Combining Surface Plasmon Resonance and Quartz Crystal

Aug 10, 2016 - We used multiparametric surface plasmon resonance (MP-SPR) and a quartz crystal microbalance with dissipation energy monitoring (QCM-D)...
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Combining Surface Plasmon Resonance and Quartz Crystal Microbalance To Determine Hydration of Dendrimer Monolayers Barbara Jachimska* and Karolina Tokarczyk J. Haber Institute of Catalysis and Surface Chemistry, PAS, Niezapominajek 8, 30-239 Cracow, Poland ABSTRACT: Polyelectrolytes are abundant in nature and crucial due to their versatility in biological systems. The controlled assembly of polyelectrolytes has potential applications in the formation of nanostructured and microstructured materials of desired structure and functionality. Dendrimers are a special class of polyelectrolytes, which are characterized by their densely branched and well-defined spherical geometry. Amino-terminal dendrimers resemble spheres, whose uniform surface charge densities can be continuously modulated by pH or ionic strength. The present study focuses on the dendrimer monolayer structure on gold surfaces. We used multiparametric surface plasmon resonance (MP-SPR) and a quartz crystal microbalance with dissipation energy monitoring (QCM-D) to investigate the conformational behavior of the sixth generation of PAMAM molecules. Both the kinetics of dendrimer deposition and the maximum surface concentration were determined. The dependence of the maximum coverage on the pH, ionic strength, and the experimental kinetic runs were quantitatively interpreted in terms of the random sequential adsorption (RSA) model using the concept of effective hard particles. It is shown that the MP-SPR measurements can be used to determine the mechanisms of dendrimer adsorption, e.g., the reversibility and orientation of molecules at interfaces. Additionally, this method can be used for a precise determination of dendrimer coverage, hence, its concentration in the bulk solution at levels of 0.05 ppm or less. The extent of hydration of dendrimer films was estimated from the combination of the QCM-D and MP-SPR data, with the assumption that the excess mass measured in QCM-D compared to MP-SPR mass is due to trapped water molecules. The structure of the resulting films is strongly dependent on the deposition conditions.

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INTRODUCTION Dendrimers have a range of applications, including biomedical applications such as drug delivery, gene delivery, cancer therapy, and diagnostic agents.1−5 Their unique properties originate in their macromolecular structure. It is known that dendrimers can undergo irreversible swelling and that this process is directly connected with the degree of protonation of the dendrimer molecule. The hydration of dendrimer films seems to be a crucial aspect in their implementation. In all biological systems, the conformational stability is intrinsically connected with natural hydration.6 Therefore, the estimation of the degree of hydration in dendrimer films on a model surface is essential for the study of their potential applications. To characterize patterned surface nanostructures, it is necessary to use techniques that can distinguish features at the molecular scale. Several new experimental techniques are now available for studying the adsorption of polymers or proteins on solid surfaces, including ellipsometry, surface plasmon resonance (SPR), atomic force microscopy (AFM) in the force spectroscopy mode, and monitoring by a quartz crystal microbalance (QCM). One of the advantages of these techniques is the possibility to directly follow interface process in situ.7−12 Optical methods with high 3D resolution are required for a proper description of the surface-mediated organization of macromolecular systems in hierarchical structures of a target architecture and functionality. © 2016 American Chemical Society

The PAMAM dendrimer is a weak polyelectrolyte whose charge can be tuned using the pH or ionic strength of the solution. The molecule is completely charged, partially charged, and uncharged at pH 4.0, 6.0, and 10.0, respectively.13 The present study focuses on the influence of pH on the dendrimer monolayer structure on the gold surface. Therefore, surface plasmon resonance and quartz crystal microbalance with dissipation energy monitoring are used to investigate the conformational behavior of the sixth generation of PAMAM molecules. MP-SPR and QCM-D are powerful methods that enable highly sensitive, quantitative, real time, label-free, and noninvasive detection of molecules adsorbed on a solid surface. MP-SPR allows the determination of the mass of the adsorbed polymer from changes in the dielectric constant at the interface. QCM-D probes the variation of shear of an oscillating piezoelectric sensor, caused by changes in the total mass due to the adsorption of molecules, which further couple with water on the surface of the film. A combination of the above techniques can provide significant information on the mechanisms of surface-adsorption of dendrimers, the associated structural changes, and the extent of hydration.14,15 These investigations lead to a more profound understanding of the self-assembling behavior of branched polyelectrolytes, Received: May 18, 2016 Revised: August 10, 2016 Published: August 10, 2016 19678

