Human Serum Albumin Adsorption Kinetics on Silica: Influence of


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Human serum albumin adsorption kinetics on silica: influence of protein solution stability Monika Wasilewska, Zbigniew Adamczyk, Agata Pomorska, Malgorzata Nattich-Rak, and Marta Sadowska Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03266 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 30, 2019

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Human serum albumin adsorption kinetics on silica: influence of protein solution stability

Monika Wasilewska, Zbigniew Adamczyk*, Agata Pomorska, Małgorzata Nattich-Rak, Marta Sadowska

Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Science, Niezapominajek 8, 30-239 Cracow, Poland

*Corresponding author Zbigniew Adamczyk e-mail: [email protected] Phone: +4812 6395104 Fax: +4812 4251923

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ABSTRACT Adsorption kinetics of human serum albumin (HSA) on silica substrates was studied using the optical waveguide lightmode spectroscopy (OWLS) and the quartz microbalance (QCM) techniques. Measurements were performed at pH 3.5, 5.6 and 7.4 for various bulk suspension concentrations and ionic strengths. The diffusion coefficient measurements showed that for pH 3.5 the HSA molecules are stable for NaCl concentration 10-3 to 0.15 M. This allowed to precisely determine the mass transfer rate coefficients for both the QWLS and QCM cells. The experimental data were adequately interpreted in terms of a hybrid random sequential adsorption model. The OWLS maximum coverage of HSA at pH 3.5, which is equal to 1.3 mg m-2, agrees with the QCM result and with previous results derived from streaming potential measurements. Thus, the results obtained at pH 3.5 served as reference data for the analysis of adsorption kinetics at larger pHs. In this way it was confirmed that the adsorption kinetics of HSA molecules at pH 5.6 and 7.4 was considerably slower than at pH 3.5. This effect was attributed to aggregation of HSA solutions and interpreted in terms of a theoretical model combining the Smoluchowski aggregation theory with the convective diffusion mass transfer theory. New analytical equations were derived adequately describing the kinetics of HSA adsorption

at larger pHs that can be used for the interpretation of other protein

adsorption from unstable solutions.

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1.

Introduction Human serum albumin (HSA), present in the blood at the level of 40-50 g L-1 is a single

non - glycosylated, α-chain protein consisting of 585 amino acids having the molar mass equal to 66,439 Da [1]. Its crystalline structure consists of 69% α-helix and 23% -sheet also comprising 17 disulfide bonds.1-3 In aqueous solutions the molecule exhibits positive electrophoretic mobility at pH < 5, otherwise the molecule exhibits a negative mobility, therefore its isoelectric point is reported to occur at pH ca. 5.14. The shape of the HSA molecule is rather irregular resembling a heart shape, which was recently approximated by triangular model5-6. In Ref.7 a more flexible bead model of albumin molecule was proposed allowing efficient modeling of its adsorption in terms of the random sequential adsorption (RSA) approach. In organism, HSA is responsible for osmotic pressure regulation and for transport of various substances such as fatty acids, ions (calcium) drugs and hormones.1,8 In practice, HSA is often used for drug delivery and for coating of medical devices: haemodializer membranes, pacemakers, orthopedic titanium implants and catheters that can prevent adhesion of other proteins, platelets and bacteria9-11. It is also exploited as blocking agents by preparing immunoglobulin covered latexes used in agglutination immunoassays12. HSA also serves as a biomarker of many diseases such as cancer, ischemia, rheumatoid arthritis, obesity, etc.13 Because of an essential significance, numerous works were performed in order to determine the adsorption kinetics and isotherms of HSA and analogous bovine serum albumin (BSA) at various substrates. Malmsten14 studied the diffusion-controlled adsorption of HSA adsorption on methylated silica (at pH 7.4, 0.15 M NaCl) using the ellipsometric method. The maximum coverage of the protein determined in this work was equal to 0.5-0.8 mg m-2 depending on the bulk protein concentration varied between 100 and 1000 mg L-1. 3 ACS Paragon Plus Environment

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Thorough measurements of albumin (BSA)

