Human Serum Albumin Monolayers on Mica: Electrokinetic

Oct 11, 2012 - HSA also binds many therapeutic drugs and controls their active concentration. .... Ruby muscovite mica supplied from Continental Trade...
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Human Serum Albumin Monolayers on Mica: Electrokinetic Characteristics Maria Dąbkowska and Zbigniew Adamczyk* J. Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Science, Niezapominajek 8, 30-239 Cracow, Poland ABSTRACT: Adsorption of human serum albumin (HSA) on mica from 0.15 M NaCl solutions and various pH values was studied using in situ streaming potential measurements, AFM imaging, and XPS. The results obtained by the streaming potential were consistent with AFM measurements and theoretical predictions based on the random sequential adsorption model. This allowed one to determine both the kinetics of adsorption and the maximum coverage of HSA as a function of pH. At pH 3.5, the maximum coverage of HSA was 0.45 (which corresponds to 1.4 mg m−2 neglecting hydration). This decreased monotonically with the increase in pH, attaining 0.30 (pH 5.1) and 0.25 (pH 7.4). At pH >10.5, the adsorption of HSA on mica was negligible. Further experimental studies performed for HSA monolayers of well-controlled coverage revealed their stability against pH cycling. It was found in these experiments that at pH 8 the electrokinetic properties of HSA monolayers approached the reference data pertinent to the bulk. However, for an intermediate pH range, deviations from the bulk reference data were observed, suggesting a dipolar (heterogeneous) charge distribution over adsorbed HSA molecules. This caused a slight shift in the isoelectric point of the monolayer determined to be 4.7 compared to the bulk value of 5.1. However, for the HSA coverage below 0.2, the effect of the substrate was significant, making the zeta potential more negative and shifting the apparent isoelectric point to more acidic values. It was suggested that these results obtained for planar and smooth interfaces could be used as reference data for interpreting albumin adsorption on colloid carrier particles. spheroid having dimensions of 9.5 × 5 × 5 nm3 12,13 with the effective cross-sectional area in the side-on orientation equal to 37 nm2. This spheroidal model is useful for the interpretation of experimental results concerning HSA adsorption kinetics and streaming potential measurements. Because of its significance, especially for immunological assays, HSA adsorption on various surfaces was widely studied by a multitude of techniques4,14−20 with respect to both kinetic and equilibrium aspects. For example, Norde and Lyklema14 determined adsorption isotherms of HSA at negatively charged polystyrene latexes. The roles of pH (4.0−7.0) and temperature (5−37 °C) were systematically studied. Interestingly, for the highly charged latex and a temperature of 22 °C, the protein coverage in the limit of zero bulk concentration approached 1 mg m−2, which indicates a high degree of irreversibility of HSA adsorption. For the higher bulk concentration range (10−500 ppm, referred to further on as parts per million), the HSA coverage increased to ca. 2 mg m−2. The kinetics of adsorption on glass slides at pH 4−7.4 and an ionic strength of 0.001−0.1 M was studied by the radioisotope techniques by Van Dulm and Norde.15 The maximum coverage was ca. 1 mg m−2 at pH 7.4 and I = 0.1 M and ca. 2 mg m−2 at pH 4.0 and I = 0.1 M.

1. INTRODUCTION Controlled protein adsorption on various surfaces is a prerequisite of efficient separation and purification by chromatography, filtration, biosensing, bioreactors, and immunological assays. One of the most extensively studied proteins is human serum albumin (HSA) and analogous bovine serum albumin (BSA), abundant at a high level of ca. 4 to 5% in blood plasmas.1,2 HSA synthesized in the liver is a single non-glycosylated, α-chain protein consisting of 585 amino acids. Its molar mass calculated from this amino acid composition is 66 439 Da.1 The crystalline structure consists of 69% α-helix and a large number of disulfide bonds (17 in total).3−6 The molecule consists of three structurally similar repeating domains, each divided into two six-helix subdomains.6 The BSA molecule exhibits a chemical structure and composition analogous to those of HSA. Albumins (HSA or BSA) are mainly responsible for osmotic pressure regulation and for the transport of numerous compounds such as fatty acids, drugs, metals, and hormones. In addition to the transport role, HSA is involved in the inactivation of a group of compounds (e.g., several exogenous toxins). HSA also binds many therapeutic drugs and controls their active concentration.7,8 In addition to its important physiological role, HSA is often used for drug delivery and for medical device coating, which prevents the adhesion of other proteins, platelets, and bacteria.9−11 Although the molecular shape of albumins is rather irregular, characterized by no symmetry axis, one can approximate it by a © 2012 American Chemical Society

Received: September 12, 2012 Revised: October 10, 2012 Published: October 11, 2012 15663

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maximum coverage of HSA on mica precisely, confirming previous results obtained by the streaming-potential method. These studies27,28 were carried out for a fixed pH of 3.5, where HSA molecules are on average positively charged, opposite to the substrate surface. Hence, the adsorption of HSA could be quite naturally interpreted in terms of the classical DLVO mean-field electrostatic interactions. Additionally, the decrease in the maximum coverage with the ionic strength could be explained using the random sequential adsorption (RSA) model as being due to lateral electrostatic interactions, as previously confirmed for microparticles29 and nanoparticles.30−32 However, these experimental data do not allow one to explain interesting observations reported in the literature21 showing a significant adsorption of HSA on negatively charged surfaces for the range of pH close to and above its isoelectric point where the molecules are on average neutral or negatively charged. Thus, the goal of this work is to study the role of pH in albumin adsorption on mica systematically. Using a combination of AFM, XPS, microelectrophoretic, and in situ streaming potential measurements, we formulated reliable clues in the adsorption mechanism of albumins for this pH range. Another important aspect of our work is a systematic study of the properties of albumin monolayers on mica, especially their stability against pH cycling.

