Mechanism of immonoglobulin G adsorption on mica-AFM and electrokinetic studies

Colloids and Surfaces B: Biointerfaces 118 (2014) 57–64

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Mechanism of immonoglobulin G adsorption on mica-AFM and electrokinetic studies ˛ Maria Dabkowska, Zbigniew Adamczyk ∗ J. Haber Institute of Catalysis and Surface Chemistry Polish Academy of Science, Niezapominajek 8, 30-239 Cracow, Poland

a r t i c l e

i n f o

Article history: Received 14 December 2013 Received in revised form 10 February 2014 Accepted 26 February 2014 Available online 19 March 2014 Keywords: AFM measurements of immunoglobulin adsorption Immunoglobulin G adsorption on mica Monolayers of immunoglobulin G on mica Streaming potential of immunoglobulin G covered mica Zeta potential of immunoglobulin G covered mica Stability of IgG monolayers on mica

a b s t r a c t Adsorption of immunoglobulin G (IgG) from aqueous NaCl solutions of the concentration 10−3 –0.15 M on mica was studied. Initially, the kinetics was evaluated at pH 3.5 by direct AFM imaging. A monotonic increase in the maximum coverage of IgG with NaCl concentration was observed. These results were interpreted in terms of the theoretical model postulating an irreversible adsorption of the protein governed by the random sequential adsorption (RSA) model. Additionally, IgG adsorption and desorption was studied under in situ conditions, with streaming potential measurements. These measurements revealed that the maximum coverage of irreversibly adsorbed IgG varies from 0.37 mg m−2 for 10−3 M, NaCl to 1.2 mg m−2 for 0.15 M, NaCl. The significant role of ionic strength was attributed to the lateral electrostatic repulsion among adsorbed IgG molecules, positively charged at this pH value. These experimental results confirmed that monolayers of irreversibly bound IgG can be produced by adjusting ionic strength of the protein solution. In further experiments the stability and acid base properties of such monolayers were studied using the streaming potential method. It revealed that the monolayers were stable against pH cycling for the range from 3.5 to 9.5. The isoelectric point of mica supported IgG monolayers was 5.9, similar to derived from the micro-electrophoretic measurements in the bulk (5.8). Beside significance for basic sciences, the results indicate that thorough characteristics of IgG can be acquired via streaming potential measurements using microgram quantities of the protein. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Antibody adsorption on solid interfaces is the most relevant step in preparation of various immunoassays, immuno-biosensor, chromatographic immuno-affinity columns and biomaterials development. Immunoglobulin G (IgG), synthesized and secreted by plasma B cells, is an abundant protein present in blood at a concentration of ca. 1 g L−1 [1]. Its molar mass calculated from this amino acid composition is 150 kDa [2]. Most of the information about the crystal structure of intact human IgG has been obtained by electron microscopy studies of its various fragments and has been determined by atomic resolution in numerous investigations [3]. The interpretation of electron density map allowed to establish that IgG molecule has a Y-shape, and consists of two Fab fragments used for binding specific antigens and Fc fragment containing antibody effector function (see Table 1) [4]. Because the amino acid sequence in the arms differs

∗ Corresponding author. Tel.: +48 12 6395104; fax: +48 12 4251923. E-mail address: [email protected] (Z. Adamczyk). http://dx.doi.org/10.1016/j.colsurfb.2014.02.053 0927-7765/© 2014 Elsevier B.V. All rights reserved.

for various antibody molecules, each antibody can specifically bind to one epitope, i.e., the part of an antigen that is recognized by the immune system. An important factor initiating activation process is the spatial orientation of the domains of the IgG molecule [5]. Flexibility in the hinge region allows the two Fab arms to assume a variety of orientations in space relative to the Fc part depending on the IgG subclasses: IgG1, IgG2, IgG3, IgG4. Because of its significance, especially for biosensing and immunological assays, IgG adsorption on various surfaces was widely studied by a multitude of techniques [6–18] both in kinetic and equilibrium aspects. For example, adsorption kinetic measurements for two monoclonal mouse IgGs on pure and methylated silica surface were performed by Buijs et al. [12,13] using FTIR and reflectometry. Ortega-Vinuesa et al. [9], studied adsorption of polyclonal IgG on silicon plates by ellipsometry as a function of the solution pH and ionic strength, for the bulk concentration of 20 mg L−1 . Awsiuk et al. [15,16] studied adsorption of rabbit and mouse IgGs on silicon Si3 N4 surfaces modified with (3aminopropyl)triethoxysilane coated with for 0.05 M phosphate buffer, at pH 7.4 using XPS.

