Adsorption, Aggregation, and Desorption of Proteins on Smectite

Article pubs.acs.org/Langmuir

Adsorption, Aggregation, and Desorption of Proteins on Smectite Particles Krzysztof Kolman,†,‡ Marcin M. Makowski,†,§ Ali A. Golriz,† Michael Kappl,† Jacek Pigłowski,‡ Hans-Jürgen Butt,† and Adam Kiersnowski*,†,‡ †

Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany Polymer Technology and Engineering Division, Wroclaw University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland § Institute of Physics, Poznan University of Technology, Nieszawska 13A, 60-965 Poznan, Poland ‡

S Supporting Information *

ABSTRACT: We report on adsorption of lysozyme (LYS), ovalbumin (OVA), or ovotransferrin (OVT) on particles of a synthetic smectite (synthetic layered aluminosilicate). In our approach we used atomic force microscopy (AFM) and quartz crystal microbalance (QCM) to study the protein−smectite systems in water solutions at pH ranging from 4 to 9. The AFM provided insights into the adhesion forces of protein molecules to the smectite particles, while the QCM measurements yielded information about the amounts of the adsorbed proteins, changes in their structure, and conditions of desorption. The binding of the proteins to the smectite surface was driven mainly by electrostatic interactions, and hence properties of the adsorbed layers were controlled by pH. At high pH values a change in orientation of the adsorbed LYS molecules and a collapse or desorption of OVA layer were observed. Lowering pH to the value ≤4 caused LYS to desorb and swelling the adsorbed OVA. The stability of OVT− smectite complexes was found the lowest. OVT revealed a tendency to desorb from the smectite surface at all investigated pH. The minimum desorption rate was observed at pH close to the isoelectric point of the protein, which suggests that nonspecific interactions between OVT and smectite particles significantly contribute to the stability of these complexes.

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INTRODUCTION Adsorption of proteins on solid surfaces is a major concern in a number of fields such as biology, medicine, biotechnology, and food processing. Adsorption of specific proteins is often considered as a strategy of surface engineering, when obtaining catalytic, antibacterial, or edible coatings is a goal.1 Optimizing the properties of such protein-coated materials demands a thorough understanding of interactions between proteins and solid substrates. Hence, studying adsorption of proteins at surfaces of metals,2,3 ceramics,4,5 polymers,6,7 or SAM layers8,9 to explain binding mechanisms and structural stability of adsorption complexes is scientifically important and technologically relevant. The smectitesa group of clay mineralsare known as a source of sheet (two-dimensional) nanoparticles for applications in e.g. polymer nanocomposites.10 They are also used as sorbents in a wide range of technological and biotechnological processes.11,12 Generally, smectite particles are layered assemblies (stacks) of negatively charged, approximately 1 nm thin, platelets bound by cations located in interlayer spaces balancing the total charge of stacks.13 Because of the high surface area ranging around 1000 m2/g and ionic character, the © 2014 American Chemical Society

smectites can serve as effective substrates for selective adsorption and immobilization of proteins.14−18 Recently, protein−smectite complexes have attracted interest due to their potential in medicine-related applications19,20 as, among others, drug delivery systems,21,22 tissue engineering,23,24 and biosensors.25 Proteins can be bound to smectite particles on the outer surfaces of the stacks18,26 but may also be adsorbed in the interlayer spaces, which often leads to intercalation of the stacks.15,18 Under some conditions adsorption of proteins may cause exfoliation of the smectite stacks into individual platelets.14,16 The adsorbed or intercalated proteins not only may retain their structure and functions27,28 but, sometimes, like in the case of β-glucuronidase, the adsorption may enhance their enzymatic activity.28 Adsorption or desorption of proteins on the surface of smectites is considered as a result of mainly electrostatic interactions between proteins and particles.29−31 With exReceived: July 18, 2014 Revised: September 12, 2014 Published: September 12, 2014 11650

dx.doi.org/10.1021/la502840s | Langmuir 2014, 30, 11650−11659

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Table 1. Basic Properties of the Studied Proteins protein LYS OVA OVT a

molar mass (kg/mol)38 14.3 45 76.6

shape

isoelectric point, pI38

av hydropathy (GRAVY)