Interesting kinetics measurements for HSA adsorption on polystyrene-covered silicon wafers and glass slides were also performed by Elgersma et al.16 using reflectometry and streaming potential measurements. The maximum coverage was determined as a function of pH and protein bulk concentration (1 to 100 ppm). It was demonstrated that at pH 4.0 the amount of adsorbed protein, equal to 0.8 mg m−2, was independent of the bulk concentration of the protein. Malmsten17 determined the kinetics of HSA adsorption on methylated silica for physiological conditions (i.e., pH 7.4 and I = 0.15 M using ellipsometry). The maximum coverage of the protein in these measurements varied between 0.5 and 1 mg m−2 for HSA bulk concentrations changing from 50 to 1000 ppm. Kurrat et al.18,19 studied the kinetic of HSA adsorption on hydrated Si and Ti oxides (optical waveguides) using the integrated optics technique (pH 7.4). The maximum coverage of irreversibly bound protein determined in these experiments was 1.7 mg m−2. Systematic measurements of the adsorption of HSA were performed by Ortega-Vinuesa et al.,20 who measured the thickness of the protein layer on silicon plates by ellipsometry as a function of the solution pH and ionic strength for a bulk concentration of 20 ppm. At pH 4, the coverage of HSA varied between 2 and 2.7 mg m−2 depending on the ionic strength, whereas at pH close to the isoelectric point (4.7) the maximum coverage of HSA approached 3 mg m−2, fairly independent of the ionic strength. Similar values were obtained in precise kinetic measurements of BSA adsorption on silicon and modified glass surfaces forming parallel-plate channels using the in situ fluorescence TIRF technique.21 The maximum coverage varied from 2.5 to 3.5 mg m−2 for the bulk concentration of BSA that changed from 10 to 50 ppm. HSA adsorption on mica was also studied using electron spectroscopy for chemical analysis (ESCA), also called X-ray photoelectron spectroscopy (XPS), by measuring the photoelectric signal of nitrogen, stemming from the protein monolayer, and that of potassium, stemming from mica.22−24 After a tedious calibration, the amount of adsorbed albumin was calculated. It varied from 0.5 mg m−2 (protein bulk concentration of 1 ppm) to 3.1 mg m−2 (protein bulk concentration of 1000 ppm). Recently, the AFM technique was used25,26 to obtain information about the binding strength of albumin and the density of monolayers. However, no quantitative data on HSA coverage and adsorption kinetics were given in these works. As noticed, a significant spread in the maximum coverage of albumins was reported in these works, with the lowest and highest values equal to 0.5 and 3.5 mg m−2, respectively. To elucidate this discrepancy and reveal albumin adsorption mechanisms, systematic measurements were performed in ref 27 using mica as a model hydrophilic substrate with welldefined and reproducible surface properties. Using the in situ streaming-potential measurements, the adsorption and desorption kinetics of HSA for a fixed pH of 3.5 were determined. It was shown that the amount of irreversibly adsorbed albumin increased systematically with ionic strength, attaining 1.4 mg m−2 for I = 0.15 NaCl. However, a significant amount of reversibly adsorbed protein was also revealed, which could be removed upon prolonged washing. In ref 28, additional experiments were performed at pH 3.5 using the XPS method, which allowed one to determine the

2. MATERIALS AND METHODS In our studies, HSA lyophilized powder 99% (Sigma) having a nominal fatty acid content of 0.02% was used. The purity of the albumin samples was checked by dynamic surface tension measurements carried out using the pendant drop shape method. No measurable changes in the surface tension of supernatants acquired by ultrafiltration or dialysis of HSA solutions were observed over a prolonged time period reaching 12 h. The presence of high-molecular-weight components in the HSA solutions was determined via SDS-PAGE electrophoresis in a Laemmli system33 using a nonreducing sample buffer and 12% polyacrylamide gel. The appropriate amount of albumin (e.g., 15 μL of 1, 2.5, and 5 ppm solutions and fresh solution in 0.15 M PBS at pH 7.4) was applied to each well of the polyacrylamide gel, and molecular protein markers were added to one well. Gel electrophoresis was run at room temperature in 0.025 M Tris, 0.192 M glycine, and 0.1% SDS. After the run, gel was fixed and silver stained.34 No lower-molecular-weight components other than the 67 kDa were found, as confirmed by gel electrophoresis in reducing buffer.28 However, a band at 2 times higher molecular weight appeared, which can be attributed to the HSA dimer commonly appearing in commercial HSA samples.35 The amount of dimer was quantitatively evaluated via special Quantity One program (Biorad) software using the intensity of the bands. The average value taken from six measurements was 13.5% (by weight), which corresponds to 6.7% by number. The effective bulk concentration of HSA after dissolving the powder in appropriate electrolyte solutions at pH 7.4 and after filtration was determined using a high-precision densitometer (Anton Paar, type DMA 5000M). The density of the HSA solutions (ρs) of a nominal weight concentration of 500−2000 ppm was measured as well as the supernatant solutions (ρe) acquired by membrane ultrafiltration using a regenerated cellulose filter (Milipore, NMWL 30 kDa). The effective bulk concentration of the HSA solution (w) was determined by applying the equation

w=

ρp (ρ − ρ ) s e ρs (ρp − ρe )

(1)

where ρp = 1.35 g cm−3 is the specific density of HSA. 15664

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scans were collected at a pass energy of 200 eV. The mica sheets were immersed in the protein solution containing 10 ppm HSA for 1−20 min at 0.15 M NaCl at pH 3.5 or 7.4 in the channel. After incubation, the mica samples were rinsed with ultrapure water for 1 min to remove any nonadsorbed molecules in the fluid film and air-dried prior to analysis. High-resolution spectra for N 1s were obtained. Elemental atomic percentages of nitrogen were determined. Because of instrumental factors, uncertainties in atomic cross sections, and electron inelastic mean free paths, the systematic error of the XPS method for quantitative purposes is about 10% .23