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Table 1 Shapes of the IgG molecule and corresponding geometrical cross-section areas Sg for various orientations. Model, shape of molecule, dimensions in nm

Sg head on, end on [nm2 ]

Sg side on [nm2 ]

Remarks, Refs.

Chemical model

Mw = 148–150 kD Refs. [21–23]

Crystallographic shape

Ref. [21]

Equivalent sphere model

Cylindrical model

95

95

107 (max.)a

Ref. [24]

130

Ref. [2]

163

Ref. [25]

39 (min.)b

Crystallohydrodynamic model

102 (max.)a 51 (min.)b

a b

Fab fragment approximated by an ellipsoid of dimenisons 11.3 nm × 7.1 nm × 4.6 nm Fc fragment approximated by an ellipsoid of dimenisons 10.3 nm × 9.3 nm × 5.0 nm

For the expanded conformation. For the collapsed conformation.

Kinetics of IgG adsorption on methylated silica for pH 7.4 and I = 0.15 M was studied in Ref. [17] using ellipsometry. Recently, the AFM technique was used [18–20] to derive information about the binding strength of IgG and the density of monolayers. However, no quantitative data on IgG coverage and adsorption kinetics were given in these works. Adsorption of various IgGs on polymeric carrier particles, which is the most relevant step in preparing immunological tests [26] was also widely studied [2,27,28]. It should be mentioned that a significant spread in the maximum coverage of IgGs was reported in these works with the lowest and highest value equal to 0.9 and 4.5 mg m−2 , respectively. The discrepancy is probably due to use of indirect experimental methods and diversity of the substrate surfaces varying in respect to surface roughness and hydrophobicity. This prevented one from interpreting these interesting experimental data in terms of theoretical models. Therefore, despite of the considerable

experimental effort, the mechanism of IgG adsorption is still not fully understood, mainly because of the lack of experimental methods providing direct information about the adsorption kinetics under in situ conditions. In view of this deficiency, the motivation this work is to evaluate the mechanism of IgG adsorption on mica representing a model of hydrophilic substrate of well-defined and controlled surface properties. The main task would be to elucidate the irreversibility issue by determining the IgG adsorption and desorption kinetics a function of ionic strength using the streaming potential measurements. The use of mica also allows one to directly determine the absolute coverage of IgG via AFM imaging of single protein molecules. In this way, one can properly calibrate the streaming potential measurements that can be effectively used for determining the acid base properties of IgG monolayers and their stability. This has a vital significance since analogous bulk measurements done for example by micro-electrophoresis require orders of

˛ M. Dabkowska, Z. Adamczyk / Colloids and Surfaces B: Biointerfaces 118 (2014) 57–64

magnitude higher quantities of the protein than the streaming potential measurements. It should be mentioned that systematic measurement of this type were not reported in the literature.

