8−10 can be written as [P1(*, 0)] = [P1]0 ,

[PS 1 ]I = [PS 1 ]II = 0

at

t=0 (11)

The surface fractions of the (P1S)I and (P1S)II denoted by [S[P1S]I] and [S[P1S]II], respectively, are related by eq 11 through the area occupancy of individual protein [S[P1S]I] = a[P1S]I [S[P1S]II] = a(1 + c)[P1S]II

(12)

where a denotes the surface area of protein per molecule occupied by (P1S)I on the substrate. The total surface area is sum of the area occupied by the proteins and empty area. In terms of fraction, we can write as [S[P1S]I] + [S[P1S]II] + [S] = 1

M w × 105 [S[P1S]I] 17.7a t

ΔF[P1S]II = −

M w × 105 [S[P S] ] 17.7a t(1 + c) 1 II

(14)

where, ΔF[P1S]I and ΔF[P1S]II are the estimated frequency change due the adsorbed protein in the state (P1S)I and (P1S)II, respectively; at is the surface area per unit mole of protein; Mw is the molecular weight of protein; and 17.7 ng·cm−2·Hz−1 is mass and frequency conversion factor. The sum of ΔF[P1S]I and ΔF[P1S]II is the predicted ΔFn/n. The experimentally obtained ΔFn/n versus time data for various systems of our studies are fitted using fminsearch and ODE45 functions in MATLAB to obtain kh, a, c, and ki. The values of kh and a can be approximated as follows. The reported values33 of diffusion coefficient of BSA and Fb are 5.93 × 10−11 and 2.02 × 10−11 m2/s, respectively. Using a thickness of the boundary layer as 20 μm, the calculated value of kh is 29.7 × 10−7 and 10.1 × 10−7 m/s for BSA and Fb, respectively. For calculation, of a, we have used already reported dimensions of BSA and Fb, which are 4.0 × 4.0 × 14 nm3 and 5.0 × 5.0 × 47 nm3, respectively.34 The a values are 16 and 25 nm2 for end on orientated approach of BSA and Fb, respectively, and 96 and 235 nm2 for side on orientated approach of BSA and Fb, respectively. We expect that the value of a is in between 16 and 96 nm2 and 25 and 235 nm2 for BSA and Fb, respectively. The longest dimensions of BSA and Fb are 14 and 47 nm, respectively, and are used as δ values for the two cases. The variations of ΔFn/n with time, during adsorption of BSA and Fb on four different surfaces are shown in Figure 2A and B, respectively. The figures indicate that initial adsorption of protein cannot be detected until the sensor gets the proteins corresponding to −ΔFn/n of about 0.15 Hz. After this, we distinctly see that adsorption depends on the type of surface. Time of start of adsorption of BSA and Fb is similar for the most of the surfaces. This indicates that the protein adsorption is not strongly restricted by diffusion of solutes. The experimentally observed equilibrium −ΔFn/n of both proteins on all four surfaces is shown in the first line of Table 1. The equilibrium adsorbed amount of BSA on the hybrid surface is less than that on the mixed surface and lies in between that on the octyl and amine surfaces. The equilibrium adsorbed amount of Fb is the least on the hybrid surface. The equilibrium adsorbed amount of both BSA and Fb is the highest on the octyl surface due to strong hydrophobic interaction. The initial rate of adsorption of BSA and Fb on various substrates is shown in second row of Table 1. The adsorption rate of BSA is the lowest on hybrid surface and the highest on octyl surface. The adsorption rate of Fb is the highest on octyl surface. Initially, the rate of adsorption of Fb on amine surface is the slowest, but after about 500s the adsorption rate on hybrid surface has crossed that on amine surface. Adsorption rate of Fb is higher on mixed and hybrid surfaces as compare to BSA.34 Initial adsorption rate is the fastest on octyl surface for both the proteins. The similar behavior of the two proteins at octyl surface is due to interactions between hydrophobic groups present at the surface and on the proteins. The difference in behavior of the two proteins on other surfaces is due to the difference of interactions between protein−surface combination and are explained below.

k1

P1 + S ↔ (P1S)I

ΔF[P1S]I = −

(13)

We can get the time evolution of area fractions by solving eqs 8−12. We use eqs 1 and 12 to get the change in frequency due to (P1S)I and (P1S)II, respectively, and get the following relations: 6154