nonspherical nonspherical nonspherical

10.7 4.5 6.1

−0.150a −0.001a −0.364a

Stokes radius (nm) 17

1.5−2.0 2.8−3.017 4.7−5.139

Grand average of hydropathy (GRAVY) of the protein molecule, after ref 40. Proteins. In adsorption experiments we used lysozyme (referred to as LYS, from chicken egg white, provided by SERVA Electrophoresis, activity 100 000 units/mg), ovotransferrin (OVT, from chicken egg white, purity 82.4% provided by Fordras S.A.), and ovalbumin (OVA, 98%, from chicken egg white, provided by Neova Technologies). Before adsorption experiments 0.125 g of each protein was dissolved in water (25 mL) and centrifuged to remove traces of undissolved residues for 300 s at 5000 rpm, and the supernatant was diluted to 0.1 g/L. Basic properties of studied proteins are listed in Table 1. Buffers. In order to control pH of protein solutions, four buffers at concentration of 50 mM/L were used: phosphate-citrate (CIT, pH = 4−5), 4-morpholineethanesulfonic acid (MES, pH = 6), 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, pH = 7), and 2-amino-2-(hydroxymethyl)-1,3-propanediol buffer (TRIS, pH = 8−9). They were all provided by Sigma-Aldrich. Reagents, Substrates, and Solvents. (3-Mercaptopropyl)trimethoxysilane (MPTS, 95% Alfa Aesar) or 3-aminopropyltrimethoxysilan (APTMS, from Merck) was used to treat gold-coated ATcut quartz crystals (Q-sense, 5 MHz resonant frequency) and silicon wafers (Si-Mat, 0.7 mm). In all the experiments in this work the 18.2 MΩ Milli-Q water was used. All other chemicals, not mentioned here, were purchased from Sigma-Aldrich and used as received. Preparation of Quartz Crystals for QCM Measurements. The quartz crystals for QCM measurements were cleaned at 85−90 °C for 30 min in a solution of ammonia (2 mL, 28%) and H2O2 (2 mL, 35%) in 25 mL of water followed by subsequent washing with water and methanol and drying. The clean QCM crystals were treated at room temperature in the following way: First, the crystals were immersed for 2 h in 2 mM solution of MPTS in methanol; MPTS methoxy groups at surface of the crystals were then hydrolyzed for 20 min with 0.5 M HCl.41 Then the MPTS-treated crystals were held for 20 h in the solution APTMS in anhydrous toluene (10 μL/20 mL) with stirring under an argon atmosphere.42 MPTS/APTMS-treated QCM crystals were stored in 0.1 M HCl solution in order to protonate amino groups of APTMS. Directly before measurements, the APTMS/MPTS-treated QCM crystals were rinsed with water and mounted in the flow chamber. Deposition of SOMASIF Particles on Quartz Crystals and QCM Measurements. The E1 quartz crystal microbalance with dissipation monitoring (QCM-D) equipped with 100 μL flow chamber from Q-Sense was used. The QCM data were analyzed using QTools 3 software. All the measurements were performed in water environment under controlled pH and temperature (25 °C) and flow rate set at 100 μL/ min. In order to equilibrate the instrument, water was pumped through the chamber until a stable quartz crystal frequency was reached. SOMASIF particles were deposited on top of the APTMS/ MPTS-treated QCM crystals by pumping 0.27 g/L dispersion of SOMASIF through the flow chamber for 30 min (time between t1 and t2; see Figure 2). Loose SOMASIF particles were rinsed off by washing with water for 30 min (t2−t3 time range). The deposited SOMASIF layers were imaged by SEMselected micrographs are available in the Supporting Information. Analysis of the micrographs indicates that SOMASIF particles cover approximately 65% of the QCM crystal surface (Figure S2 in the Supporting Information). The SOMASIFcoated QCM crystals were further used for protein adsorption experiments. Adsorption of proteins was performed by pumping protein solutions (0.1 g/L) through the flow chamber (time between t3 and t4; see Figure 5). After 30 min, the adsorbed protein layer was rinsed using a selected buffer solution for 1 h (time between t4 and t5; see Figure S5 in the Supporting Information).

ception to highly acidic environments (pH < 3) smectite particles in aqueous media carry a negative charge that prevents adsorption of proteins under pH above their isoelectric point (pI). The adsorption efficiency of some proteins at fixed pH depends on the interlayer cations of a smectite due to variation in the net charge of the silicate.31 Apart from electrostatic interactions, the adsorption depends on other interactions such as hydrogen bonding and van der Waals attraction.28 In this work we present study of adsorption of lysozyme (LYS), ovalbumin (OVA), and ovotransferrin (OVT)the main components of egg whiteson particles of a SOMASIF fluoromicaa synthetic analogue of naturally occurring smectites. LYS and OVT have antimicrobial properties and additionally reveal exceptional heat resistance.32 The particular reason to investigate adsorption of LYS, OVA, and OVT was, however, their ability to exfoliate smectite particles, which was reported previously.33 The exfoliated smectite particles functionalized with proteins may be considered as nontoxic, antimicrobial fillers for applications in, for instance, bionanocomposites, food packaging materials, etc.34 This study is aimed at gaining an insight into binding, adhesion, and stability of the protein layer adsorbed on smectite particles. In a broader context, our results should enable predicting stability of protein−smectite complexes and therefore finding new applications for this class of materials. In our approach we studied the adsorption and behavior of protein−smectite complexes under pH ranging from 4 to 9 using quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques. The analysis of the adsorption behavior of the isolated proteins onto relatively uniform silicate particles35 in combination with interface-sensitive analytical techniques36,37 was supposed to provide a quantitative insight into phenomena occurring during the exfoliation of montmorillonite described in our previous paper.33 To date, combination of AFM and QCM methods has not been employed to investigate adsorption of proteins on surface of smectite particles. So this paper shows also a potential of a research strategy involving a combination of AFM and QCM in studying adsorption of proteins on colloidal particles immobilized on gold or silicon substrates. To the best of our knowledge, this is also the first report on the adsorption of lysozyme, ovalbumin, and ovotransferrin on the synthetic smectite. As, generally, little is known about the stability of the protein−smectite adsorption complexes, we report here an influence of pH on adsorption, aggregation, and desorption of proteins on smectite particles.