The effective bulk concentration of HSA was also determined spectrophotometrically using BCA (bicinchoninic acid) protein assays.36 HSA samples were incubated for 30 min at 37 °C with a BCA protein assay, and the purple color (product reaction of the reduction of Cu2+ by protein to Cu+ in an alkaline medium) was measured at a wavelength of 562 nm using a Shimadzu spectrophotometer. The concentration of HSA was calculated using a prepared calibration curve with known concentrations of dissolved standard protein (BSA, Sigma) and was correlated with the spectrophotometric absorbance measurement at 280 nm. These concentrated stock solutions of HSA were diluted to the desired bulk concentration (usually 1−10 ppm) prior to each experiment without using any filtration procedure. Ruby muscovite mica supplied from Continental Trade was used as a substrate for HSA adsorption. The solid pieces of mica were freshly cleaved to thin sheets prior to every experiment. Water was purified using a Milipore Elix 5 apparatus. Chemical reagents (sodium chloride and hydrochloric acid) were commercial products of Sigma-Aldrich and were used without further purification. The experimental temperature was kept constant at 293 ± 0.1 K. The diffusion coefficient of HSA was determined by dynamic light scattering (DLS) using the Zetasizer Nano ZS Malvern instrument at protein concentration varying between 200 and 2000 ppm as described previously.13 The microelectrophoretic mobility was measured with the laser Doppler velocimetry (LDV) technique using the same Malvern device. The streaming potential of bare and HSA-covered mica was determined using a previously described homemade apparatus.37 The laminar flow of the electrolyte (or the protein suspension) was forced by a regulated hydrostatic pressure difference ΔP through the parallel plate channel with dimensions of 2bc × 2cc × L = 0.027 × 0.29 × 6.2 cm3, formed by mica sheets separated by a perfluoroethylene spacer. The resulting streaming potential Es was measured by a pair of AgCl electrodes at various pressures to obtain the slope of the Es versus ΔP dependence. The cell electric resistance Re was determined using another pair of platinum electrodes to account for the surface conductivity effect. By knowing the slope of the Es versus ΔP dependence, we can calculate the apparent zeta potential of the substrate surface (ζ) from the Smoluchowski relationship

ζ=

ηL ⎛ ΔEs ⎞ ηKe ⎛ ΔEs ⎞ ⎟ ⎜ ⎟= ⎜ 4εbcccR e ⎝ ΔP ⎠ ε ⎝ ΔP ⎠

3. RESULTS AND DISCUSSION 3.1. HSA and Mica Substrate Characteristics. The bulk characteristics of HSA comprised the diffusion coefficient and the electrophoretic mobility acquired at pH 3.5−11 (0.15 NaCl). The diffusion coefficient of HSA determined by DLS, denoted by D, was practically independent of pH and equal to 6.1 × 10−11 m2 s−1 at T = 293 K. Accordingly, the hydrodynamic diameter of albumin dH calculated from the Stokes−Einstein relationship was 7.0 nm. This is a good estimate of its geometrical size, given the structural stability of the protein and its compact shape. The dependence of the electrophoretic mobility of HSA molecules μe on pH is shown in Figure 1. As seen, at pH 3.5, μe

Figure 1. Dependence of zeta potentials on pH. (1) (▲) HSA, 0.15 M, NaCl (microelectrophoresis). (2) (●) mica, 0.15 M NaCl (streaming-potential measurements). The solid lines denote nonlinear fits.

(2)

where ε is the dielectric permittivity of water and Ke is the specific conductivity of the cell. The surface concentration of HSA monolayers on mica after the streaming potential measurements was determined by AFM imaging in air using the NT-MDT Solver BIO device with the SMENA SFC050L scanning head. All measurements were made in semicontact mode by using high-resolution silicon probes (NT-MDT ETALON, HA NC series), polysilicon cantilevers with a resonance frequency of 240 kHz ± 10% or 140 kHz ± 10%, and a force constant of 9.5 N/m ± 20% or 4.4 N/m ± 20%, respectively. The curvature radius of the tip was 10 nm, and the cone angle was less than 20°. The images with number of adsorbed HSA molecules were determined within the scanning area of 0.5 μm × 0.5 μm for 10 randomly chosen areas over the mica sheet, which ensures a relative precision of these measurements of better than 5%. All of the images were flattened using an algorithm provided with the instrument. We have also employed XPS to quantify the kinetics of HSA adsorption on mica. The XPS measurements were carried out on a hemispherical spectometer (SES R4000, Gammadata Scienta) using Mg Kα (1253.6 eV, 11 kV, 17 mA) as a X-ray source. The photoelectrons were analyzed at a takeoff angle of 90°. The base pressure in the analysis chamber was about 8 × 10−9 Pa, and it was about 3 × 10−8 Pa during measurements. The energy resolution of the system, measured as the full width at half-maximum (fwhm) for the Ag 3d5/2 excitation line, was 0.9 eV. The analysis area of the samples (rectanglar plates of mica) was approximately 4.8 mm2. All survey

= 1.39 μm cm s−1 V−1. At pH 5.1, the electrophoretic mobility vanishes and becomes negative afterward, attaining −0.84 μm cm s−1 V−1 at pH 7.4 and −1.26 μm cm s−1 V−1 at pH 9.5. Thus, pH 5.1 can be treated as the isoelectric point (iep) of the protein. By knowing the electrophoretic mobility, one can calculate the electrokinetic charge per molecule Qc from the Lorenz− Stokes relationship13,37

Q c = 3πηdHμe

(3)

where η is the dynamic viscosity of the solvent (water). The number of electrokinetic charges per molecule Nc can be calculated by considering that |e| = 1.602 × 10−19 C. Using eq 3, one can calculate that at pH 3.5, Nc = 5.7, at pH 7.4, Nc = −2.1, and at pH 9.5, Nc = −4.5 (Table 1). The zeta potential of HSA, ζp, was calculated from the wellknown Henry relationship η μ ζ= εf (κa) e (4) 15665