DOI: 10.1021/acs.jpcc.6b05020 J. Phys. Chem. C 2016, 120, 19678−19685

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The Journal of Physical Chemistry C

Figure 1. (A) SEM image of the G6 PAMAM dendrimers. Inset image presents magnification. (B) SEM image of Au sensor and static water contact angle. The dimensions of the scale bars: 100 nm.

(Krechmer mode) with two independent channels and an integrated peristaltic pump. The MP-SPR apparatus worked in a wide angular scan range (40−78°). The initial parameters for the bare sensor were obtained by fitting the measured curve using the Viewer program. The immobilization of molecules on the sensor surface can be followed by monitoring the intensity changes at a fixed angle or angular position over time. The excess concentration of PAMAM on the surface was calculated according to eq 119

which are interesting candidates for reversibly tunable swelling materials for drug delivery or smart molecules for surface functionalization.

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EXPERIMENTAL SECTION Materials. Sixth-generation poly(amido amine) (G6 PAMAM, M = 58 kDa, diagnostic grade) dendrimers in aqueous solutions (9.4% concentration) were obtained from Dentritech (Midland). The size and monodispersity of the dendrimers were analyzed by scanning electron microscopy (SEM) (see Figure 1A). The physicochemical properties of G6 PAMAM dendrimers are presented in Table 1.13,16−18 Dendrimer solutions were prepared by diluting the starting solution. The ionic strength was adjusted using NaCl, and the pH was adjusted by adding HCl or NaOH.

ΓSPR =

characteristic

value, unit

remarks

256 254 510 9156 3.75 ± 0.05 nm 3.45 ± 0.5 nm 3.45 ± 0.01 nm 8.0 ± 0.02 10.0 ± 0.1 10.5 ± 0.02 510

calculated calculated calculated calculated DLS13 TEM17 SAXS16 potentiometric titration18 electrophoretic mobility13 potentiometric titration18 calculated

28

calculated from electrophoretic mobility13

dn dc

(1)

where ΔΘSPR is the change in the MP-SPR angle, k is an MPSPR instrumental constant obtained after calibration, dG6 is the thickness of the adsorbed layer, dn/dc is the refractive increment. The refractive index used in this work was estimated by an Atago refractometer RX-50000α. For BioNavis MP-SPR instruments, the k value is 1.0 × 10−7 nm/deg for λ = 670 nm and 1.9 × 10−7 nm/deg for λ = 785 nm. The value of dn/dc for the dendrimer solution was 0.239 cm3/g. All experiments were performed at a constant flow rate of 50 μL/min. MP-SPR sensors with a glass slide base (Schott D 263, size 20 mm × 12 mm × 0.5 mm) and with a thin layer of ca. 50 nm of gold were produced by BioNavis. Scanning electron microscopy (SEM) and static water contact measurements were used to characterize the sensor roughness and hydrophobicity (see Figure 1B). SEM measurements were performed using a JEOL-3700F field emission scanning microscope at an operating voltage of 15 keV. Quartz Crystal Microbalance with Dissipation Energy Monitoring (QCM-D). The QCM-D measurements were performed with a Q-sense E4 instrument (Västra Frölunda, Sweden). In the QCM-D experiment, the changes in the frequency (Δf) and the energy dissipation (ΔD), resulting from the adsorption on the sensor, are simultaneously measured. From the Sauerbray equation, a decrease in the frequency of the crystal oscillation indicates that adsorption is occurring on the sensor surface. The Sauerbray equation is valid only for rigid films absorbed on the surface. For flexible films, the adsorbed mass can be calculated using the Voigt viscoelastic model.20 The second parameter monitored during the QCM-D experiment is the energy dissipation, which is related to the viscoelastic properties of the adsorbed film. A QCM-D sensor (Q-sense) with a thin surface layer of gold was used in all experiments as a support for adsorption.