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MATERIALS AND METHODS

Smectite. In the study we used the synthetic sodium fluoromica SOMASIF ME100, in short SOMASIF, from CO-OP Chemical Japan. The specific surface area of SOMASIF was 900 m2/g. The cation exchange capacity (CEC) was 1.15 mequiv/g.35 The smectite was purified by 24 h sedimentation from 10 g/L dispersions water. Afterward, the supernatant containing 2.7 g/L of SOMASIF (determined by centrifugation) was separated from the residue and diluted 10 times to obtain a stock dispersion with a concentration of 0.27 g/L. 11651


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Figure 1. AFM topology images obtained in (A) liquid and (B) air. (C) Height profile of a 2-platelet SOMASIF stack measured along the blue line shown in (B). layer allows to extract the apparent areal mass (mf) consisting of the masses of the adsorbate and the solvent. Since the SOMASIF particles were actually the substrate for adsorption of proteins, the changes of Δf and ΔD due to SOMASIF particles in analysis of protein adsorption were neglected. Atomic Force Microscopy. A Veeco Multimode AFM with a Nanoscope III controller (Bruker AXS, Santa Barbara, CA) equipped with standard tapping mode liquid cell was used to perform the topographic imaging of SOMASIF particles attached on top of silicon wafers and force spectroscopy on particles before and after adsorption of proteins. DNP type A cantilevers (Bruker AFM Probes) with a resonance frequency of 65 kHz in air and 8 ± 2 kHz in aqueous solutions. The average spring constant was k = 0.42 ± 0.04 N/m as determined by the thermal tune method.46 15 ×15 mm2 silicon wafers were used as substrates. The wafers were cleaned using the same protocol as for the QCM crystals. The cleaned silicon wafers were functionalized with APTMS in anhydrous toluene solution (10 μL/20 mL) at room temperature for 20 h. The APTMS-functionalized silicon wafers were stored in 0.1 M HCl solution in order to hydrolyze the remaining methoxy groups and to protonate amino groups of APTMS. SOMASIF particles were attached to the surface of APTMSfunctionalized silicon wafers by immersing the latter in 0.27 g/L dispersion of SOMASIF. Directly before measurements, the wafers were coated with SOMASIF particles and thoroughly rinsed with water to remove loose particles. To minimize the drift of the instrument, water was injected into the liquid cell 30 min before measurements. SOMASIF particles on the silicon surface were imaged in a tapping mode. When a particle was identified, the AFM tip was placed above the particle, and 300 cantilever deflection-vs-piezo position curves were recorded on a grid spanned over 150 × 200 nm2 area of SOMASIF particle with a maximum load of 5 nN. The adsorption of proteins was performed by injecting solution of proteins (0.1 g/L in water) into the liquid cell. After 30 min, the protein solution was replaced with a buffer, and after another 30 min the imaging and force spectroscopy measurements were carried out. In order to study adhesion of proteins to SOMASIF particles, the deflection-vs-piezo position curves were converted to force vs distance curves. In presentation of the results we followed a generally accepted convention that assumes that the attractive force between the cantilever and probed objects has a negative sign.47 Hence, the adhesion determined from force vs distance curve as the minimal value of the retraction part of the curve is negative as well. The linear fit of baseline (zero force region) was subtracted from the detector voltage vs piezo position over the whole range. The conversion factor between the detector signal in volts and deflection signal in nanometers was calculated from the constant compliance region. Actual force was calculated by dividing deflection voltage signal by the conversion factor and multiplying it by the spring constant of the cantilever. The distance was obtained by subtracting the cantilever deflection from the piezo position.36,48 In order to improve quality of AFM images of the SOMASIF-coated silicon wafers and QCM crystals as well as to demonstrate the stability of attached SOMASIF particles, AFM tapping mode imaging and a nanowear experiment were carried out on Veeco Dimension 3100 AFM (Bruker AXS, Santa Barbara, CA) with a Nanoscope IIIA controller at ambient conditions. The standard AFM images of

The changes in frequency and dissipation were measured simultaneously for the fundamental frequency and five overtones (n = 3, 5, 7, 9, and 11). Frequency shift data at each overtone were recorded as the fundamental value divided by the overtone number (Δf n/n, normalized frequency shift). Depending on the observed energy dissipation shifts either Sauerbrey43 or Voigt44 models were used to determine the adsorbed mass. The Sauerbrey equation (1) is a linear relation between the mass (Δm) adsorbed at the surface with the area A and measured changes in resonant frequency (Δf): Δf Δm = CQCM × A n