2. Materials and methods In our studies, a polyclonal sheep immunoglobulin (IgG) was used (Abcam, United Kingdom) supplied as an aqueous stock suspension having the nominal concentration of 1.82 g L−1 . The purity of the IgG samples was checked by dynamic surface tension measurements and the SDS-PAGE electrophoresis in Laemmli system [29] using non-reducing sample buffer and 12% polyacrylamide gel [30]. These filtered stock solutions of IgG were diluted to a desired bulk concentration (usually 1-500 mg L−1 ) prior to each experiment. Ruby muscovite mica obtained from Continental Trade (Poland) was used as a substrate for polyclonal immunoglobulin G adsorption measurements. The solid pieces of mica were freshly cleaved into thin sheets prior to every experiment. Water was purified using a Millipore Elix 5 apparatus. Chemical reagents (sodium chloride, hydrochloric acid) were commercial products of Sigma–Aldrich (USA), and used without further purification. The experimental temperature was kept constant at 293 ± 0.1 K. The diffusion coefficient of IgG was determined by dynamic light scattering (DLS), using the Zetasizer Nano ZS Malvern instrument (United Kingdom). The micro-electrophoretic mobility of protein solution was measured using the Laser Doppler Velocimetry (LDV) technique using the same Malvern device. Additionally, the isoelectric point of IgG was determined using isoelectric focusing (IEF) with PhastGel media which is a homogeneous polyacrylamide gel containing Pharmalyte carrier ampholytes (pH range 3–9), and silver staining (Sigma–Aldrich). Streaming potential of bare and IgG covered mica was determined using a home-made apparatus previously described [31–33]. Knowing the streaming potential, the zeta potential of the substrate covered by protein molecules ( i ) was calculated from the Smoluchowski’s formula. The procedure for determining the zeta potential of protein covered mica consisted of three stages: measuring the reference zeta potential of bare mica in pure electrolyte, formation of IgG monolayer of controlled coverage in situ under the diffusion controlled conditions, rinsing the cell by pure electrolyte and measuring the streaming potentials for IgG covered mica. The surface concentration of IgG in monolayers adsorbed on mica was determined by AFM imaging in air using the NT-MDT Solver BIO (Russia) device with the SMENA SFC050L scanning head. All measurements were performed in semi-contact mode by using high resolution silicon probes (NT-MDT ETALON probes, HA NC series, polysilicon cantilevers. The number of adsorbed IgG molecules was determined using scan areas of 0.5 ␮m × 0.5 ␮m. Typically, 1000 IgG molecules were counted over a few randomly chosen areas over the mica sheet, which ensures a relative precision of these measurements below 5%. The AFM measurements were performed under air conditions, therefore considerable attention was devoted to elaborate an appropriate experimental procedure of drying the immunoglobulins adsorbed on mica. The water film could be evaporated from such hydrophilic monolayers under controlled humidity and temperature in a continuous way without forming drops. This minimized the action of meniscus forces and possible deformation of monolayers.

59

3. Results and discussion 3.1. IgG and mica substrate characteristics The diffusion coefficient of IgG was determined by DLS in NaCl solutions for concentration range 10−3 –0.15 M and for pH range from 3.5 to 11, regulated by an addition of HCl or NaOH (T = 293 K). For pH 3.5 the diffusion coefficient of IgG assumed an average value of 3.9 × 10−7 cm2 s−1 . An almost identical value of 3.89 × 10−7 cm2 s−1 was reported by Jøssang et al. [24] for human IgG at pH 7.6. The hydrodynamic diameter of the protein was calculated using the Stokes–Einstein dependence dH =

kT 3D

(1)

where dH is the hydrodynamic diameter, k is the Boltzmann constant, T is the absolute temperature,  – is the dynamic viscosity of water and D is the diffusion coefficient of IgG. Using the above diffusion coefficient one can calculate from Eq. (1) that the hydrodynamic diameter of IgG equals to 11 nm. Similar values can be derived from the sedimentation coefficient measurements of Warner and Schumaker [34] from which one can predict that for rabbit IgG, dH = 11.4–11.8 nm for pH 3–4, and from Carrasco et al. measurements [25] where dH = 11.5 nm for IgG1 and dH = 12 nm for IgG2. The electrophoretic mobility e of IgG solutions was measured using the micro-electrophoretic method. Values of e for various ionic strength and pH are collected in Table 2. As can be seen, the electrophoretic mobility of IgG at pH 3.5 is positive and equal to 1.25 and 0.68 ␮m cm (Vs)−1 for NaCl concentration of 10−3 and 0.15 M, respectively (see Table 2). Knowing the electrophoretic mobility, one can calculate the average number of free (electrokinetic) charges per molecule from the Lorenz–Stokes relationship [32,35] Nc =

30 dH e 1.602

(2)

where dH is expressed in nm, e is expressed in ␮m cm (Vs)−1 and Nc is expressed as the number of elementary charges (e) per molecule. It should be noted that |e| = 1.602 × 10−19 C. It should be mentioned that Eq. (2) remains especially accurate if the double-layer thickness is comparable or smaller than the protein characteristic dimension, e.g., its hydrodynamic diameter. Using Eq. (2) one can calculate that for pH 3.5, Nc = 6.3 for I = 10−3 M NaCl, and Nc = 4.5 for I = 0.15 M NaCl (see Table 2). Knowing the electrophoretic mobility, the zeta potential  p of IgG can be calculated from the Henry’s relationship =

 e εf (a)