Table 1. Physical Characteristics of G6 PAMAM Dendrimers primary amine groups tertiary amine groups total amine groups number of atoms hydrodynamic radius RH radius R radius R pKa i.e.p. isoelectric point i.e.p. isoelectric point bare charge Nm from structural formula max effective charge Nc

ΔΘSPR kdG6

Deionized water with a conductivity of ca. 1 μS/cm was used in the preparation of all solutions. Sodium chloride (NaCl), hydrochloric acid (HCl), and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich. NaCl was used as the supporting electrolyte. All electrolyte solutions were filtered using a 0.22 μm Millipore filter to eliminate aggregates. All experiments were performed at a constant temperature of 298 ± 0.1 K. Methods. Multiparametric Surface Plasmon Resonance (MP-SPR). The MP-SPR measurements were performed using a MP-SPR model Navi 200 (BioNavis Ltd., Finland), which consists of a goniometer and prism coupling-based device 19679

DOI: 10.1021/acs.jpcc.6b05020 J. Phys. Chem. C 2016, 120, 19678−19685

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Figure 2. (A) MP-SPR sensogram illustrating the immobilization of G6 PAMAM dendrimer on Au sensor. (B) Adsorbed mass ΓMP‑SPR [ng/cm2] calculated using eq 1. The G6 PAMAM concentration c = 5 ppm in NaCl solution at I = 1 × 10−2 M at different pH values.

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κa, were a is the dendrimer radius in the adsorbed state and κ is the inverse Debye length. For ionic strength I = 1 × 10−2 M, κa is equal 1.21 nm. Theoretically, the surface coverage can be estimated by a random sequential adsorption model (RSA).22 The RSA model assumes that the dendrimers are adsorbed irreversibly, in a sequential manner, at random positions and without overlapping. For charged particles such as dendrimers, the radius in the RSA model can additionally take into account the electrostatic repulsion between the charged dendrimers. The mass of the adsorbed dendrimers can be evaluated per unit of the geometrical surface, ΓAd [ng/cm2]. ΓAd can be estimated from the following formula

RESULTS AND DISCUSSION Adsorption of PAMAM Followed by MP-SPR Method. In the surface plasmon resonance method the resonance angle strongly depends on the local reflective index of the medium in the close vicinity of the metal surface. Therefore, this method is an extremely sensitive technique for studying the immobilization or interactions of macromolecules at a metallic surface. SPR can be used for real time and label-free detection of many types of biological recognition phenomena, such as ligand− receptor interactions, protein−protein interactions, or polymer−protein interactions. The adsorption of dendrimers was followed, using the MPSPR technique, on a gold sensor surface at an ionic strength of 1 × 10−2 M. The changes in the resonance angle (ΔΘ) are illustrated in Figure 2A and, consequently, the adsorbing mass, ΓSPR, as a function of time in Figure 2B. In the first stage, the baseline was obtained for the supporting electrolyte solution; in this case, the NaCl solution. In the next stage, the dendrimer solution of the concentration 5 ppm, in the pH range of 3.0 to 10.0, was allowed to flow through the MP-SPR cell. It caused the increase of the ΓSPR signal with time, until it reached a plateau. After 30 min of adsorption, the film was rinsed with the supporting electrolyte solution. Slight changes were observed in ΓSPR, which indicates that the dendrimer molecules were irreversibly adsorbed onto the sensor surface. The mass of the dendrimer films adsorbed onto the Au surface is very sensitive to the pH of the dendrimer solution. An increase in the pH results in a corresponding increase in the mass of the adsorbed film. The adsorbed mass increases slowly as the pH is increased from 4.0 to 9.0. A significant change in the adsorbed mass is observed between pH 9.0 and 10. The maximum value is obtained near the isoelectric point (i.e.p.) (pH = 10 for sixth generation13) of PAMAM dendrimers. When the pH is coincident with the polymer i.e.p., the macromolecules present the most compact structure and possess minimal charge. The adsorption mass decreases with decreasing pH, from 270 ± 0.06 ng/cm2 at pH 10.0 to 25 ± 0.06 ng/cm2 at pH 4.0, i.e., the amount of mass adsorbed increased four times with an increase in pH from 4.0 to 9.0, and ten times when the pH was increased from 4.0 to 10.0. This phenomenon of increasing adsorption with an increase in pH may have an electrostatic origin. Generally, the adsorbed mass per layer depends on the molecular packing at the surface. The packing-related surface coverage is sensitive to many factors of which the most important are molecular size, shape, charge, and features of the surface (chemical composition).21,22 The surface coverage of charged nanoparticles is a function of the screening parameters