(1) −2

−1

where CQCM (17.7 ng × cm × Hz at f = 5 MHz) is the mass sensitivity constant and n is the overtone number. As the Sauerbrey formula is only valid for thin films rigidly attached to the QCM crystal surface (ΔDn/(Δf n × n−1) ≪ 4 × 10−7 Hz−1 37), it is not appropriate for viscoelastic films like polymers, proteins, etc. Hence, the QCM behavior of “soft” films in our study was described using a conventional Voigt model comprising parallel arrangement of spring (elastic behavior) and dashpot (viscous behavior). In the Voigt model, the changes of resonant frequency Δf and energy dissipation ΔD are given by eqs 2 and 3, respectively: Δf =

Im(β) 2πtqρq

ΔD = −

(2)

Re(β) πftqρq

(3)

where tq, ρq, and f denote thickness and density of quartz as well as the oscillation frequency, respectively. β is a complex expression (4−7) including tq, ρq, and f as well as the elastic modulus of the adsorbed film (μf), the viscosity of the adsorbed film (ηf), the density of the adsorbed film (ρf), the thickness of the adsorbed film (df), the viscosity of bulk liquid (ηl), and the density of bulk liquid (ρl).

β = ξ1

α=

2πfηf − iμf 1 − α exp(2ξ1df ) 2πf 1 + α exp(2ξ1df )

ξ1 2πfηf − iμf ξ2 2πfηl

+1

ξ1 2πfηf − iμf ξ2 2πfηl

−1

ξ1 =

−

ξ2 =

i

(4)

(5)

(2πf )2 ρf μf + i 2πηf

(6)

2πfρl ηl

(7)

In the Voigt model, the density and viscosity of bulk fluid and the density of adsorbed layer were assumed as 1000 kg/m3, 0.001 kg/ms, and 1000 kg/m3, respectively. Such values are justified as it is generally accepted that highly hydrated protein layers have a density close to the density of water.45 Knowing the thickness and density of adsorbed 11652


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SOMASIF-coated silicon wafers and QCM crystals were recorded in tapping mode using OMCLAC 160 TS-W2 silicon cantilever with resonant frequency of 300 kHz. The AFM images were analyzed using Gwyddion software.

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RESULTS AND DISCUSSION Deposition of SOMASIF Particles on Gold and Silicon Substrates. Obtaining uniform layers of smectite particles is relatively difficult as they tend to agglomerate in water dispersions and on solid substrates.49 In our approach, therefore, we applied an intermediate layer of thiols and silanes on gold or silanes alone on silicon. In this way we obtained surfaces functionalized with positively charged end groups (NH3+) that effectively bound and immobilized SOMASIF particles. Deposition of particles from highly diluted dispersions enabled formation of relatively uniform flat layers of evenly distributed, exfoliated SOMASIF platelets and, in some places, stacks (Figure 1). The average surface coverage determined from SEM was 65% (Figure S2 in the Supporting Information). The morphology of the SOMASIF particles deposited on the substrates was investigated by AFM (Figure 1) and SEM (Figure S1 in the Supporting Information). Both in the case of gold and silicon we observed a flat arrangement of smectite platelets on the surface. Topographies obtained in water (Figure 1A) and air (Figure 1B) show selected, representative SOMASIF particles having diameter in the range of 0.5−2 μm and height in the range of 1−10 nm. Although SOMASIF dispersions were purified, smaller particulate impurities, occurring due to breaking off of the smectite particles, were present in the immobilized silicate layers (Figure 1A,B). The height profile of a 2-platelet stack of SOMASIF visible in Figure 1B is presented in Figure 1C. The steps on the height profile correspond to the platelet thickness (1.1 nm) and the sum of the platelet thickness and spacing between subsequent platelets in the stack (1.1 nm + 0.3 nm). Note that the platelet thickness and an average platelet-to-platelet spacing determined for hydrated SOMASIF powder by means of X-ray diffraction were found to be 0.85 and 0.4 nm, respectively.35 The attachment of particles on the surface of QCM crystals was monitored in situ by measuring frequency shift Δf (Figure 2A) and energy dissipation ΔD (Figure 2B). After equilibration of the QCM signal under constant water flow, the SOMASIF dispersion was pumped over the surface of the functionalized QCM crystal for 30 min (t1−t2 in Figure 2A). The attachment of SOMASIF particles was manifested by a decrease in Δf by approximately −110 Hz and increase of ΔD by approximately 55 × 10−6 at the time t2 (Figure 2B). At the time t2 the apparent mass of the adsorbed SOMASIF particles was found 1.65 × 10−4 kg/m2. Stability of the attached SOMASIF particles was verified by washing the QCM crystal with water for 30 min (from the time t2 onward, Figure 2). As during the washing no increase in Δf was observed, we assumed that there were no SOMASIF particles loosely adsorbed on the QCM crystal. Adsorption of Proteins on SOMASIF Particles in Water. To investigate properties of protein layers adsorbed on SOMASIF, we used AFM imaging and force spectroscopy (Figure 3). First, let us discuss the interaction of the AFM tip with pure SOMASIF particles. On the approach curve shown in Figure 3A we see a long-range attractive force plus a shortrange repulsion. These interactions are observed typically at 5− 10 nm and 1−2 nm, respectively. The attraction can be attributed to van der Waals forces approximated by eq 8

Figure 2. Adsorption of SOMASIF particles (t1) followed by washing (t2) recorded by QCM-D: (A) normalized frequency shift Δf and (B) energy dissipation ΔD of fifth overtone.