(3)

where f(a) is the dimensionless Henry function, a is the characteristic protein dimension, for example the hydrodynamic radius dH /2, 1/2



−1 = (εkT/2e2 I) is the double-layer thickness, I = 12 i ci zi2 is the ionic strength, ci are the ion concentrations and zi are the ion valences. The dependence of the  p on pH for 10−3 M, 10−2 M and 0.15 M NaCl is graphically presented in Fig. 1. As can be seen, for all ionic strengths,  p vanishes at pH = 5.8 (Fig. 1), which can be identified as the isoelectric point of the IgG (denoted by iep, hereafter). Slightly lower values of iep (5.6) reported in the literature [27,28] are probably due to use of various subclasses of polyclonal antibodies. On the other hand, the zeta potential of the mica substrate, denoted by  i , was determined via the streaming potential measurements. It varied between −60 and −30 mV for 10−3 and 0.15 M,

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60

Table 2 Electrophoretic mobilities, electrokinetic charges and zeta potentials (calculated from the Henry’s model) of IgG for various ionic strengths and pHs. pH

NaCl concentration [M]

Number of electrokinetic charges

Electrophoretic mobility [␮m cm (Vs)−1 ]

Zeta potential [mV] (Henry model)

3.5

10−3 10−2 0.15

1.25 0.98 0.68

6.3 5.6 3.8

7.4

10−3 10−2 0.15

−0.65 −0.63 −0.53

−3.7 −3.6 −3.1

−13 −11 −8.5

9.1

10−3 10−2 0.15

−0.79 −0.8 −0.68

−4.5 −4.6 −3.9

−16 −14 −11

NaCl (at pH 3.5). Knowing the zeta potential, one can calculate the electrokinetic (uncompensated charge) of mica using the Gouy–Chapman relationship [32,35] 1/2

0 =

(8εkTnb ) 0.1602

sin h

 e 

(4)

2kT

where  0 is the electrokinetic charge density expressed in e nm−2 and nb is the number concentration of ions expressed in m−3 . The zeta potentials and the electrokinetic charge densities of mica for various ionic strength and pH are given in Table 1S (supporting information). 3.2. Kinetics of IgG adsorption The direct AFM imaging method was used to quantitatively determine IgG adsorption kinetics on mica. Its monolayers were produced under diffusion controlled transport for low bulk concentration of the protein (1–2 mg L−1 ) and various ionic strength according to the above described procedure. Typical micrograph of IgG monolayer obtained for pH 3.5 and 0.15 M NaCl are shown in the inset of Fig. 2. As can be seen, IgG molecules appear as isolated entities that enables their enumeration by an image analyzing software. In this way the number of molecules per unit area (surface concentration denoted by N) can be directly determined by taking averages from various surface areas randomly chosen over the mica substrate. It is convenient to express N as the number of molecules per a square micrometer, i.e., in ␮m−2 . It should be mentioned that by determining the

24 18 13

surface concentration in this way no information about protein size, shape and surface area is required, which makes the above approach simple and reliable. By determining N as a function of adsorption time, one obtains experimental kinetic dependencies, which are shown in Fig. 2 for various ionic strengths. Because IgG adsorption occurred under the diffusion controlled transport, the experimental data are expressed as the dependence of N on the square root of adsorption time t1/2 . As can be seen, these results exhibit two characteristic features: the surface concentration increases linearly with t1/2 for adsorption time below 25 min (t1/2 = 5 min. ½ ) and the maximum coverage attained for longer times increases significantly with ionic strength. Accordingly, it was determined that the maximum coverage equals 1500 ␮m−2 for 10−3 NaCl and 2400 ␮m−2 for 10−2 M, NaCl. However, because of a limited AFM image resolution, resolution, the coverage for 0.15 M, NaCl could not be determined, although it is definitely higher than 4000 ␮m−2 . As previously done in the case of the adsorption of human serum albumin on mica [36], the results shown in Fig. 2 were theoretically interpreted in terms of the coarse-graining model [37]. According to this approach, the bulk transport of proteins is described by the phenomenological continuity equation. The boundary conditions for this equation at the substrate surface are formulated in an analytical form by interpolation of the Monte-Carlo type simulation performed according to the random sequential adsorption (RSA) model [38]. The non-linear mass transfer equation