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different parts of the protein try to be at different sides on the surfaces, depending on its characteristics. The amine surface (whose measured zeta potential is positive) is expected to bind the negative domain of BSA quickly leading to its strong and rapid binding with BSA. This leads to the quick saturation of the surface and lesser equilibrium adsorbed amount on the surfaces. Fb has two sets of three polypeptide chains, which are linked by disulfide bonds. The unique combination gives two Ddomains with carboxylic groups about four negative charges, a dangling positively charged αC-domain,36,38 and E-domain at the middle with amine groups. Because of the lesser charge density on the D-domain, the movement of Fb is the slowest on the amine surface. The slow movement of Fb is also coupled with its bulkiness and lesser charge density of the domains than that of BSA. The lesser charge density on Fb requires a greater number of Fb to neutralize the surface charge of the amine surface, which can accommodate more number of Fb molecules than BSA molecules. This leads to higher equilibrium adsorbed amount of Fb than BSA on amine surface. The hydrophobic parts of mixed and hybrid surfaces interact with the protein’s hydrophobic parts. Presumably, these types of interactions are moderate for both the proteins. Due to high charge density on BSA than Fb following trend of in equilibrium adsorption are found. The equilibrium adsorbed amount of BSA is lesser on amine surface than that on hybrid and mixed surfaces. The equilibrium adsorbed amount of Fb on amine surface is higher than that on hybrid and mixed surfaces. Figure 2 also shows the fitted data (dotted lines). The rate constants ki (i = 1−4) obtained are listed in third to sixth row in the Table 1. The ratio k1/k2 is the highest for octyl surface for both the proteins. The ratio k3/k4 (listed in the eighth row) for both the proteins on all the substrates is higher than the k1/ k2 ratio. This suggests that the (P1S)II state is highly preferable compared to the (P1S)I state on all the substrates. The k1 values are expected to depend on the first binding event, which is expected to follow Arrhenius type behavior, that is, k1 ∝ exp[−γ/kT]. Here kT is the thermal energy and γ is the activation energy barrier37 of the protein’s adsorption in the first step. The energy barrier is expected to depend on the surface energy of the adsorbing surface (γs). All other k values are expected to depend on the protein’s packing on the surface and on interactions of protein on the surfaces. We have plotted a variation of ln(k1) with surface free energy (γs) of different surfaces as shown in Figure 3. It is found that ln(k1) varies

Figure 2. (A) Variation of ΔFn/n with time during adsorption of BSA on various substrates; symbols are experimental points obtained from QCM and dotted lines are fitted data, which were calculated using the rate constants determined from the proposed model as discussed in the Results and Discussion section. (B) Variation of ΔFn/n with time during adsorption of Fb on various substrates; symbols are experimental points obtained from QCM and dotted lines are fitted data, which were calculated using the rate constants determined from the proposed model as discussed in the Results and Discussion section.

BSA has three homologous domains with different charges −10, −8, and 0 electronic charges at pH 7.4.35 Apparently the

Table 1. Analysis of Variation of ΔFn/n with Time to Find Equilibrium −ΔFn/n, Initial Slope, Rate Constants, and Fraction Area Covered Fb protein substrates −ΔFn/n at equilibrium initial rate of decrease of ΔF (Hz·s−1) k1 k2 k3 k4 k1/k2 k3/k4 At