F≈

AHR 6D2

(8)

in which AH is Hamaker constant, R is the tip radius, and D is the tip−surface distance. Note that we assumed that attractive forces have a negative sign (Figure S3 in the Supporting Information). The van der Waals force calculated for Si3N4 tip interacting with mica across water (AH = 2.45 × 10−20 J,50 R = 50 nm, see Figure S3 in the Supporting Information) was found very similar to the attraction force observed in our experiments. The contact between SOMASIF surface and the AFM tip is established at a distance of 1−2 nm, where the minimum of force curve is observed (Figure 3A). From this point on, the SOMASIF particle with connecting APTMS molecules were compressed. This compression leads to the short-range repulsion. Electrostatic double layers forces are not observed. First, they are too long-ranged; in distilled water the decay length of the exponentially decaying force is above 100 nm.51 Second, probably, the surface charge of the Si3N4 at neutral pH is most likely not strong. Because of oxidation of the tip surface the isoelectric point can be below 9, likely in the range from 6 to 7.13,52 After adsorption of proteins on SOMASIF (Figure 3B−D), the interactions between the cantilever and SOMASIF particles were reduced. When adding OVA or LYS, the long-range force decreased (Figure 3B,C). Proteins adsorb to the surface of the platelet. At this stage we can only speculate that the adsorbed layer acts like a spacer. The protein layer in itself will cause only a weak van der Waals attraction but hinders the tip from getting closer to the platelet. In addition, it causes a short-range steric repulsion once the tip starts to penetrate the layer. The effect is even stronger in the case of OVT (Figure 3D). OVT forms the thickest adsorbed layers. In the force curves the long-range attraction is negligible. Conversely, rather than a very shortrange repulsion (< 1 nm) we observe a strong repulsion over the last 2 nm. This is interpreted again a steric force required to push the adsorbed protein to the side when the tip approaches closer to the platelet surface. The adhesion is correspondingly relatively weak. These observations together with QCM results suggest that decrease in attraction is proportional to the 11653


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Figure 3. Force vs distance curves (red curve: approach; black curve: retract) for the pure SOMASIF platelet (A) and after adsorption of OVA (B), LYS (C), and OVT (D). Curves are averaged of 11 experimental curves (error bars denote the standard deviation). The insets show distribution of adhesion force histograms.

quantity of adsorbed proteins. In our study we used the same cantilevers for imaging and force spectroscopy, and therefore we used cantilevers with a medium spring constant. The sensitivity of such cantilevers was, however, found too small to detect the thickness of soft protein layer. The adhesion was determined from retract curves, when the tip was in contact with the surface and the cantilever force has not overcome the adhesive tip−sample interaction yet. The average adhesion force between the tip and the pure SOMASIF platelet was 3.1 nN. For either protein layer deposited on SOMASIF platelets the average adhesion was weaker, reaching 2.1, 0.9, and 0.25 nN for OVA, LYS, or OVT, respectively. Similarly as observed on the cantilever approach, the adhesion force between the cantilever and the surface was suppressed due to protein adsorption and resulted mainly from van der Waals interactions. The decrease of adhesion force is related to amount of adsorbed proteins recorded by the QCM method. In many force curves recorded when retracting the tip, additional minima (jump-out events) were observed aside of the main adhesive peak. A representative example recorded for OVT layer adsorbed on SOMASIF is shown in Figure 4. Depending on protein force of jump-out events ranged from −0.3 to −0.1 nN, while average jump-out distances ranged mainly from 8 to 30 nm from the SOMASIF surface. The jumpout events may result from stretching of individual protein molecules bridging the tip and the particle surface until the cantilever force overcomes the force tethering the protein to the tip or the surface of SOMASIF particles.53 At this point the protein is denatured by the tip. The jump-out event shown in Figure 4 occurred at distance of 25−30 nm, which, compared to the size of OVT, may indicate unfolding of OVT molecules

Figure 4. Exemplary force vs distance curve (red curve: approach; black curve: retract) obtained on SOMASIF particles after adsorption of OVT layer. A jump-out event is indicated with a dashed circle.