8000

40 1

30

N [um-2]

2 3

20

0.45 4000

10

0.30

2

0

2000

3

0.15

p

[mV]

0.60

1

6000

-10

0

-20

0

5

10

15

20

25

30

t1/2 [min1/2]

-30 -40 2

3

4

5

6

7

8

9

10

11

pH Fig. 1. Zeta potential  p of IgG as a function of pH at T = 293 K. The points denote experimental values determined by micro-electrophoresis: 1 – (䊉) 10−3 M NaCl, 2 – () 10−2 M NaCl, 3 – () 0.15 M NaCl. The solid lines denote non-linear fits. The error bars are calculated as standard deviation of ten various measurements.

Fig. 2. Dependence of the surface concentration of polyclonal IgG, N (␮m−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 IgG molecules for I = 0.15 M, cb = 2 mg L−1 for 1 – () I = 0.15 M NaCl, 2 – () I = 10−2 M NaCl, and 3 – (䊉)I = 10−3 M NaCl. The solid line shows the exact theoretical results obtained by the numerical solution of the diffusion equation using the random sequential adsorption model. The inset shows the IgG monolayer on mica for N = 1470 ␮m−2 . The error bars show the standard deviation of the Poisson distribution of IgG molecule numbers over various surface areas.

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Mw N Av

0.25

0.50

0.75

1.00

1.25

60 "a"

40 bulk

20

0

-20

-40 mica

-60 0

1000

2000

3000

4000

5000

-2

N[ m ]

(5)

where Av is the Avogadro’s constant and Mw is the molar mass of the protein. However, a disadvantage of using compared to the surface concentration N is that its calculation requires the knowledge of the effective molecular mass of a protein, which depends on the unknown degree of hydration. By neglecting hydration, one obtains from Eq. (5) the maximum coverage of IgG equal to 0.37 and 0.56 mg m−2 for the ionic strength of 10−3 and 10−2 M, NaCl, respectively, whereas for 0.15 M one can only state that the maximum coverage exceeds 1.0 mg m−2 (see Table 3). Because of the limited precision of the AFM data, some series of streaming potential measurements were performed in order to determine the adsorption and desorption kinetics of IgG under in situ conditions. This allows one to determine in a reliable way the maximum coverage of IgG for various ionic strengths. 3.3. Streaming potential of IgG – covered mica

-2

[mg m ] 0.25

0.50

0.75

1.00

1.25

60

"b"

40

bulk

20

[mV]

= 1015

[mg m-2]

[mV]

formulated in this model is numerically solved using the generalized finite-difference scheme as described in Refs. [37,39]. One can observe in Fig. 2 that the theoretical results derived by numerical solution of the governing mass transfer equation well describe the experimental data. This suggests that the direct AFM enumeration of adsorbed IgG molecules is a valid procedure for determining its surface concentration. The significant role of ionic strength demonstrated in Fig. 2 can be explained in terms of the lateral electrostatic repulsion among adsorbed IgG molecules, positively charged at this pH 3.5. Analogous effects were previously observed for colloid particles deposition on solid substrates, for example, gold nanoparticles [40], dendrimers, [41], HSA [36] and fibrinogen [42]. These effects were quantitatively interpreted in terms of the RSA model by exploiting the effective interaction range concept [36,42]. It is useful to express the amount of adsorbed protein in terms of the coverage, expressed in mg m−2 and denoted by . This unit, commonly applied in the literature, is connected with N in ␮m−2 through the relationship