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Table 1. Electrokinetic Properties of HSA and Mica Determined by Microelectrophoresis and Streaming Potential Measurements for Various pH Values and an Ionic Strength of 0.15 NaCla electrophoretic mobility (μm cm s−1 V−1)

pH

a

3.5

mica HSA

1.39

5.1

mica HSA

0

7.4

mica HSA

−0.52

9.5

mica HSA

−0.89

10.5−11

mica HSA

−1.1

zeta potential (mV)

electrokinetic surface charge (e nm−2)

number of electrokinetic charges per Sg (e)

27 −30 0 −45 −10 −58 −17 −59 −21 −60

0.15 −0.18 0 −0.31 −0.058 −0.375 −0.10 −0.415 −0.12 −0.42

5.7 −6.7 0 −11.3 −2.1 −13.9 −3.7 −15.4 −4.5 −15.5

Sg is the characteristic cross section of the HSA molecule, equal to 37 nm2.

where f(κa) is the dimensionless Henry function and a is the characteristic protein dimension. For example, the hydrodynamic radius is RH = dH/2, κ−1 = (εkT/2e2I)1/2 is the doublelayer thickness, I = (1/2)(∑icizi2) is the ionic strength, and ci is the ion concentration. For thin double layers where κa ≫1, f(κa) approaches unity, and for thick double layers where κa ≪ 1, f(κa) approaches 3/2. In our case, for the ionic strength of 0.15 M, substituting a = κdH/2, one obtains κa = 4.5. Using eq 4, it was determined that the zeta potential of albumin ζp varied between 27 mV at pH 3.5 and −21 mV at pH 10.5−11 (Table 1). The mica substrate zeta potential, denoted by ζi, was determined via the streaming potential measurements according to the above-described procedure. The dependence of ζi on pH for various ionic strengths is also shown in Figure 1. As seen, the zeta potential of mica was negative over the entire range of pH, varying from −30 mV at pH 3.5 to −58 mV at pH 7.4 and to −60 mV at pH 10.5−11 . Using these zeta potential values, we calculated the electrokinetic (uncompensated for) surface charge of mica using the Gouy−Chapman relationship13,37 σ0 =

⎛ eζ ⎞ (8εkTnb)1/2 ⎟ sinh⎜ ⎝ 2kT ⎠ 0.1602

quantitatively studied using direct AFM imaging in semicontact mode in air. This proved to be more efficient for determining the coverage than the in situ imaging of adsorbed molecules under wet conditions. Albumin monolayers were produced for low bulk concentration over a desired time period under diffusion transport in a thermostatic cell. Typical micrographs of such monolayers obtained at pH 3.5 and 0.15 M NaCl and a bulk protein concentration of 1 ppm are shown in Figure 2. As observed, HSA molecules appear as isolated entities, which enables their enumeration by image analyzing software. In this way, the number of molecules per unit area (surface concentration, denoted by N) can be determined by taking averages from various surface areas randomly chosen over the

(5)

where σ0 is the electrokinetic surface charge (2D charge density) expressed in e nm−2 and nb is the number concentration of ions expressed in m−3. Using the above zeta potential values, one can calculate from eq 5 that for mica, σ0 = −0.18 e nm−2 at pH 3.5, σ0 = −0.375 e nm−2 at pH 7.4, and σ0 = −0.420 e nm−2 at pH 10.5 −11. (For the sake of convenience, these values are collected in Table 1.) It should be observed, however, that even the lowest value of σ0 (−0.42 e nm−2) is still much higher than the lattice charge of the basal plane of mica, equal to −2.1 e nm−2.38 This effect is caused by the specific adsorption of cations, including H+, which is often referred to as the ion condensation effect4,14 extensively studied by Scales et al.39,40 Considering that the characteristic cross-sectional area of the HSA molecule, Sg, equals 37 nm2, one can calculate the average number of negative charges on mica within this area to be equal to −6.7 at pH 3.5, which approximately matches the number of positive charges per HSA molecule (Table 1).27 However, at pH 7.4, the number of charges on mica per Sg equals −13.9, and at pH 10.5−11, it equals −15.7 (Table 1). 3.2. HSA Adsorption Kinetics. To calibrate the streaming potential measurements, HSA adsorption kinetics on mica was

Figure 2. AFM micrographs (semicontact mode, imaging in air) of HSA monolayers on mica. Adsorption conditions: pH 7.4, 0.15 M NaCl, cb = 1 ppm. (A) N = 861 μm−2, θ = 0.03. (B) N = 3444 μm−2, θ = 0.13. 15666

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mica substrate. For the sake of convenience, N is expressed hereafter as the number of molecules per square micrometer(i.e., μm−2). It should be mentioned that by determining the surface concentration via direct counting no information about protein size and shape is required, which makes this procedure reliable. The results obtained using this method for various pH values (3.5, 5.1, and 7.4) are shown in Figure 3 as the dependence of N on the square root of albumin adsorption t1/2.