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adsorption kinetics on modified silica

substrates under flow conditions were performed by Wertz and Santore15,16 using the TIRF method. Both the initial mass transfer rates and maximum coverages were determined as a function of the bulk protein concentration and the flow shear rate in the cell. It was demonstrated that the initial adsorption kinetics abruptly decreased for smaller flow rates. On the other hand, the maximum coverage of HSA increased with the bulk concentration that was reflected in the appearance of a pseud-isotherm. This behavior was interpreted as due to a time-dependent molecule spreading upon adsorption. In Ref.17 the influence of pH and ionic strength on the maximum coverage of HSA and other proteins at oxidized silicon wafers was studied using ellipsometry and atomic force microscopy (AFM) imaging. It was observed that the maximum adsorbed amount occurred at pH 4.7 independently of ionic strength. For 0.15 M solution and pH 4.7 the maximum coverage was equal to 3 mg m-2 and. At pH 7 the maximum coverage decreased to 0.7 mg m-2. The adsorption kinetics of HSA on mica under various ionic strengths and pHs was studied in Refs.18-19 by the streaming potential measurements calibrated by AFM and XPS. At pH 3.5 a systematic increase in the maximum coverage of HSA with NaCl concentration from 0.42 to 1.3 mg m-2 was observed, which was attributed to the decreasing range of lateral electrostatic interactions among adsorbed molecules. Kubiak-Ossowska et al21 determined the maximum coverage of bovine serum albumin (BSA) at a silica substrate using the surface plasmon resonance (SPR) technique. The coverage attained a maximum of 1.2 mg m-2 at pH 5-6, and systematically decreased for lower and larger pH values, for example, at pH 7.4 it was equal to 1.0 mg m-2. In a few works, the kinetics of albumin and other protein adsorption was studied using the quartz crystal microbalance (QCM), which enables and in situ measurements at various

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substrates under flow conditions.6 For example, Pomorska et al22 quantitatively determined adsorption/desorption kinetics of HSA at a SiO2 sensor at various ionic strengths (regulated by NaCl) and pH 3.5. The maximum coverage of HSA was equal to 0.35, 0.65, and 1.4 mg m-2 for NaCl concentration equal to 10-3, 10-2, and 0.15 M, respectively A useful technique directly furnishing the dry mass of adsorbed solutes is the optical light mode waveguide spectroscopy (OWLS). This method is characterized by a large sensitivity of the order of 10-2 mg m-2 and allows for a direct online monitoring of adsorption kinetics.23-31 Kurrat et al.29 used this method to determine the kinetics of adsorption of albumins (bovine and human) on the Si(Ti)O2 substrate. The irreversibly adsorbed mass of HSA at pH 7.4 (HEPES buffer, 0.01 M) was equal to 1.7 mg m-2. Höök F. et al.30 measured the adsorption kinetics of three model proteins – human serum albumin, hemoglobin, and fibrinogen on TiO2 surface for 10 mM HEPES buffer, pH 7.4. Three different experimental techniques have been used including OWLS. The average OWLS/ellipsometry mass for HSA was equal to 1.88 mg m-2. Sander et al.31 combined QCM and OWLS techniques to characterize protein adsorption on bare and poly-L-lysine (PLL) modified silica (SiO2). The bovine serum albumin (BSA) was used as reference protein. The maximum ’wet’ mass on SiO2 surface derived from QCM measurements was equal to 5.8 mg m-2 at pH 5, and 2 mg m-2 at pH 7 (for a 10 mM NaCl concentration). The adsorbed mass from OWLS was equal 2.1 and 0.18 mg m-2 at pH 5 and 7, respectively. It is worth underlining that in this interesting study the mass transfer rate for the OWLS cell was determined. However, despite this extensive effort, the understanding of albumin adsorption at solid substrates, especially silica, still remains incomplete. Severe discrepancies persist concerning both the kinetic aspects (initial adsorption rates), the maximum coverage and reversibility of adsorption. For example, at neutral and basic pH range (5-9) the kinetic of