or a multilayer arrangement of the adsorbed protein. The latter would be in agreement with the QCM data (Figure 5); the mass of adsorbed OVT was found the highest of all investigated proteins. The occurrence of jump-out events (ne) expressed as the percentage of force vs distance measurements in which jump-outs were identified (in relation to the total number of measurements) is presented in Table 2. The highest occurrence of jump-outs was found for OVA. This is most likely due to flexible structure and negative charge of OVA molecules. This makes the protein layer relatively loosely bound to the SOMASIF surface and more accessible to the AFM tip. The ne values observed in the case of LYS and OVT were smaller because of their positive net charge and relatively rigid structure.54 To demonstrate the robustness of the deposited SOMASIF layer and also to benchmark the substrate stability in force spectroscopy measurements, an additional nanowear experiment was performed. We tested 500 × 500 nm2 area on a 11654


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Table 2. Apparent Areal Mass Change (Δmf) and Jump-Out Events (ne, the Count of Events per 100 Force vs Distance Measurements) for OVA, LYS, and OVT as a Function of pHa pH (H2O)

4

5

Δmf [%] ne [%]

30 80

20 14

28 29

Δmf [%] ne [%]

−1 6

−9 2

3 6

Δmf [%] ne [%]

−14 11

−29 10

−27 5

6 OVA −35 63 LYS 9 3 OVT −27 13

7

8

9

−22 16

−88(S) 6

−86(S) 5

11 8

17 9

21 6

−42 11

−63 39

−64 36

a

Superscript (S) index denotes the mass at time t5 calculated using Sauerbrey’s equation. The negative Δmf indicates decrease in the apparent areal mass.

SOMASIF particle after adsorption of OVT. A load of 20 nN in a contact mode at ambient conditions resulted in partial removal (sweeping) of adsorbed proteins by the cantilever tip. AFM imaging of the area (2 × 2 μm2 including the tested particle) revealed that the shape and structure of SOMASIF particle did not change. This indicates that SOMASIF particles were stable during measurements of protein adhesion, as the forces applied in the latter were significantly smaller. Exemplary results of nanowear experiments are presented in the Supporting Information (Figure S4). In QCM, the adsorption was performed directly after washing away loosely attached SOMASIF particles, i.e., right after the t3. The amount of adsorbed proteins was monitored in real time by simultaneously measured frequency change (Δf) and energy dissipation (ΔD) (Figure 5). Protein adsorption leads to a decrease in Δf (Figure 5A) and increase in ΔD (Figure 5B), meaning that protein layers clearly exhibit a viscoelastic behavior. Note that larger objects induce usually a stronger decrease of Δf and increase of ΔD.55 In our case, the strongest response was measured for the largest protein OVT. However, the smallest changes in Δf and ΔD were recorded for OVA and not for LYS having the smallest molecular weight and dimensions (cf. Table 1). Such an anomalous behavior of OVA can be attributed to unfavorable electrostatic interactions between OVA and SOMASIF as well as to relatively low hydrophilicity of the protein. In contrast to positively charged LYS, OVA has a relatively strong negative net charge. In addition, considering from the GRAVY values (Table 1), OVA is the least hydrophilic protein.40 Therefore, the adsorbed OVA layer binds the smallest amount of water, which may cause smaller than expected changes in Δf and ΔD. To extract quantitative information about the adsorbed masses, we employed the Voigt model (Figure 5C). After 30 min of adsorption, the apparent areal masses (mf) of OVA, LYS, and OVT were 1.3 × 10−5, 4.3 × 10−5, and 10.4 × 10−5 kg/m2, respectively. The fundamental principle of QCM relies on shift of the crystal resonance frequency due to changes of the surface mass. However, in the case of viscoelastic layers, the important quantity describing damping of quartz crystal oscillations is the dissipation factor (D).56

Figure 5. Adsorption of ovalbumin (□), lysozyme (○), and ovotransferrin (△) on SOMASIF functionalized QCM crystal surface (t3): (A) normalized frequency shift Δf5/5, (B) energy dissipation ΔD5, and (C) the apparent areal mass (mf). The inset in (A) shows decrease of Δf5/5 during the first 15 min of protein adsorption on APTMS/MPTS functionalized gold (no SOMASIF particles).

D=

ED 2πES

(9)

ED in eq 9 is energy dissipated during oscillations while ES denotes stored energy. In order to gain an insight into organization of adsorbed molecules, we plotted the energy dissipation ΔD-vs-normalized frequency shift Δf for the adsorption of each protein (Figure 6).57 The adsorption was divided into regions characterized by different ΔD/Δf ratios. Initially, the ΔD/Δf ratios recorded for OVA, LYS, and OVT were 0.18 × 10−6, 0.15 × 10−6, and 0.1 × 10−6, respectively. This region represents low surface coverage and strong interactions between the surface and adsorbed proteins (the adsorbed mass behaves rigidly). On the adsorption progress, the ΔD/Δf values of OVA, LYS, and OVT changed to 0.3 × 10−6, 1.5 × 10−6, and 0.63 × 10−6, respectively. As more proteins approach and adsorb at the substrate, their interactions with the surface get weaker, and as a consequence, the subsequent protein layers are easier penetrated by water. This is manifested by damping of quartz oscillations and increase in the ΔD/Δf values. In the case of LYS and OVT, a decrease of ΔD/Δf was observed at the end of adsorption (1.05 and 0.44, respectively), meaning saturation of the adsorbed protein layer. 11655