proteins) via the AFM measurements of adsorption kinetics. This is feasible using the experimentally determined slope of the N versus t1/2 dependence, denoted by sa = ΔN/Δt1/2. By knowing this parameter, one can calculate from eq 7 the protein concentration in the bulk. Alternatively, by knowing the bulk concentration of the protein and its molar mass, one can determine its diffusion coefficient using eq 7. The agreement of experimental and theoretical results observed in Figure 3 suggests, therefore, that albumin adsorption was irreversible for all pH values studied, that is, 3.5, 5.1, and 7.4 (at an ionic strength of 0.15 M NaCl). This was independently confirmed in a separate series of desorption experiments, where the HSA monolayers on mica were conditioned for a prolonged time period (up to 24 h) in pure electrolyte solutions. The coverage determined by AFM prior to and after these experiments was the same within experimental error bounds. Considering the electrokinetic charge data shown in Table 1, we conclude that such behavior should be expected only at pH 3.5, where HSA molecules and mica bear opposite signs of electrokinetic charge. Irreversible adsorption at pH 5.1 and especially at pH 7.4, where both mica and HSA molecules bear negative charges, should be treated as anomalous from the viewpoint of conventional theories. However, this behavior is quite common for protein solutions. For example, in the case of fibrinogen, its adsorption on negatively charged substrates for the pH range above isoelectric point (pH 5.8) was reported in many works (e.g., Zembala and Dejardin,41 Malmsten,17 Ortega-Vinuesa et al.,20 Toscano and Santore,42 Kalasin and Santore,43 and Wasilewska and Adamczyk37). Such anomalous adsorption behavior was explained in terms of a heterogeneous (dipolar) charge distribution over protein molecules. This also seems probable in the present case of HSA given its heterogeneous charge distribution that is theoretically predicted in various computer simulations.44,45 To gather more evidence to support this hypothesis, additional measurements were performed using the XPS and in situ streaming potential methods. The advantage of XPS measurements is that they can be used for a broad coverage range of protein and their sensitivity increases with the coverage. Hence, in this respect, the XPS method is complementary to the AFM measurements. The experimental XPS procedure was described elsewhere.28 The surface area of the nitrogen peak (at 400 eV) was used as the measure of the HSA coverage of monolayers produced under diffusion-controlled adsorption on mica directly in the streaming potential cell. Afterward, one of the mica sheets was examined under AFM to determine the coverage, and the second was examined using XPS. The signal was determined from a few surface areas selected at random over the entire surface area covered by the albumin molecules, which rendered the procedure rather tedious and time-consuming. The results of these measurements are shown in Figure 4, where the coverage of albumin previously determined by AFM is compared to XPS data at pH 3.5 and 7.4. As seen, the agreement is quite satisfactory in the region where both methods overlap. Additionally, using XPS one determines the maximum coverage of HSA, which is a parameter of basic significance. For pH 3.5, Θmax determined by XPS was 0.45, and at pH 7.4, Θmax was 0.28. This corresponds to the commonly used unit of mass per unit area of Γ = 1.4 and 0.92 mg m−2, respectively.

Figure 3. Dependence of the surface concentration of HSA, N (μm−2) on the square root of adsorption time t1/2 (min1/2). The points denote experimental results obtained by the direct AFM enumeration of adsorbed HSA molecules for I = 0.15 M, cb = 1 ppm for (○) pH 3.5, (Δ) pH 5.1, and (●) pH 7.4. The solid line shows the exact theoretical results obtained by the numerical solution of the diffusion equation using the random sequential adsorption model.

The advantage of this transformation compared to the straight dependence of N on adsorption time t is that for irreversible adsorption under diffusion-controlled transport the initial kinetics for bare surfaces is described by the equation ⎛ D ⎞1/2 N = 2⎜ ⎟ t 1/2nb ⎝π⎠

(6)

where nb is the bulk concentration of protein expressed in m−3. One can alternatively formulate eq 6 in terms of the protein concentration expressed in ppm (denoted by cb) and N expressed in μm−2 N = 2 × 10−12

A v ⎛ D ⎞1/2 1/2 ⎜ ⎟ t cb Mw ⎝ π ⎠

(7)

where Av is Avogadro’s constant and Mw is the molar mass of the protein. Often, to facilitate comparisons of experimental data with theoretical approaches, it is convenient to express the albumin surface concentration in terms of the dimensionless coverage (2D density) defined as Θ = 10−6NSg

(8) 2

where Sg is expressed in nm . As seen in Figure 3, the experimental data are well reflected by the theoretical results calculated from eq 7 for a broad range of N up to 6000 μm−2, which corresponds to Θ = 0.22. However, for higher coverage, the direct AFM enumeration of adsorbed albumin molecules became inaccurate. The results shown in Figure 3 have interesting practical implications, indicating that one can determine in a convenient way the unknown bulk concentration of albumin (or other 15667

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Figure 5. Dependence of the zeta potential of mica ζ on the coverage of HSA, θ. The points denote experimental results obtained from the streaming potential measurements for I = 0.15 M at various pH values: (1) (●) pH 3.5, (2) (▲) pH 5.1, (3) (⧫) pH 7.4, (4) (○) pH 9.5, and (5) (■) pH 10.5−11. The solid lines denote exact theoretical results calculated from the 3D electrokinetic model using eqs 11 and 13. The dashed lines show the bulk zeta potential of HSA.

Figure 4. Kinetics of HSA adsorption on mica shown as the dependence of θ on the square root of the adsorption time t1/2. The points denote experimental values obtained by (1) (●) AFM, pH 3.5 and (■) XPS, pH 3.5; (2) (○) AFM, pH 7.4 and (□) XPS, pH 7.4. The solid lines denote exact theoretical results calculated by solving the diffusion transport equation with the RSA blocking function.

By knowing the maximum coverage, one can theoretically predict the surface blocking effects using the random sequential adsorption (RSA) model.46 This allows one to calculate HSA adsorption kinetics for the entire range of times by the numerical solution of the governing diffusion transport. This procedure was previously applied to the interpretation of fibrinogen adsorption on mica.47,48 Theoretical results calculated using this approach are shown in Figure 4 by the solid lines. As observed, the experimental data obtained by AFM and XPS agree with theoretical predictions for the entire range of time. The results shown in Figures 3 and 4 prove, therefore, that it is feasible to determine reliably the coverage of HSA on mica via AFM (lower-coverage range) or XPS measurements. Hence, these results can be exploited for the calibration of more efficient streaming-potential measurements. These measurements are carried out under wet, in situ conditions, which eliminates conformation changes and the desorption of protein upon drying. Thus, they can be efficiently used to study albumin adsorption for wide range of pH. This is vital in explaining anomalous albumin adsorption at negatively charged surfaces at pH ≥5.1(Figure 3). Streaming potential measurements for HSA-covered mica were carried out according to the above-described procedure. Briefly, the cell was filled with the HSA solution of a prescribed concentration for various time periods ranging from 5 to 1600 min. Afterwards, the cell was flushed with pure electrolyte, and the streaming potential of HSA-covered mica was determined. The measured streaming potential signal was converted to the apparent zeta potential of an HSA monolayer on mica ζ using eq 2. In this way, the dependencies of ζ on the adsorption time were obtained for various pH values, which served as primary experimental data. These results can be converted to the ζ versus Θ relationship using the above AFM or XPS kinetic results (Figures 3 and 4). Alternatively, to facilitate comparison with theoretical predictions, the nominal coverage of HSA calculated from eqs 6 and 8 was used. The experimental data acquired in the streaming potential measurements at pH 3.5, 5.1, 7.4, 9.5, and 10.5−11 are shown in Figure 5 as the dependence of the zeta potential of mica on the nominal coverage of albumin Θ.