61

0

-20

mica

-40

-60 0

1000

2000

3000

4000

5000

-2

Streaming potential measurements for IgG-covered mica were carried out as above described. First, the cell was filled with IgG solution and the protein was adsorbed in situ to a desired surface concentration under diffusion transport. Afterward, the cell was rinsed with pure electrolyte (NaCl) of the same concentration and the streaming potential of the protein covered mica was measured. Zeta potential was calculate from the Smoluchowski equation. After completion of a measurement, the actual surface concentration of IgG on mica was determined by the AFM imaging for N < 4000 ␮m−2 . It was shown that the surface concentrations obtained in this way agree with previously determined in direct adsorption measurements performed in the diffusion cell and shown in Fig. 2. In this way dependencies of  on N are obtained, which are shown in Fig. 3 (parts a–b) for NaCl concentrations of 10−3 , 10−2 and 0.15 M. One can observe that in all cases,  increases abruptly with the IgG coverage . This results in the inversion of the negative potential of mica for N > 1000 ␮m−2 ( > 0.25 mg m−2 ). Afterwards, for higher IgG surface concentration, changes in the zeta potential become rather minor. From the data shown in Fig. 3, one can estimate that the limiting surface concentrations where the zeta potential of mica does not change, are 1800, 2600 and 4000 ␮m−2 for NaCl concentrations of 10−3 , 10−2 and 0.15 M, respectively. This corresponds to = 0.38, 0.68 and 1.0 mg m−2 (see Table 2). The experimental data shown in Fig. 3 were interpreted in terms of the extended electrokinetic model, previously used for

N[ m ] Fig. 3. The dependence of the zeta potential of mica  on the surface concentration of IgG expressed in ␮m−2 (lower axis) and the coverage expressed in mg m−2 (upper axis). (a) I = 10−2 M, (b) I = 0.15 M. The solid lines denote exact theoretical results calculated from the 3D electrokinetic model, Eqs. (6) and (8). The error bars show the standard deviation stemming from the electrode asymmetry potential.

describing colloid particles, [31] fibrinogen [32] and HSA [36] adsorption on mica. In this approach, the three-dimensional fluid velocity and electrostatic potential distributions near a particle attached to a solid/electrolyte interface and immersed in a simple shear flow are rigorously considered. It was shown that the net effect induced by adsorbed particles is due to (i) the flow damping in the vicinity of the interface and (ii) additional charge outflow from the double-layer adjacent to particles. The following equation quantitatively describing these effects was derived [36] ( ) = Fi ( )i + Fp ( )p

(6)

where  ( ) is the zeta potential of protein covered substrate and Fi ( ), Fp ( ) are dimensionless functions of the absolute (dimensionless) coverage that describe these two effects, respectively. The coverage is defined as

= 10−6 Sg N

(7)

where N is expressed in ␮m−2 and Sg is the characteristic crosssection area of the protein expressed in nm2 (see Table 1).

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The functions Fi ( ), Fp ( ) were theoretically determined using the multipole expansion method [44,45] and approximated by the analytical expressions [48]. Fi ( ) = e

0.5

−C 0  i

√ 1 − 2Cp0  Fp ( ) = √ (1 − e ) 2

(8)

where Ci , Cp are functions of the dH parameter. They were numerically calculated in Ref. [49] for 0.1 < dH < 200. As shown, these functions become practically constant for dH > 1 (thin doublelayers) approaching the limiting values of Ci0 = 10.2 and Cp0 = 6.51 for a  1. It is interesting to mention that in the limit of higher coverages, the Fi ( ) function vanishes, and the Fp ( ) approaches the value of 0.71. Results calculated using Eqs. (6)–(8) are plotted in Fig. 3a–b (solid lines). In accordance with the most elaborate crystallohydrodynamic model [25], it is assumed that Sg = 102 nm2 (for the end on or the side on orientations, see Table 1). It should be mentioned that this value is close to the equivalent sphere model where Sg = 95 nm2 and the cylindrical model [2] where Sg = 107 nm2 for these molecule orientations. As can be observed in Fig. 3, the theoretical results properly reflect the experimental data for the entire range of IgG coverage. It should be mentioned that this high ionic strength adsorption regime of IgG, pertinent to physiological conditions, is fully analogous to the previously studied case for positive latex particles deposition [31] and HSA adsorption on mica [36] where the 3D electrokinetic model also proved adequate. This confirms the hypothesis of a particle-like and irreversible adsorption of IgG at pH 3.5 driven by electrostatic interactions. It should also be mentioned that the sensitivity of the streaming potential measurements in respect to IgG coverage is especially high for < 0.3. This can be exploited for a reliable and sensitive determination of the coverage of IgG using the streaming potential measurements, without relying on tedious AFM enumeration procedure. The dependence of