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albumin adsorption was much slower than theoretically expected [6,28,30]. On the other hand, the maximum coverage attains values above the ones predicted for a closely packed monolayer and is subject to a considerable spread.14,15,29,30 Thus, at pH ca. 7 the maximum coverage reported in the literature varies between 0.18 and 3.5 mg L-1. No satisfactory physical explanation of this discrepancy was provided. Therefore, the main goal of this work is to quantitatively evaluate albumin adsorption kinetics at silica substrates using two complementary techniques, i.e., the OWLS method providing the ‘dry’ mass and the QCM method yielding the wet mass. The experimental data are interpreted in terms of a hybrid theoretical approach where the bulk and surface transport of molecules are simultaneously considered in an exact way. This allows to determine the mass transfer rates for the OWLS cell and in consequence the dynamic hydration function for HSA at silica sensor. Additionally, a new theoretical model is formulated for describing the kinetics of HSA adsorption at larger pHs by combining the Smoluchowski theory with the convective diffusion mass transfer theory. Useful analytical equations are derived that can be used for the interpretation of other protein adsorption from unstable solutions.

2. Experimental section The experimental data presented hereafter were obtained for the human serum albumin (HSA) and bovine serum albumin (BSA) both supplied in the form of a lyophilized powder 99% (Sigma) having the nominal fatty acid content of 0.02%. All chemical reagents such as sodium chloride, hydrochloric acid were commercial products of Sigma Aldrich and were used without additional purification. Ultrapure water, was obtained using the Milli-Q Elix&Simplicity 185 purification system from Millipore. The composition of albumin solutions was determined via Gel Filtration Chromatography technique using Superdex 200 - column and via SDS-PAGE electrophoresis in Laemmli system.31 6 ACS Paragon Plus Environment

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The effective bulk concentration of albumin molecules in the stock solutions was spectrophoretically determined according to the procedure described in Ref.33 On the other hand, the bulk concentration of the diluted albumin solutions (1- 10 mg L-1) directly used in the QCM measurements was determined by AFM imaging and enumeration of single molecules adsorbed at mica.18-19 The diffusion coefficient of albumin molecules and microparticles was determined by dynamic light scattering (DLS) using the Zetasizer Nano ZS instrument (Malvern). The hydrodynamic diameter was calculated from the Stokes-Einstein equation. The electrophoretic mobility of albumin molecules was measured using the Laser Doppler Velocimetry (LDV) technique also using the Zetasizer Nano ZS. The zeta potential was calculated from the Henry equation. The pH 3.5 and 7.4 was adjusted by the addition of HCl or NaOH, and the solution ionic strength was regulated by the addition of NaCl. The kinetics of albumin adsorption was determined using the Optical Waveguide Lightmode Spectroscopy (OWLS) and the quartz microbalance (QCM) techniques. The former experiments were performed using the OWLS 210 instrument (Microvacuum Ltd., Budapest, Hungary), equipped with a laminar slit shear flow cell, holding a silica-coated waveguide (OW2400, Microvacuum). A diffractive grating on the surface of the waveguide incouples He−Ar laser light at two well-defined incident angles for the transverse electric (TE) and magnetic (TM) polarization modes33. Adsorption to the waveguide surface alters the interfacial refractive index and, therefore, the incoupling angles of the laser light are monitored. Assuming an optically uniform adsorbed layer, the ‘dry’ mass of adsorbed protein, mOWLS is given as Feijter’s formula34

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mOWLS  ladlayer

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nadlayer  nsolution

 dnadsorbate

dcadsorbate 

(1)

where ladlayer (cm) and nadlayer is the thickness and the refractive index of the adlayer, respectively, dnadsorbate / dcadsorbate is the refractive index increment of the respective adsorbate 0.182 cm3 g−1 for the proteins, and nsolution is the refractive index of the solutions. The adsorbing substrates were planar optical waveguides (OW 2400 from MicroVacuum, Budapest, Hungary) made of a glass substrate (refractive index nS = 1.526) covered by a film of Si0.78Ti0.22O2 of the thickness 170 nm, and the refractive index nF =1.8. A grating embossed in the substrate enables the light to be coupled into the waveguide layer. The sensor surface was coated with an additional layer (10 nm) of pure SiO2. The QCM measurements were carried out according to the standard procedure described in Ref.35-37 using the quartz/silicon dioxide (SiO2) sensors delivered by QSense, Gothenburg, Sweden. The sensors were cleaned before each experiment in a mixture of 95% sulfuric acid (H2SO4) and hydrogen peroxide (30%) in volume ratio 3:2 for 10 minutes. In the next step, the sensor was rinsed by deionized water at 80° C for 30 min. and dried out in a stream of a nitrogen gas. The roughness of sensors was examined by atomic force microscopy (AFM) imaging carried out in a semi-contact mode under ambient conditions. It was confirmed that the sensors were smooth exhibiting the root mean square roughness equal to 0.80 nm. The adsorbed HSA molecule mass per unit area (coverage), hereafter referred to as the QCM mass (coverage) was calculated from the Sauerbrey equation35-37

m  CQ

f no

(2)