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Figure 6. Plots of ΔD5 as a function of Δf5/5 for (A) ovalbumin, (B) lysozyme, and (C) ovotransferrin. Dashed lines show regions characterized by different ΔD/Δf ratios. (D) Possible arrangement of protein molecules at different stages of adsorption.

molecules.58 The swollen protein layer has a higher surface area and is more accessible to the AFM tip. Additionally, the increase of pH enhances protein−tip electrostatic interactions and decreases protein−SOMASIF interactions. Above pH = 6 the ne decreases, indicating desorption or increased rigidity (more compact structure) of adsorbed OVA molecules. For LYS, the force spectroscopy results do not show any significant change of jump-out event occurrence (ne) in the function of pH (Table 2). In the investigated pH range LYS has a net positive charge, which makes the protein strongly adsorbed at the negatively charged SOMASIF surface. The strong interactions between LYS and SOMASIF were confirmed by QCM results discussed later. It seems that interactions of the adsorbed LYS with the AFM tip are weaker than these of LYS-SOMASIF, and therefore almost no jumpout events were observed. The pH-dependent force spectroscopy measurements of OVT layer revealed that the minimum value of ne appears at pH = 5, slightly below the pI of OVT (see Table 1). At pH close to pI, the total net charge of protein molecule is close to zero; thus any electrostatic interactions are greatly suppressed. The maximum values of the jump-out events occurrence are observed at pH ranging from 8 to 9, where electrostatic repulsion between OVT molecules and SOMASIF particles as well as between OVT molecules within the adsorbed layer affects the stability of OVT/SOMASIF complexes. The low stability is manifested by increase of the ne. The changes in the apparent areal mass mf of the adsorbed proteins were studied by analysis of ΔD and Δf recorded between the times t4 and t5 (buffer rinsing; see Figure S5 in the Supporting Information). To present the measured data more quantitatively, the apparent areal mass change Δmf was defined:

As surfaces of QCM crystals were not fully covered by SOMASIF platelets, it was necessary to check the adsorption of proteins on APTMS/MPTS-treated QCM crystals without SOMASIF particles (Figure 5A, inset). Such control experiments were performed in order to determine the contribution of proteins adsorbed outside of SOMASIF particles in the QCM results. It was found that LYS did not adsorb at all on the surface without SOMASIF particles. In the case of OVT the Δf was over 3-fold lower when the protein was adsorbed on the APTMS/MPTS-treated QCM crystal without SOMASIF. Since Δf is proportional to the mass, the mass of OVT was therefore over 3-fold higher on the SOMASIF-coated QCM crystals. Considering now the surface fraction occupied by SOMASIF (0.65), one can estimate that the mass of OVT adsorbed outside of the SOMASIF platelets is approximately 10 % of the total amount of the adsorbed OVT. So the QCM data for OVT are not much confused by the influence of the protein adsorbed out of the smectite surface. In the case of OVA the Δf measured in the main and the control experiments were similar. In this case the amount of protein adsorbed on SOMASIF is, therefore, directly proportional to the surface fraction of the smectite. In other words, 35% of OVA is adsorbed outside of the SOMASIF particles. This is the main limitation for the accuracy of the QCM data recorded for OVA. Influence of pH on Aggregation and Desorption of Proteins on SOMASIF Surface. In order to study the influence of pH, we treated the adsorbed protein layers with buffers having pH ranging from 4 to 9. To characterize the adsorbed protein layers during the washing process, the same techniques as described for the adsorption of proteins in water were used. The results of QCM and AFM analysis for all proteins are summarized in Table 2. The occurrence of jump-out events (ne) as a function of pH was measured using force spectroscopy. High ne indicates that the protein layer can be easily penetrated by the AFM tip and protein/AFM tip interactions are strong enough to make them stick to each other. In the case of OVA the occurrence of jump-out events (ne) in AFM experiments was found related to pH (Table 2). As pH increases, the ne rises to reach maximum at pH 6. This increase of the occurrence of jump-outs can be attributed to swelling of protein layer due to rearrangement of adsorbed protein

Δmf =

mf (t5) − mf (t4) × 100% mf (t4)

(10)

where mf(t4) and mf(t5) denote the apparent areal mass at the times t4 and t5, respectively. The protein layers were rinsed by buffers with pH in range from 4 to 9. In this way we obtained a change in adsorbed mass as a function of pH (see Table 2). At pH below 6, where OVA molecules are electrically neutral, an increase in apparent areal mass is observed. Since no 11656