octyl 26.7 0.145 6.67 × 7.75 × 5.32 × 6.55 × 8.61 × 8.11 0.996

amine 15.3 0.040

10−5 10−4 10−3 10−4 10−2

2.10 × 6.64 × 8.13 × 3.13 × 3.16 × 2.60 × 0.738

BSA mixed 11.0 0.080

10−7 10−4 10−5 10−4 10−4 10−1

1.68 × 1.32 × 6.46 × 1.36 × 1.27 × 4.74 0.721

hybrid 9.5 0.060

10−5 10−2 10−4 10−4 10−3

1.25 × 1.53 × 9.54 × 7.89 × 8.17 × 1.21 0.401 6155

octyl 16.3 0.269

10−5 10−2 10−4 10−4 10−4

2.18 × 4.77 × 4.71 × 7.94 × 4.58 × 5.93 × 0.996

amine 9.9 0.168

10−5 10−4 10−3 10−3 10−2 10−1

2.76 × 9.77 × 2.44 × 3.44 × 2.82 × 7.11 × 0.995

mixed 11.6 0.049

10−6 10−5 10−2 10−4 10−2 101

4.87 × 5.24 × 2.43 × 1.39 × 9.30 × 1.74 0.902

hybrid 11.1 0.022

10−7 10−4 10−2 10−2 10−4

1.57 × 4.98 × 3.46 × 1.02 × 3.16 × 3.38 × 0.688

10−7 10−4 10−4 10−3 10−4 10−1



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Fb on octyl surface (Figure 5A) and for adsorption of BSA on octyl, amine, and mixed surfaces (Figure 5B). At the initial stage, (PS)I converts to (PS)II and the absence of (PS)II makes the ratio increase. At a later stage, (PS)II decreases as the backward rate constant is higher than the forward rate constant, which is required to fit the adsorption data. The higher backward rate constants lead to rapid increase in unadsorbed concentration of proteins as shown in Figure 5C and D, which depicts the variation of concentration of proteins in the surface layer with time. The backward directional movement is indicated in our previous studies of aggregation of insulin,30 where we have shown the surfaces assist the aggregation behavior of insulin in solution. Figure 5C indicates that the adsorptions of Fb on hybrid and mixed surfaces are strongly limited by its diffusion. For other cases of adsorptions of the proteins are not strongly dependent on the diffusion limitation, probably due to higher backward rate of transformations of (PS)II. 3.3. Viscoelastic Properties of Adsorbed Layer. The experimentally obtained ratio of ΔDn/(ΔFn/n) characterizes the viscoelastic properties of the surface. A more rigid layer has a lower slope22,39,40 of the ΔDn versus ΔFn/n plot. The ratio of ΔDn/(ΔFn/n) at the end of adsorption is always greater than 0.2 × 106 Hz−1, which confirms that the adsorbed protein layer is viscoelastic.41 The typical variation of ΔFn/n with ΔDn is shown in Figure 6 for BSA and Fb on the octyl surface. Two different slopes can be defined during the adsorption. During the initial period of adsorption, that is, at low ΔFn/n values, the slope is defined as the first slope, and at a higher ΔFn/n value the slope is defined as the second slope. The value of the first slope is smaller than the value of the second slope for all the cases except the BSA adsorbing on the hybrid surface. This suggests that, during initial adsorption, the proteins adsorbed strongly on the surface. From the above view, we conclude that protein in the (P1S)I form goes rapidly to the (P1S)II form. However, depending on the surface, the (PS)II behaves differently. This leads to the different slope for all other surfaces as listed in Table 2. Both the slopes are very high for BSA on the hybrid surface, possibly due to its weak binding to the hybrid surface. The slightly higher first slope than the second slope may be due to strong protein−surface interaction. The viscoelastic properties (μ1, η1, τr) and the corresponding adsorbed amounts (Δm, Δmw) of proteins are listed in Table 3. These are determined using the procedure discussed in Materials and Methods section. The adsorbed proteins have the highest elasticities and the lowest viscosities on the hybrid surface in comparison to that on other surfaces. The relaxation time, which is ratio of the viscosity to the elasticity of the adsorbed protein, is the smallest on the hybrid surface. The relaxation time in general increases with adecrease in surface energy of the adsorbing surface. This is explained below. The proteins adsorbs strongly on a hydrophobic surface due to its strong hydrophobic−hydrophobic interaction. This leads to tight binding between the surface and protein and longer relaxation time. The only discrepancy is seen for the synthesized hybrid surface. The proteins bind through hydrophilic groups with hydrophilic moiety present in the hybrid surface. Apparently, this binding is weaker than the hydrophobic−hydrophobic binding. The weaker binding leads to less compact structure and short relaxation time. To find the exact change of structure in proteins responsible for these types of behaviors on the surfaces, we found the change in secondary structures in the next section.

linearly with surface free energy for adsorbing surfaces except for amine surface.

Figure 3. Variation of rate constant, k1, with Surface energy. (circles, BSA; squares, Fb; octyl surface, red; amine surface, green; mixed surface, purple; hybrid surface, black).

The fractions of the total surface occupied, At, by both the proteins on various surfaces as predicted by the fitted model is shown in the ninth row of the Table 1. The value of At is the highest for both proteins on the octyl surface. This may be due to strong favorable interaction between the hydrophobic groups of proteins and the hydrophobic octyl surface. The area occupied by Fb on the hybrid surface is the lowest. The area occupancy depends on the number of molecules at the surface and spreading of the adsorbed proteins. The fitted parameter, c, can be viewed as the spreading factor. In general, a larger At is expected for a high c value. We plot the variation of c with surface energy, and this is shown in Figure 4. The c decreases

Figure 4. Variation of c with surface energy (circles, BSA; squares, Fb; octyl surface, red; amine surface, green; mixed surface, purple; hybrid surface, black).