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where m is mass change, f is the frequency change, no is the overtone number and CQ is the mass (coverage) sensitivity constant equal to 0.177 mg m-2 Hz for the 5 MHz AT-cut quartz sensor.35-40 All experiments were performed at the temperature of 298 K. 3. Results and discussion 3.1. Bulk HSA and substrate characteristics The physicochemical properties of HSA molecules comprising the electrophoretic mobility and zeta potential for various ionic strengths and pHs were acquired by the LDV method. At pH 3.5 the electrophoretic mobility was equal to 2.6 and 1.1 m cm (s V)-1 for NaCl concentration of 10-3 and 0.15 M, respectively, which corresponds to the zeta potential of 49 and 19 mV, respectively (calculated from the Henry equation). At pH above 5.6 and 7.4 the electrophoretic mobility of HSA molecules becomes negative (for details see the Supporting Information section). Also, the stability of HSA solutions was determined via the DLS measurements of the diffusion coefficient that was recalculated for the hydrodynamic diameter. These data available in the Supporting Information section confirm that the HSA solutions are only stable at pH 3.5 and NaCl concentration 10-3 to 0.15 M. In this case, the diffusion coefficient was equal to 6.5×10-7 cm2 s-1 independently of NaCl concentration that corresponds to the hydrodynamic diameter equal to 7.5 nm (calculated from the Stokes–Einstein equation). At pH 5-6 and 7.4 the hydrodynamic diameter of HSA molecules monotonically increased with time. One should mention that an exact kinetic of the hydrodynamic diameter changes in this cases could not be acquired because of the limitation of the DLS technique. The zeta potential of silica substrates was determined using the streaming potential measurements according to the procedure described in Ref.19 At pH 3.5 it was equal to -30 and -15 mV for NaCl concentration of 10-3 and 0.15 M, respectively. At pH 7.4 it was equal to -75 and -40 mV for NaCl concentration of 10-3 and 0.15 M, respectively. 9 ACS Paragon Plus Environment

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Thorough AFM characteristics of the QCM and OWLS sensor topology were also performed (Supporting Information Section). For the OWLS, the average height and width of the grooves was equal to 10 nm, and 320 nm, respectively whereas the average distance between the groove center lines was equal to 460 nm. By virtue of this geometry, the roughness factor of the OWLS sensors characterizing the increase in the effective surface area was equal to 1.02.

3.2. The kinetics of HSA adsorption – low coverage regime The first series of OWLS measurements was devoted to determining the mass transfer rate constant kc for the cell that is a parameter of basic significance for the interpretation of experimental data obtained at different pHs for unstable (aggregating) systems. Primarily, in these experiments, the dependence of the HSA coverage expressed in mg m-2 (denoted hereafter as Γ ) on the adsorption time was measured at pH 3.5, various bulk concentration of the solution, i.e., 3, 5 and 10 mg L-1 and NaCl concentration of 10-3,10-2 and 0.15 M. It was confirmed that after a short transition time (lasting typically a few seconds), the coverage vs. the time dependencies became linear and proportionally increased with the bulk protein concentration. In order to precisely determine kc, such primary kinetic runs are expressed as the dependence of     tr  / cb on the normalized adsorption time t  ttr (where  tr is the coverage of HSA molecule adsorbed during the transient state lasting ttr ). The experimental data for various bulk HSA concentrations and ionic strengths transformed in this way are presented in Fig. 1. One can observe that these results can be fitted by a linear relationship characterized by the slope equal to 1.2×10-3 ± 5×10-5 L m-2 s-1 = 1.2×10-4 ± 5×10-6 cm s-1 that corresponds to the mass transfer rate constant kc . It should be mentioned that because more than ten independent experimental runs were performed in order to determine the masse

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transfer constant, the precision of its determination is relatively large, about 4% as indicated above. Such a behavior indicated that HSA adsorption at the silica sensor for this low coverage regime was bulk transport controlled.