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rheology of adsorbed layers were obtained using QCM. AFM measurementsatomic force spectroscopyrevealed information about adhesion and interactions between proteins, AFM tip, and SOMASIF particles. The adsorption of LYS and OVT on SOMASIF was driven mainly by electrostatic interactions between the proteins and negatively charged SOMASIF surface. The adsorption of OVA resulted mainly from nonspecific interactions and was observed despite repulsive electrostatic interactions between OVA molecules and SOMASIF particles. Changing pH environment over the adsorbed layer induced either structural reorganization or release (desorption) of protein molecules. At pH as low as 4−5 the adsorbed OVA layer was swollen, likely due to incorporation of water. Increasing pH to the level of 8−9 causes the OVA layer to collapse or partially desorb. In the case of LYS−SOMASIF we identified end-on to side-on reorientation of molecules at the surface and an increase in the apparent areal mass upon pH increasing from 5 to 9. The OVT− SOMASIF complexes were found the least stable: under all investigated pH proteins were desorbing from the smectite surface. The minimum desorption rate was, however, observed for pH corresponding to pI, suggesting that the stability of OVT on the SOMASIF surface relies on nonspecific protein− smectite interactions, while desorption is due to electrostatic repulsion between OVT molecules.

additional proteins were added to the system, the increase in Δmf suggests rearrangement of adsorbed molecules causing swelling of the protein layer. In the pH range between 6 and 9 the apparent areal mass of adsorbed OVA layer decreased. This decrease can be attributed to desorption of proteins from surface or expelling of water molecules and hence collapse of the adsorbed protein layer. Changing pH of the OVA layer environment above 8 causes an increase of Δf, overlapping of Δf profiles measured at 5th and 7th overtones (t4−t5, see Figure S5A in the Supporting Information) and a drop in ΔD to zero at the end of experiment (t5, see Figure S5B in the Supporting Information). These results are characteristic for rigid layers, which suggests that at pH range from 8 to 9 the OVA molecules collapse or partially desorb, making the Sauerbrey equation (1) applicable to calculate the apparent mass (mf) for both pH 8 and 9. This observation is consisted with AFM measurements, where the occurrence of jump-out events decreased at high pH values (8−9). In the case of LYS a decrease in apparent areal mass is observed under pH of 5, while above we observe an increase in the mf. The structure of LYS molecules within the adsorbed layer depends on charge density distribution on the surface of proteins and hence on pH. Thus, at low pH (4−5), LYS molecules carry a strong net positive charge and adsorb end-on to the SOMASIF surface because of the strong electrostatic repulsion within the protein layer. At pH values above 6, LYS adsorb side-on due to decreased repulsion between molecules in the layer.59 Although density of the side-on orientation is lower, the increase of apparent areal mass is observed most likely because protein layer would be easier penetrated by water. As more solvent try to penetrate the protein layer, it starts to swell. For OVT, the most prominent drops of the adsorbed mass were observed for the pH above the pI of OVT (pH 7−9, Table 2), whereas the minimum decrease was found at pH = 5 (Table 2). This indicates that electrostatic repulsion between the protein molecules in the layer significantly reduces binding of OVT molecules at the SOMASIF surface. Close to the isoelectric point the total net charge of protein macromolecules is largely suppressed. Therefore, surprisingly somewhat, we conclude that the stability (the integrity) of OVT-SOMASIF complexes is mainly a result of nonspecific interactions the smectite particles and protein molecules. The release of proteins, manifested by the apparent areal mass drop, occurs when pH is changed from the optimum due to electrostatic repulsion between molecules within adsorbed layer. It is especially prominent at pH values above pI, where not only the protein molecules repel each other within the layer but also there is an electrostatic repulsion between the proteins and SOMASIF particles. Occurrence of the AFM jump-out events (Table 2) is in line with this reasoning. The largest number of jump-outs is observed at high pH values, where electrostatic interactions between the cantilever and OVT layer become stronger.

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ASSOCIATED CONTENT

S Supporting Information *

SEM imaging and micrographs, force spectroscopy−van der Waals interactions, AFM nanowear experiments, and QCM studies of the adsorption of OVA. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected] (A.K.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Fordras and the Neova Technologies Companies for providing ovotransferrin and ovalbumin, respectively. K.K. thanks Dr. Jagoba Iturri from Max Planck Institute for Polymer Research in Mainz for help with interpretation of QCM data. This work is cofinanced by the European Union as part of the European Social Fund. M.M. and K.K. also thank the International Max Planck Research School for Polymer Materials at Max Planck Institute for Polymer Research in Mainz. A.K. and J.P. acknowledge the support through the statutory subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Chemistry of Wroclaw University of Technology.

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CONCLUSION In this work we applied QCM and AFM techniques to study the adsorption of ovalbumin (OVA), lysozyme (LYS), and ovotransferrin (OVT) on the surface of smectite particles (SOMASIF). The research strategy combining results of AFM and QCM measurements was found very effective in studying interactions of protein molecules with solid flat particles of the smectite. An apparent areal mass change and information about

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Adsorption, Aggregation, and Desorption of Proteins on Smectite

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