As seen, at pH 3.5, 5.1, and 7.4 the zeta potential of mica increased abruptly with Θ attaining saturation values close to the bulk zeta potentials of albumin, determined by electrophoresis (marked by dashed lines in Figure 5). For pH 9.5, the zeta potential of mica increased only slightly from the initial value of −58 mV to ca. −40 mV (bulk value of albumin equal to −30 mV). For pH 10.5−11, there was practically no change in the zeta potential of mica. Qualitatively, these results indicate that there was a significant adsorption of albumin at pH 3.5, 5.1, and 7.4, less adsorption at pH 9.5, and no adsorption at pH 10.5−11. The experimental results were interpreted in terms of the recently developed electrokinetic model.49,50 Two main effects are considered in this approach: (i) flow damping in the vicinity of adsorbed molecules, which diminishes the ion flux from the double layer adjacent to the substrate surface and (ii) enhanced ion flux from the double layer adjacent to the adsorbed protein molecules. Using this approach, the expression for the streaming potential of an interface covered with protein molecules can be formulated as27 ζ(Θ) = Fi(Θ)ζi + Fp(Θ)ζp

(9)

where ζ(θ) is the zeta potential of the protein-covered substrate and Fi(Θ) and Fp(Θ) are dimensionless functions accounting for effects i and ii, respectively. It should be mentioned that eq 9 does not involve any fitting parameters because the functions Fi(Θ) and Fp(Θ) were theoretically determined. For example, in the limit of low coverage, Fi(Θ) = 1 − Ci(Θ) and Fp(Θ) = Cp(Θ),49 thus eq 9 assumes a linear form ζ(Θ) = ζi + (Cpζp − Ciζi)Θ

(10)

where the Ci and Cp functions approach the limiting values of C0i = 10.2 and C0p = 6.51, respectively, for thin double layers.49 Theoretical results were also reported in refs 49 and 50, which allowed one to determine the Fi(Θ) and Fp(Θ) functions for the entire range of coverage in the limit of thin double layers. These results were obtained by numerically evaluating the flow field in the vicinity of adsorbed particles using the multipole expansion method. The exact numerical results can 15668

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latter pH well correlates with the pKa values of the most abundant basic amino acids in the molecule (i.e., lysine having pKa 10.5−1145). Otherwise, if albumin adsorption was governed by van der Waals interactions or hydrogen bonding, then the change in its adsorption efficiency with pH would be negligible or much less abrupt than experimentally observed. However, as seen in Figure 5, the slopes of the ζ versus Θ dependencies for the higher-coverage range become rather small, prohibiting a precise determination of the maximum coverage of albumin as a function of pH. One can only estimate with a precision of ±0.03 that at pH 3.5 Θmax = 0.45, at pH 5.1 Θmax = 0.30, and at pH 7.4 Θmax = 0.25. This agrees quite reasonably with previous data derived from XPS (Table 2).

be approximated by the following analytical interpolation functions with a precision of better than 1%.37 Fi(Θ) = e−Ciθ

Fp(Θ) =

1 (1 − e− 2

2 C pθ

)

(11)

It should be noted that according to eqs 9 and 11 the limiting value of the monolayer zeta potential for the higher-coverage range, where the function Fi(Θ) vanishes and the Fp(Θ) function approaches 1/√2 is 1/√2 ζp = 0.71ζp. This means that in the case of uniformly charged molecules the zeta potential of their saturated monolayers should be 30% lower than the bulk zeta potential determined by microelectrophoresis. This unexpected result was experimentally confirmed for model colloid systems such as polystyrene latex particles,51 nanoparticles,52 and polyelectrolytes.53 This is significant because any deviations from this limiting behavior indicate a nonuniform distribution of charge over adsorbed molecules. As seen in Figure 5, the experimental dependence of the HSA monolayer zeta potential on the coverage is adequately reflected by the above theoretical model, expressed by eqs 9 and 11 at pH 3.5. The limiting monolayer zeta potential is 20 mV, which is comparable to the theoretical value of 0.71ζp = 19 mV. This suggests that HSA molecules are uniformly charged at this pH. However, at pH 5.1 and 7.4 the experimental data are well reflected by the theoretical model using bulk zeta potentials lower than those determined by microelectrophoresis. Thus, at pH 5.1 the effective zeta potential was −8 mV (compared to the bulk value close to zero), and at pH 7.4 it was −15 mV (the bulk value was −10 mV). This suggests that the part of the HSA molecule exposed to flow is noticeably more negative than the average value determined from microelectrophoretic measurements. Thus, the results shown in Figure 5 confirm a significant adsorption of HSA on mica for the range of pH where the molecules are either neutral or negatively charged. This is in accordance with previous experimental evidences gathered for other types of substrate surfaces.21,42,43 This anomalous HSA adsorption can be explained, analogously as done for fibrinogen,37,54 in terms of a heterogeneous charge distribution. Thus, for the pH range of 5.1 to 9.5 there exist positively charged patches on the HSA molecule, despite the fact that it is on average neutral or negatively charged. Hence, the albumin molecule effectively behaves as a dipole. The positively charged areas of the molecule are directed to the mica surface, which creates attractive electrostatic interactions ensuring their efficient attachment. Given the high charge density of mica at I = 0.15 M and pH 5.1−7.4 (Table 1), one can estimate as previously done for fibrinogen37 that the presence of a few positive charges on albumin would suffice to produce the minimum energy depth of ca. −15kT. Combined with the van der Waals attraction amounting to a few kT units, this ensures irreversible albumin adsorption on mica for this pH range. It should be mentioned that the existence of a similar dipolar charge distribution was also observed in the case of chymotrypsin,54 using analogous streaming potential measurements. The decisive role of electrostatic interactions is further confirmed by the fact that at pH 9.5 albumin adsorbs less efficiently and at pH 11 its adsorption vanishes (Figure 5). The