Fig. 1. Dependence of the normalized HSA coverage at the silica sensor (Γ - Γtr) / cb on the normalized adsorption time t – ttr determined by OWLS for different protein bulk concentrations, pH 3.5, NaCl concentrations equal to 0.01 and 0.15 M, flow rate equal to 2.5×10-3 cm3 s-1. The solid line shows the linear fit of experimental data (points).

In Refs.15,16 analogous dependencies as that shown in Fig. 1 were obtained in the case of BSA adsorption at modified silica substrates for the TIRF flow cell. At initial adsorption times the adsorption rate was proportional to the bulk concentration of the albumin and to the cube root of the wall shear rate. It is also interesting to mention that Sander et al31 determined for the OWLS cell of the same geometry the mass transfer rate constant equal to 50.9 ng cm

-2

min

-1

for a 10 mg L-1

solution and flow rate of 0.05 ml min-1. This value corresponds to the mass transfer rate constant kc equal to 8.5×10-5 cm s -1 that corresponds to 1.2×10-4 cm s-1 for the flow rate of

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0.15 ml min-1. As can be noticed, this value matches the mass transfer rate constant experimentally determined in this work for HSA. In other series of experiments the entire HSA adsorption/desorption kinetic measurements were performed at pH 3.5 and different ionic strengths whose main goal was determining the maximum coverage of irreversibly adsorbed molecules. A typical kinetic run acquired for 0.15 M NaCl concentration and the bulk protein concentration of 5 mg L-1 is shown in Fig. 2. It is seen that for the HSA coverage up to 1.1 mg m-2 the adsorption kinetic remains a linear function of time. For t > 10 min the kinetics becomes considerably slower, hence the limiting coverage of ca. 1.4 mg m-2 is attained after 15 min adsorption time. Then, the desorption run was initiated where pure electrolyte having the same ionic strength and pH was flushed through the cell with the same flow rate. One can observe in Fig. 2 that the amount of desorbed HSA is practically negligible amounting to ca. 0.05 mg m-2 during the desorption run lasting 20 min.

1.6

desorption

1.4 1.2 1.0 -2

 [mg m ]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.8 0.6 0.4 0.2 0.0 0

10

20

30

40

time [min] Fig. 2. The kinetics of HSA adsorption on silica determined by OWLS, bulk protein concentration 5 mg L-1, pH 3.5, NaCl concentration 0.15 M, flow rate 2.5×10-3 cm3 s-1. The arrow shows the beginning of desorption run.

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The red solid line shows the theoretical results derived from the RSA model for the irreversible side-on adsorption model and stable solutions.

In order to increase the precision of the maximum coverage determination additional adsorption/desorption kinetic runs were performed that gave the value of 1.3± 0.1 mg m-2 at 0.15 M NaCl (see Table 1). It is interesting to mention that this agrees within error bounds with the maximum HSA coverage previously determined by QCM for a silica sensor equal to 1.4±0.05 mg m-2 [21] and with theoretical predictions for a side-on adsorption of HSA molecules equal to 1.2 mg m-2.41 Analogous measurements were carried out for NaCl concentration of 10-3 and 10-2 M where the irreversibly bound HSA coverage was equal to 0.5 ± 0.1 and 0.65 ± 0.1 mg m-2, respectively (see Table 1). As suggested in previous works18-20,22,41 the decrease in the maximum coverage of HSA on silica for low NaCl concentration (ionic strength) can be interpreted in terms of the increasing range of the lateral electrostatic interactions among adsorbed molecules. Table 1. The maximum coverage of irreversibly adsorbed HSA determined by various methods, pH 3.5, various NaCl concentrations.