with increase in surface energy for both the proteins. The c value of Fb is higher than that of BSA on the same substrates. We note that the spreading rate34 of Fb and BSA are reported to follow the above-mentioned trend. The c value on the hybrid surface is the lowest for both proteins. This indicates that protein undergoes the least deformation on the hybrid surface. We have observed the relative contribution of (PS)II and (PS)I to total value of −ΔF at any instant by plotting (PS)II/ (PS)I with time for different cases. The ratio increases smoothly with time for adsorption of Fb on amine, mixed, and hybrid surfaces (Figure 5A) and for adsorption of BSA on hybrid surfaces (Figure 5B). This is a typical behavior one can expect for sequential adsorption behavior, with a backward rate constant lesser than the forward rate constant at any step. However, the ratio passes through maximum for adsorption of 6156



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Figure 5. Variation of (PS)II/(PS)I and unadsorbed protein concentration at the surface, [P1(*,0)]/[P1]0 with time: (A) (PS)II/(PS)I of Fb, (B) (PS)II/(PS)I of BSA, (C) [P1(*,0)]/[P1]0 of Fb, and (D) [P1(*,0)]/[P1]0 of BSA.

on all other surfaces except in the following two cases, where no change in α-helix is found. These are adsorption of Fb on the hybrid surface and adsorption of BSA on the octyl surface. There are reports of a decrease in α-helix due to adsorption of BSA on germanium45 and polyelectrolyte surface.46 The increase in α-helix of proteins on the amine surface is probably due to increased hydrogen bonding between the amine of the substrate and acid groups of proteins. We note that formation of α-helix requires hydrogen bonding between amine and COOH group. Fb contains a greater number of proline and glycine, which are prone to β-turn formation. Because of this, the percentage of β-turn increases at the adsorbing surface for Fb adsorption. The amount of β-sheet increases and β-turn decreases for BSA on all the surfaces. The proportion of random parts for both proteins increases on all the surfaces, except for BSA on the octyl surface, where the proportion of random parts is found to decrease. It is reported that α-helix can transform to β-sheet through coil structure47,48 in the case of protein that is above critical size. The present analysis shows that the amount of relative proportions of secondary structure depends on the substrate−protein interaction. Increases in random coil for all the cases suggest that it might be the most stable structure. We pointed out earlier that we get a relative proportion of secondary structures from FTIR. Total content of secondary structures is proportional to the total adsorbed mass of the proteins on the substrates and calculated by multiplication of relative proportion of the secondary structures with corresponding adsorbed mass (Δm). Total content of α-helix and βsheet increases with an increase in corresponding adsorbed mass on different substrates (see Figures S3 and S4). The interfacial viscosity and elasticity depends on the amount,

Figure 6. Variation of ΔD with ΔFn/n during adsorption of Fb and BSA on octyl surfaces (lines are least-squares straight line fit, and slopes of the lines are written above it).

3.4. FTIR Analysis of Adsorbed Protein. We have obtained FTIR spectra of Fb and BSA in solution and after their adsorption on four substrates (octyl, amine, mixed and hybrid). These are shown in the Supporting Information (Figures S1 and S2). The figures are for proteins in solution and adsorbed proteins on octyl, amine, mixed, and hybrid surfaces. The obtained FTIR curves are fitted with Gaussian curves to determine various secondary structures. The peak positions of various secondary structures of proteins, that is, αhelix, β-sheet, turn, random, and side chain component, in the amide-I region are already reported in literature42−45 for various proteins and are listed in Table S1 in the Supporting Information. From our spectra, we see the amide-I peak band of protein shifts from the range 1575−1725 to 1600−1700 cm−1 due to adsorption on substrates. The relative content of α-helix, β-sheet, turn, random, and side chain components are determined and shown in Figure 7. The proportions of α-helix of both the proteins increase on the amine surface but decrease Table 2. Slope of ΔFn/n versus ΔD Plot Fb

BSA

protein substrates

octyl

amine

mixed

hybrid

octyl

amine

mixed

hybrid

1st slope 2nd slope

−0.011 −0.219

−0.142 −0.455

−0.433 −0.843

−0.199 −0.577

−0.198 −0.387

−0.063 −0.437

−0.075 −0.275

−1.486 −1.144

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Table 3. Sauerbrey Mass (Δm), Wet Mass (Δmw), and Viscoelastic Properties of Adsorbed Proteins at Equilibrium SAM

protein

Δm (mg/m2)

μf (Pa·s × 103)

ηf (Pa × 10−3)

Δmw (mg/m2)

octyl amine mixed hybrid octyl amine mixed hybrid

BSA BSA BSA BSA fibrinogen fibrinogen fibrinogen fibrinogen

2.7 1.7 2.1 1.9 4.8 2.7 2.0 1.7

1.6 1.5 1.8 1.5 1.9 1.6 1.8 1.5

15 13 14 30 12 21 25 25

4.8 3.2 3.5 3.0 7.7 4.5 3.0 2.7

relaxation time (s) 1.07 1.15 1.29 5.00 1.58 7.62 7.20 6.00

× × × × × × × ×

10−7 10−7 10−7 10−8 10−7 10−8 10−8 10−8

Figure 8. (A) Variation of elasticity (η) of adsorbed proteins (BSA and Fb) with total content of α-helix as discussed in the section 3.4. (B) Variation of viscosity (μ) of adsorbed proteins with total content of βsheet. Total content of secondary structures is calculated by multiplication of relative proportion of the secondary structures (shown in Figure 7) with corresponding adsorbed mass (Δm).