Table 2. Maximum Coverage of HSA on Mica at Various pH Values and 0.15 M NaCl pH

XPS

3.5 5.1 7.4 9.5 11

0.45 ± 0.05

streaming potential (adsorption)

streaming potential (desorption)

± ± ± ± 0

0.43 ± 0.03 0.32 ± 0.02 0.26 ± 0.02 0.16 ± 0.02 0.04 ±0.02

0.45 0.30 0.25 0.15

0.28 ± 0.03 0

0.03 0.03 0.03 0.02

The results shown in Figure 5 indicate that it is feasible to determine unknown coverages of albumin monolayers precisely for lower and moderate coverage via streaming potential measurements. This can be done by inverting eqs 9 and 11 to the form of Θ vs ζ dependencies. A useful analytical result can be derived by iteratively solving these equations.28 The first iteration yields the following expression for the protein coverage as a function of the experimental value of the zeta potential 1 Θ = − ln X(ζ ) Ci (12) where X1(ζ) can be treated as the normalized zeta potential given by ζ− X(ζ ) = ζi −

ζp 2 ζp 2

=

ζ − ζ∞ ζi − ζ∞

(13)

where ζ∞ = ζp/√2 is the limiting zeta potential. Using eqs 12 and 13, we transformed the experimental data shown in Figure 5 at pH 3.5, 5.1, and 7.4 to the universal relationship of Θ versus −(1/Ci) ln X(ζ). As noticed in Figure 6, this resulted in a straight line dependence that was valid over a wide range of albumin coverage. This has practical implications, indicating that one can determine the coverage of albumin in situ, which allows the study of its adsorption and desorption kinetics. This approach was applied in ref 28 to determine the influence of ionic strength on the maximum coverage of HSA at pH 3.5. In this work, we applied this method to evaluate more accurately the role of pH for a fixed ionic strength of 0.15 M NaCl. The experimental procedure was as follows: (i) HSA monolayers with a fixed coverage of 0.4 were produced in situ in the streaming potential cell at pH 3.5, (ii) the cell was flushed with pure electrolyte (0.15 M NaCl) having an appropriate pH and the streaming potential variations were measured as a function of the desorption time, and (iii) the 15669

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Hence, these results confirm that the adsorption of HSA on mica is mainly governed by electrostatic interactions. Moreover, the amount of irreversibly adsorbed protein decreases systematically with pH, exhibiting no maximum at the isoelectric point (pH 5.1). The maximum in HSA adsorption (Θmax = 0.45) occurs at pH 3.5, where the entire molecule is positively charged. This finding can be exploited to produce irreversibly bound HSA monolayers on mica for a desired, well-controlled coverage within the range from zero to 0.45. Various properties of such monolayers can then be studied, for example, the dependence of the zeta potential on pH, enabling the determination of the isoelectric point, which is discussed below. 3.3. Characteristics of HSA Monolayers on Mica. This series of experiments was aimed at determining the stability of HSA monolayers against pH cycling and their acid−base properties, especially the isolectric point. The experimental procedure implemented in these experiments was as follows: (i) HSA monolayers of a desired coverage (varying between 0.05 and 0.4) were produced using in situ adsorption followed by a washing step at pH 3.5 and 0.15 M NaCl. (ii) The pH in the cell was increased in a stepwise manner by an appropriate addition of NaOH (buffers were avoided because of their specific adsorption on monolayers). (iii) After the stabilization of the pH (a few minutes), the streaming potential was measured. (iv) The entire sequence was reversed after attaining pH 9.7 by the addition of an appropriate amount of HCl. It should be mentioned that variations in the ionic strength induced by these pH changes were negligible and were limited to a maximum of 2%. A few cycles were performed according to this procedure. The results of these measurements, made for initial monolayer coverages of 0.1 and 0.4, are shown in Figure 8.

Figure 6. Universal graph showing the dependence of the HSA coverage on −1/Ci ln[(ζ − ζ∞)/(ζi − ζ∞)]. The points denote experimental results derived using the streaming potential measurements for 0.15 M NaCl at various pH values of (○) 3.5, (Δ) 5.1, and (●) 7.4. The solid line denotes exact theoretical results calculated from the 3D electrokinetic model in eqs 9 and 11.

corresponding coverage of albumin was calculated from eqs 12 and 13. In Figure 7, the results of these experiments performed at pH 5.1, 7.4, 9.5, and 11 are shown.

Figure 7. Desorption kinetics of HSA expressed as the dependence of the coverage θ on the desorption time t. All monolayers were produced at pH 3.5 and 0.15 M NaCl. The points denote experimental results calculated from eqs 12 and 13 using the experimental streaming potential data obtained at various pH values: (1) (▲) pH 5.1, (2) (⧫) pH 7.4, (3) (○) pH 9.5, and (4) (■) pH 11. The dashed lines denote interpolations of experimental data.

As noticed, the initial coverage decreased with desorption time, attaining final steady-state values after 400 min. These coverages, which can be treated as maximum values, decreased monotonically with pH, thus Θmax = 0.32 at pH 5.1, Θmax = 0.26 at pH 7.4, and Θmax = 0.16 at pH 9.5. At pH 11, the maximum coverage was 0.04, which is comparable to the experimental error bounds. It should be mentioned that this desorption method is more precise than the previously described method on the basis of the analysis of the streaming potential versus albumin coverage curves (Figure 5). For the sake of convenience, the maximum coverages determined by XPS, streaming potential adsorption, and desorption runs are collected in Table 2. As seen, the results obtained by various methods are in reasonable agreement.