NaCl concentration [M]

κ dH/2

Maximum coverage OWLS

Maximum coverage QCM*

[mg m-2]

[mg m-2]

Maximum Maximum coverage coverage streaming theoretical*** potential** [mg m-2] [mg m-2]

10-3

0.44

0.50± 0.1

0.35± 0.05

0.42

0.35

10-2

1.2

0.65± 0.1

0.70± 0.05

0.66

0.62

0.15

4.3

1.3± 0.1

1.4± 0.05

1.3

1.2

κ -1 is the electric double-layer thickness * Ref.22, silica QCM sensor ** Ref.19, mica. *** Theoretical results calculated using the effective hard particle, random sequential adsorption (RSA) model.7,41

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The experimental data derived from OWLS were theoretically interpreted using the hybrid random sequential adsorption (RSA) model where the coupling of the bulk transport of HSA molecules governed by the mass transfer rate constant kc with the surface transport governed by the blocking function B    is considered in a rigorous manner.22,36,41 The results obtained by a numerical integration of the transport equation derived within the scope of this model (see the Supporting Information section) are shown in Fig. 2 as solid line. One can observe that they agree for the entire range of coverage with the experimental results derived by OWLS, which confirms a quantitative character of this technique. It is useful to compare the OWLS results with adsorption kinetic data obtained by QCM for the silica substrate under the same physicochemical conditions that can furnish quantitative information about the dynamic hydration of HSA molecules as a function of its coverage. The results obtained for pH 3.5, NaCl concentration 0.15 M, bulk solution concentration 5 mg L-1 , kc = 1.2×10-4 cm s-1 are presented in Fig. 3.

4

1

3

2

 [mg/ m ]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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2

2 1

0 0

2

4

6

8

10

12

time [min]

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Fig. 3. The kinetics of HSA adsorption on silica determined by QCM (green curve number 1, flow rate 7.5x10-4, kQ = 3.1×10-4 cm s-1, kc = 1.2×10-4 cm s-1 and OWLS (blue solid points, kc = 1.2×10-4 cm s-1), pH 3.5, NaCl concentration 0.15 M, bulk solution concentration 5 mg L-1.

One can observe that for the adsorption time below 4 min both the OWLS and QCM kinetics remain a liner function of time with the ratio of their slopes equal to 2.7. It should be mentioned that this parameter is defined as the water factor w30, i.e.,

w

Q 

(1)

where  Q is the coverage sensed by QCM, due to the dry protein mass and the specifically and hydrodynamically bound water mass. Knowing the water factor, the dynamic hydration function of HSA molecules is defined as30,35,42 H  1

   1 Q w

(2)

Hence, for the low coverage regime where both the QCM and OWLS coverages remain linear functions of time, the water factor and the hydration function are given by

w0 

kQ kc

(3)

k H0  1 c kQ where kQ is the wet mass rate constant derived from QCM measurements.

The dependencies of the water factor and the hydration function on the dry mass derived comparing the QCM and OWLS results are presented in Fig. 4 as solid points. For zero dry coverage, the water factor and the hydration function are equal to 2.7 and 0.63, respectively and these parameters monotonically decrease with the coverage. One can observe that these results agree with previously derived in Ref.22 where the QCM wet mas was compared with the dry mass theoretically calculated by solving the mass transfer equation (solid line in Fig. 4). These data were interpolated with a precision of ± 0.02 by the expression22 15 ACS Paragon Plus Environment

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H  H 0 1  C2  2 

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(4)

where H 0  0.62 is the hydration in the limit of zero coverage and C2 = 0.186 m4 mg-2.

Fig. 4. The water factor w (left hand side vertical axis) and the dynamic hydration function for HSA vs. the dry coverage  ; the triangular points show the QCM/OWLS experimental results for the water factor, the circular points show the QCM/OWLS experimental results for the hydration function and the solid line shows the interpolated QCM/RSA results for the water function, pH 3.5 and 0.15 M NaCl.

It should be mentioned that analogous decrease in the water factor and hydration function with the coverage was previously reported in Ref.35 for fibrinogen, and in Ref.42 for avidin and streptavidin adsorption at a silica sensors. This behavior was interpreted as due to the overlapping of the hydrodynamically coupled water shells surrounding adsorbed molecules. These results confirm that the adsorption kinetics of HSA at pH 3.5, where its solutions remain stable over long time period, proceeds according to the irreversible mechanism with the maximum rates governed by the bulk transport and can be described by the hybrid RSA 16 ACS Paragon Plus Environment

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model.

In consequence, these results can be exploited as useful reference data for a

quantitative analysis of HSA adsorption at other pHs, where the protein solution instability can play a significant role. This effect is well illustrated in Fig. 5 showing the OWLS adsorption kinetic runs obtained for pH 5.6 and 7.4 and NaCl concentration of 0.15 M.