Figure 7. Comparison of amount of secondary structures (α-helix, βsheet, β-turn, and random) of (A) Fb and (B) BSA in solution and on various substrates.

structure, and arrangement of materials at the interface. Literature does not have the interrelation among adsorbed mass of proteins, its secondary structures, and viscoelastic properties. The theory and simulations of elasticities of αhelix49 suggest that it obeys linear elasticity. The α-helix is structurally similar to a spring and expected to decide the elastic component of the adsorbed proteins. So we suggest that elasticity of adsorbed proteins may be dependent on α-helix content, which is shown in Figure 8a. The elasticity decreases with an increase in α-helix content. The viscous part is expected to depend on the structures which allow dissipating applied external force. β-sheet, along with β-turns, may be related to dissipate energy, which has not been verified. However, viscosity of the adsorbed proteins increases with total content of β-sheet as shown in Figure 8b. We note the theory of βsheet50 suggests that it can store energy. However, its behavior in the presence of β-turn and α-helix has not been demonstrated.

resistance in the interface to study the adsorption behavior of proteins. The rate constant, k1, is expected to depend on the first binding event and is found to depend on the surface energy of the adsorbing surface. The spreading of adsorbed proteins decreases with an increase in surface energy. It increases with a decrease in surface energy of the substrate. The spreading factor of Fb is higher than that of BSA on the same substrates. However, protein undergoes the least deformation on the hybrid surface. Adsorptions of Fb on hybrid and mixed surfaces are strongly limited by its diffusion. For other cases, adsorption of the proteins is not strongly dependent on the diffusion limitation, probably due to a higher backward rate of transformations of deformed proteins. Both proteins lie flat on mixed and hybrid surfaces. The viscoelastic properties, such as relaxation times, of adsorbed proteins are the slowest on the octyl surface, while those are the fastest on the hybrid surface. The analysis of secondary structures of proteins using ATRFTIR suggests that the behaviors of the proteins (BSA and Fb) are completely different on the amine surface than on the other three surfaces. The α-helix of the proteins increases on the amine surfaces only; but on all other surfaces, the α-helix of the protein decreases due to adsorption.

4. CONCLUSIONS We have studied the adsorption characteristics and properties of the adsorbed proteins BSA and Fb on the amine, octyl, mixed, and hybrid surfaces. The start of adsorption of a protein is not strongly restricted by diffusion of solutes. We have proposed a combined kinetic model and mass transfer 6158



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The viscoelastic properties of adsorbed proteins, determined by the QCM data, suggest that the adsorbed layer of BSA is softer than that of Fb and proteins adsorbed on the hybrid surface are softer as compared to other surfaces. The viscoelastic properties of adsorbed proteins are related to the total content of secondary structures. The elasticity of the adsorbed proteins is inversely related to the total content of αhelix, and the viscosity of the adsorbed proteins is proportionally related to the total content of β-sheet.

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ASSOCIATED CONTENT

S Supporting Information *

Appendix-1: Equations describing the kinetics of adsorption on surface; Appendix-2: Figure S1. FTIR spectra of BSA in solution (a) and adsorbed on Octyl (b), Amine (c), Mixed (d) and Hybrid (e) surfaces; Figure S2. FTIR spectra of Fibrinogen in solution (a) and adsorbed on Octyl (b), Amine (c), Mixed (d) and Hybrid (e) surfaces; Table-S1. Spectral Band position (cm−1) of proteins on various substrates in the amide-I region. Figure S3. Variation of adsorbed mass proteins (BSA and Fb) with total content of β-sheet. Figure S4. Variation of adsorbed mass proteins (BSA and Fb) with total content of α-helix. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +91 11 26591018. Fax: +91 11 26581120. sudip@ chemical.iitd.ac.in. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank department of biotechnology for their financial support (Sanction number BT/PR9683/ MED/32/16/2007) for this work.

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REFERENCES

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Properties of Adsorbed Bovine Serum Albumin and Fibrinogen on Self

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