Figure 8. Dependence of the zeta potential of the HSA monolayer (adsorbed at pH 3.5 and 0.15 M, NaCl) on pH cycling starting from 3.5 to 9.5 and back to 3.5. The points denote experimental results obtained from the streaming potential measurements for (o) θo = 0.4, first cycle, (●) θo = 0.4, third cycle, (Δ) θo = 0.1, first cycle, and (▲) θo = 0.1, third cycle. The lines denote interpolations of experimental data.

As seen, the differences between the first and third pH cycles were quite minor, especially for the initial coverage of 0.1, where the process was completely reversible. This shows that there was little desorption of HSA during pH cycling and that conformational changes in the adsorbed HSA molecules and their reorientation processes were also negligible or reversible. 15670

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ence spectroscopy. The experimentally determined value of the isoelectric point was 4.2 on the POMA hydrophobic surface and ca. 3.2 for a hydrophilic PPMA substrate. In the latter case, the coverage of the fibronectin monolayer was significantly lower. Thus, this effect is fully analogous to that observed in our case for HSA (Figure 9). However, the bulk characteristics of the protein were not given in this work, which prohibits a comparison of bulk and surface isoelectric points.

After the stability of the monolayer behavior was confirmed, few series of experiments were performed where the zeta potential of HSA monolayers characterized by various coverages was determined as a function of pH. The main goal of these studies was a comparison of the surface data obtained for monolayers with the bulk HSA characteristics acquired by microelectrophoresis. The results of these experiments are shown in Figure 9.

4. CONCLUSIONS The in situ streaming potential measurements calibrated using AFM imaging of single protein molecules and XPS furnished reliable clues as to the mechanism of HSA adsorption on mica, a model hydrophilic substrate. It was demonstrated that a quantitative interpretation of streaming potential measurements can be achieved in terms of the theoretical model, expressed in eqs 9 and 11 that postulate the 3D adsorption of HSA molecules as isolated particles. This allows one to study in situ HSA adsorption/desorption processes and determine the amount of irreversibly adsorbed HSA as a function of the pH (for 0.15 M NaCl, where the κdH/2 parameter was 4.5). At pH 3.5, where HSA molecules are positively charged, the maximum coverage of irreversibly adsorbed HSA was 0.45 (which corresponds to 1.4 mg m−2 neglecting hydration). This value decreased monotonically with the increase in pH, attaining 0.32 (pH 5.1) and 0.26 (pH 7.4). At pH ≥10.5, the adsorption of HSA on mica was negligible. An anomalous adsorption of HSA on mica observed at pH ≥5.1 (isoelectric point), where the molecules bear a net negative charge, was explained in terms of the heterogeneous charge distribution. Hence, these results demonstrated quite unequivocally that HSA adsorption on mica is mainly governed by electrostatic interactions. Further experimental studies of HSA monolayers with wellcontrolled coverage revealed their stability against pH cycling. This proved that conformational changes in the adsorbed HSA molecules or their reorientation processes were negligible or reversible. It was also shown that at pH 8 the zeta potential of HSA monolayers approaches the reference data pertinent to the bulk. For an intermediate range of pH, a quite noticeable deviation from the bulk reference data is observed, which suggests a dipolar (heterogeneous) charge distribution over adsorbed HSA molecules. This causes a slight shift in the isoelectric point of the monolayer, determined by extrapolation to be 4.7 compared to the bulk value of 5.1. However, for an HSA coverage below 0.2, the effect of the substrate remains significant, making the zeta potential more negative and shifting the apparent isoelectric point to more acidic values. These results obtained for planar and smooth interfaces can be used as convenient reference data for more complicated situations involving albumin adsorption on colloid carrier particles, as widely studied in the literature.

Figure 9. Dependence of the zeta potential of the HSA monolayers ζ (adsorbed at pH 3.5 and 0.15 M NaCl) on pH cycling starting from 3.5 to 9.7 and back to 3.5 (three cycles for each curve were made for a fixed ionic strength of 0.15 M NaCl). The points denote experimental results obtained from the streaming potential measurements for the initial coverage of the monolayer equal to (●) θo = 0.4, (○) θo = 0.2, (▲) θo = 0.1, and (■) θo = 0.05. The upper line (1) denotes the reference results for the HSA zeta potential in the bulk (smoothed, i.e., 0.71ζp) and the lower line (2) shows the smoothed results for the bare mica substrate. The dashed lines denote the fits of the experimental data.

As reference data, the corrected bulk zeta potential values of HSA (i.e., 0.71ζp) are also shown in Figure 9 (solid line 1). As seen, for the acidic range of pH 8. For the intermediate range of pH, a quite noticeable deviation from the bulk reference data is observed, which suggests a dipolar (heterogeneous) charge distribution over adsorbed HSA molecules in accordance with previous predictions derived from the data shown in Figure 5. These result in a shift in the isoelectric point of the monolayer, determined by extrapolation to be 4.7 compared to the bulk value of 5.1. The influence of the monolayer coverage on the location of the isoelectric point was also established in these experiments. As seen in Figure 9, the isoelectric point is systematically shifted to more acidic values upon decreases in the coverage. Hence for Θ = 0.2, the isoelectric point is 4.5, and for Θ = 0.1, iep = 3.8. Therefore, a significant finding derived from these measurements is the location of the isoelectric point, which becomes practically fixed for monolayer coverage above 0.2. However, for the lower range of HSA coverage, the effect of the substrate remains significant, making the zeta potential more negative and shifting the apparent isoelectric point to more acidic values. It is interesting that similar acid−base characteristics were determined by Osaki et al.55 for fibronectin, whose adsorption kinetics on maleic acid copolymers was studied by a combination of streaming potential and reflectometric interfer-

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. 15671

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ACKNOWLEDGMENTS This work was financially supported by a Ministry of Science and Higher Education (MNiSzW) grant (no. N N204 026438) and a POIG grant (no. 01-01.02-12-028/09) FUNANO. We are grateful to Dr. Jacek Gurgul for her invaluable help in performing the XPS measurements and Marta Kujda for carrying out the gel electrophoresis.

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