1.2

desorption

1.0

1

0.8

2

-2

 [mg m ]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.6

desorption

0.4

0.2

0.0 0

10

20

30

40

50

60

time [min] Fig. 5. The kinetics of HSA adsorption on silica determined by OWLS, bulk concentration cb = 10 mg L-1, NaCl concentration 0.15 M, flow rate 2.5 ×10-3 cm3 s-1: red solid points pH 5.6; blue solid points pH 7.4, the arrows show the beginning of desorption runs. The dashed line shows the maximum adsorption kinetics observed at pH 3.5, i.e. Γ = kc cb t.

One can observe that the initial adsorption kinetics significantly deviates from the limiting kinetics predicted by the bulk transport controlled mechanism (shown as dashed line in Fig. 5) especially at pH 7.4, where it is almost seven times smaller. Also, the maximum coverage of irreversibly adsorbed protein molecules equal to ca. 0.7 mg m-2 is significantly smaller than the maximum coverage determined at pH 3.5 (1.3 mg m-2). It should be mentioned that an analogous behavior was previously observed by Sander et al31 for BSA adsorption at silica senor. Also, in Ref.29 similar trends were confirmed, i.e., the initial adsorption rate of HSA and BSA at silica/titania substrate at pH 7.4 (in HEPES 17 ACS Paragon Plus Environment

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buffer) measured by OWLS was more than one order of magnitude smaller than the theoretically predicted rate for bulk transport controlled deposition. This effect was attributed to a significant desorption of albumin molecules at pH 7.4. However, given that desorption is always negligible at low coverage range, the initial deposition rate should be the same as for irreversible systems, i.e., it should be controlled by the bulk transport. We postulate that the decrease in the adsorption kinetics of albumins at pH 7.4, which was also observed in other works6,29,31, is caused by the aggregation of its solutions rather than by the desorption effect. In order to prove this hypothesis, a quantitative approach was developed in this work combining the Smoluchowski theory of aggregation with the convective diffusion mass transfer theory (see the Supporting Information section). By virtue of this approach, the following equation was formulated describing the kinetics of adsorption from aggregating solutions



2/3

   0  kc cbta   Dl  l  l 1

t ta

t0 t a

 l 1 d l 1 1   

(5)

where  0 is the net coverage of aggregates at the time t equal t0 (i.e., at the beginning of the adsorption run), ta is the characteristic aggregation time of the protein solution, Dl are the normalized diffusion coefficients of the aggregates and   t ta is the normalized adsorption time. If only single protein molecules can adsorb, D1 = 1, and the adsorption kinetics is given by

 1  t    10  kc cb

t  t0  ta  t0  ta  t 

(6)

where  10 is the coverage of single molecules at t  t0 . 18 ACS Paragon Plus Environment

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Eqs.(5-6), which describe the dry mass of molecules, are suitable for the analysis of OWLS experimental data. For the interpretation of the QCM data one should introduce the dry mass water factor defined for single molecules by Eq.(4), which gives the formula

 Q t   w  1   1 t 

(7)

It is interesting to mention that the protein adsorption rate at t  t0 is given by (Supporting Information section) ta  d  0    kc cb w1 2 2 t a  t0  dt t t0 2

(8)

where w10 is the water factor for zero coverage of single molecules. Eq.(8) can be converted to the form

 k ta  t0  Q'  kQ 

12     1   

(9)

where kQ  kc w10 is the QCM mass transfer rate constant for stable systems and kQ'  kc w10 is the QCM mass transfer rate constant for aggregating systems. Eq.(9) enables to calculate the aggregation time of the suspension if the initial time t0 is known (defined as the time from preparing the protein solution to the start of the adsorption run). In Fig. 6 (part a) the OWLS experimental data acquired at pH 7.4 (0.15 M NaCl) are compared with theoretical results derived from Eq.(6). One can observe that an adequate fit for the entire range of adsorption time up to 30 min is attained if the aggregation time is assumed equal to 5 min. It is also seen that the theoretical results predicted for stable HSA 19 ACS Paragon Plus Environment

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solutions (dashed line in Fig. 6a) considerably overestimate the experimental data. This effects suggests that the aggregation model can be useful for analyzing protein adsorption from unstable solutions.