on  can be obtained by numerically inverting Eq. (8) as described in Ref. [36]. This allows one to determine the desorption kinetic of the IgG under various conditions from its monolayers of a well-defined coverage. The utility of such measurements for determining the maximum coverage of IgG is illustrated in Fig. 4. It is interesting to mention that these measurements were performed under flow conditions at the volumetric flow rate of the suspension in the channel was equal to 0.037 cm3 s−1 . As can be seen in Fig. 4, in all cases, only a slight decrease in the IgG monolayer coverage occurred for the flushing time below 100 min. Afterward, there were practically no changes in the coverage up to 800 min. These suggest that a considerable fraction of the IgG molecules is irreversibly adsorbed. From the measurements shown in Fig. 4 one can determine that the coverages of irreversibly bound IgG are 0.14, 0.23 and 0.47 for 10−3 , 10−2 and 0.15 M, NaCl, respectively. This corresponds to the protein dimensional coverage given by the formula 21

=

0.6

10 (Mw /Av )

Sg

(9)

of 0.34, 0.56 and 1.2 mg m−2 for 10−3 , 10−2 and 0.15 M, NaCl, respectively (see Table 3). As can be noticed, these data agree with previously obtained by a direct AFM enumeration of adsorbed IgG molecules via AFM imaging. It should be mentioned that our result obtained for 0.15 NaCl agree with some previous measurements reported in the literature [6,12,13,15,16,28,46] obtained using indirect techniques, e.g., reflectometry.

0.4

0.3

0.2

0.1

0.0 0

200

400

600

800

t [min] Fig. 4. Desorption kinetics of HSA determined by streaming potential measurements and expressed as the dependence of its the coverage on the desorption time t. All monolayers were produced at pH 3.5. The points denote experimental results obtained from streaming potential measurements for various ionic strength: () 0.15 NaCl, initial IgG coverage о = 0.5, (䊉), 10−2 M NaCl, о = 0.2, () 10−3 M, NaCl, о = 0.11. The error bars show the standard deviation stemming from the zeta potential measurement uncertainty.

It is instructive to compare these results with theoretical predictions resulting from various IgG molecule models shown in Table 1. In the case of the equivalent sphere model, the maximum coverage derived from RSA simulations is 0.547 [47]. Hence, for Sg = 95 nm2 one obtains from Eq. (9), = 1.4 mg m−2 . For the cylindrical model, the theoretical maximum coverage for the expanded head-on and end-on conformations where the effective molecule cross-section can be approximated by a rectangle of the length to width aspect ratio of 5.4 is close to 0.51 [48]. Thus, for Sg = 107 nm2 one obtains from Eq. (9), = 1.2 mg m−2 . As can be noticed, this is consistent with our experimental value. A similar result of 1.3 mg m−2 is derived from the crystallohydrodynamic model if one assumes that the maximum coverage is close to 0.547, although the two ellipse case has not been theoretically evaluated in the literature. On the other hand, for the collapsed conformations, where the effective molecule cross-section can be approximated by a two touching circles, the theoretical maximum coverage is 0.547 [49]. Hence, using Sg = 39 nm2 , one obtains from Eq. (9), = 3.5 mg m−2 that is almost three times higher than our experimental result. Considering these theoretical predictions, one can conclude that at pH 3.5 adsorption of IgG occurs most probably in the expanded conformation and head on or end on orientations. Given the fact that at this pH the positive charge is mainly located at the Fab fragments of the molecule [2,49], the head on orientation with Fab fragments facing the substrate surface seems the most probable. 3.4. Characteristics of IgG monolayers on mica After establishing the conditions for obtaining IgG monolayers of well-defined coverage, series of experiments were performed with the aim of determining the stability of the monolayers against pH cycling and their acid − base properties, especially the isolectric point. This was done in terms of the streaming potential measurements carried out under wet conditions, which eliminated conformation changes and desorption of protein upon drying. These experiments were performed as follows: first, IgG monolayers of a desired coverage (varying between 0.37 and 1.2 mg m−2 ) were produced in the streaming potential cell, afterward, the pH in the cell was increased in a stepwise manner by an appropriate addition of NaOH (buffers were avoided because of their specific

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Table 3 Maximum coverages of IgG and HSA on micaa vs. the ionic strength, pH 3.5. Maximum coverages in [mg m−2 ] obtained by various methods

NaCl concentration [M]

10−3 10−2 0.15 a

dH /2

AFM

Streaming potential (adsorption)

Streaming potential (desorption)