“a”

1

0.8

2

0.6 -2

 [mg m ]

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0.4

0.2

0.0 0

5

10

15

20

25

30

t [min]

“b”

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Fig. 6. The kinetics of HSA adsorption on silica: Part a: red solid points show the OWLS experimental results obtained for the bulk protein concentration 10 mg L-1, pH 7.4, NaCl concentration 0.15 M, flow rate 2.5 ×10-3 cm3 s-1, the dashed line 1 denotes the theoretical RSA results for stable suspensions, the green solid line 2 denotes the theoretical results calculated for aggregating solutions. Part b: green solid points show the QCM results obtained for the bulk protein concentration 10 mg L-1, pH 7.4, NaCl concentration 0.15 M, flow rate 2.5×10-3 cm3 s-1, the dashed line shows the theoretical RSA results for stable suspensions, the solid line 1 denotes the theoretical results calculated for aggregating solutions (adsorption of single molecules alone) and the solid line 2 denotes the theoretical results calculated for aggregating solutions (adsorption of single and dimeric molecules).

In Fig. 6 (part b) analogous QCM experimental data acquired at pH 7.4 (0.15 M NaCl) are compared with theoretical results derived from Eq.(7). One can observe that for adsorption time below 10 min the theoretical results postulating single molecule adsorption agree with the experimental data. For lager time, a better fit is attained if the adsorption of two molecule aggregate is considered (curve 2 in Fig. 6b). It should be mentioned that analogous runs proving significant protein aggregation have been recorded for other ionic strengths and for bovine serum albumin solutions shown in the Supporting Information section. These results confirm that at pH 5.6 and 7.4 the albumin solutions are unstable that diminish their adsorption kinetics and the maximum coverages. Because the characteristic aggregation time at pH 7.4 and 0.15 M NaCl is ca. 5 min this renders the adsorption experiments under these conditions rather irreproducible. 21 ACS Paragon Plus Environment

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CONCLUSIONS OWLS measurements theoretically interpreted in terms of the hybrid random sequential adsorption (RSA) model enabled to determine the mass transfer rate constant kc, which is a universal quantity defining the maximum adsorption rates in the cell. Also, in these experiments, the maximum coverage of HSA at silica was determined, which agrees with previous results reported in the literature and with theoretical predictions. Therefore, the results obtained at pH 3.5 can be exploited as useful reference data for a quantitative analysis of HSA adsorption at other pHs. A comparison of the OWLS with the QCM results made it possible to determine the water factor and the dynamic hydration function of albumin molecules at silica sensors. It is shown in this way that the QCM/OWLS and the QCM/RSA hydration functions agree with each other for the entire range of albumin coverage. Exploiting the experimental data acquired at pH 3.5 it was shown that at pH 5.6 and 7.4 the albumin solutions are unstable that decreases their initial adsorption kinetics and the maximum coverages. This effect was quantitatively interpreted in terms of an approach, which combines the Smoluchowski theory of aggregation with the convective diffusion mass transfer theory. New analytical equations were derived adequately describing the kinetics of HSA adsorption at larger pHs that can also be used for the interpretation of other protein adsorption from unstable solutions.

ASSOCIATED CONTENT Supporting information Stability of HSA solutions at various pHs, electrophoretic mobility and zeta potential of HSA solutions, AFM characteristics of OWLS and QCM sensors, adsorption kinetics of stable and aggregating protein solutions This material is available free of charge via the Internet at http://pub.acs.org AUTHOR INFORMATION Corresponding Author: 22 ACS Paragon Plus Environment

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e-mail: [email protected] Founding sources This work was financially supported by the National Research Center Research projects: UMO-2015/19/B/ST5/00847, and UMO-2014/13/D/ST4/01846 Notes The authors declare no competing financial interest.

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Table of Contents Human serum albumin adsorption kinetics on silica: influence of protein solution stability Monika Wasilewska, Zbigniew Adamczyk*, Agata Pomorska, Małgorzata Nattich-Rak, Marta Sadowska Jerzy Haber Institute of Catalysis and Surface Chemistry Polish Academy of Science, Niezapominajek 8, 30-239 Cracow, Poland. These authors contribute equally

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