HSA/mica streaming potential (desorption)a

0.42 1.15 4.5

0.37 ± 0.02 0.56 ± 0.02 >1.0

0.38 ± 0.1 0.68 ± 0.1 1.0 ± 0.2

0.34 ± 0.03 0.56 ± 0.05 1.2 ± 0.05

0.42 0.66 1.3

Previous data from Ref. [43].

adsorption on monolayers) and after the stabilization of the pH (a few minutes), the streaming potential was measured. The entire sequence was reversed after attaining pH 9.5 by the addition of an appropriate amount of HCl. Three cycles were performed according to this procedure. The results of these measurements for various ionic strengths of NaCl, made for initial monolayer coverages of 0.56 mg m−2 , 1.2 mg m−2 are shown in Fig. 5. It can be observed that the differences between first and last cycle were practically negligible for all ionic strengths. This shows that desorption of IgG during pH cycling and

30

"a" 20

[mV]

10

adsorption

0

-10

reorientation processes in the adsorbed IgG molecules were negligible or reversible. The experimental data obtained for mica supported monolayers were compared with reference results in the bulk obtained by micro-electrophoresis and shown above in Fig. 1. According to Eqs. (6) and (8) the bulk zeta potential data should be corrected by the factor 0.71  p in order to match the corresponding values for protein covered surfaces. As can be observed in Fig. 5 (solid lines) the zeta potential vs. pH dependencies expressed in this way agree with zeta potentials acquired from the streaming potential measurements for the dense IgG monolayer (I = 0.15 M, the coverage 1.2 mg m−2 ). Consequently the IgG isoelectric point derived from these measurements as equal to 5.9, is almost identical as the isoelectric point derived from electrophoretic mobility measurements, equal to 5.8 (see Fig. 1). It should be mentioned that in order to produce the supported monolayers of IgG on mica the protein quantity of only 1 ␮g is needed. After formation, the monolayer can be thoroughly studied for a wide range of pH without additional use of the protein. In contrast, analogous measurements via the bulk electrophoretic mobility measurements would require mg quantities of the protein. Therefore, the results shown in Fig. 5 have a major practical significance, given the high costs of the IgG and similar proteins.

reversal -20

4. Conclusions -30 2

4

6

8

10

pH 30

"b" 20

[mV]

10 adsorption

0

reversal

-10

-20

-30 2

4

6

8

10

pH Fig. 5. Dependence of the zeta potential of the IgG monolayers  (adsorbed at pH 3.5) on pH cycling starting from 3.5 to 9.5 and back to 3.5 (three cycles for each curve were made for a fixed ionic strength). The points denote experimental results obtained from the streaming potential measurements: (䊉), first cycle and (o) third cycle. (a) 10−2 M, NaCl, = 0.56 mg m−2 , (b) 0.15 M NaCl, = 1.2 mg m−2 . The lines denote the results for the IgG zeta potential in the bulk normalized by the factor 0.71. The error bars are calculated as standard deviation of three various measurements.

The AFM imaging of single protein molecules and in situ streaming potential measurements furnished reliable clues on the mechanism of IgG adsorption on mica. It was demonstrated that a quantitative interpretation of streaming potential measurements can be achieved in terms of the theoretical model, expressed by Eqs. (6) and (8) postulating a 3D adsorption of IgG molecules as isolated particles. This allows one to study in situ IgG adsorption/desorption processes and determine the amount of irreversibly adsorbed protein in the limit of its low bulk concentration that varied between 0.37 and 1.2 mg m−2 for 10−3 and 0.15 M, NaCl, respectively. The maximum coverage for 0.15 M, NaCl agrees with the IgG molecule models developed in the literature, i.e., the crystallohydrodynamic and the cylinder model. Considering these theoretical predictions, our experimental measurements are consistent with the hypothesis that IgG adsorption at pH 3.5 occurs in the expanded conformation and the head on orientation. It was also revealed that the IgG monolayers on mica were stable against pH cycling for the range 3.5–9.5 and that their electrokinetic properties approach the bulk data. Accordingly, the isoelectric point of mica supported IgG monolayers was 5.9, practically the same as derived from the micro-electrophoretic measurements in the bulk. Beside significance for basic science, the results presented in this work indicate that thorough characteristics of IgG can be acquired via streaming potential measurements using ␮g quantities of the protein.

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