ΓAd =

Mw Mw Θ Θ= SG6AV πRH 2A v

(2)

where Mw is the molecular weight (PAMAM G6Mw = 58 kDa), SG6 is the geometrical cross-sectional area SG6 = πRH2, Av is Avogadro’s number, and Θ is the surface coverage. For noninteracting, spherical, uniform particles, the RSA model estimates a maximum coverage of 0.547.21−23 In the case of interacting (charged) particles, this value can be much smaller, depending on the interaction range, correlated with the thickness of the electric double layer. Using eq 2 and assuming RH = 3.75 ± 0.05 nm for a surface coverage Θ = 0.547, the adsorption mass for a monolayer is calculated as ΓAd = 123 ng/ cm2. This adsorption value will be reached if the dendrimers can form a uniform monolayer on the surface. The adsorbed mass obtained from MP-SPR for pH 4.0 to 9.0 is lower than that predicted for a monolayer (Figure 3). This can be explained by considering that in this situation, the dendrimer molecules are highly charged and thus interact with each other. Electrostatic interactions cause lower coverage than that predicted for noninteracting molecules. The mass progressively increased from pH 4.0 to pH 9.0 due to the concurrent, gradual decrease in the charge of the dendrimers.13 The MP-SPR measurements show a coverage Θ = 0.191 for pH 4.0, which is a fraction of the maximum coverage. At pH 9.0, the coverage Θ = 0.458 is close to the theoretical value, Θmax = 0.547, for spherical, noninteracting, hard particles (Figure 4A). At pH 9.0, the zeta potential of the dendrimer is 15.3 ± 0.8 mV, several times lower than at pH 4.0 (see Table 2). The electrostatic repulsion between dendrimers at pH 4.0 is very strong in comparison to that at pH 9.0 (Figure 4B). The values of zeta potential and surface coverage in relation to the pH of the solution are summarized in Table 2. At pH 10.0, the absorbed mass suddenly increases and reaches a value much higher than that for monolayer formation. This suggests that in these 19680

DOI: 10.1021/acs.jpcc.6b05020 J. Phys. Chem. C 2016, 120, 19678−19685

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related to the effective radius of the macromolecules (aeff). Figure 4 presents the surface coverages of dendrimers ΘMP‑SPR for varying pH, in relation to the zeta potential of the dendrimer and the effective radius of the dendrimers in the adsorbed film. It can be seen that the effective radius at pH 4.0 is 6.26 nm, which is nearly equal to the size of the dendrimer added to the length of a double layer. The hydrodynamic radius of dendrimers obtained from dynamic light scattering is 3.75 ± 0.05 nm,13 and the Debye length is 3.07 nm for an ionic strength of 1 × 10−2 M. As the pH is increased, the effective radius decreases. At pH 9.0, i.e., around the i.e.p., the effective radius approaches 4.04 nm. This estimate of the effective radius can be further used to extract the interaction values in order to characterize the electrostatic repulsion range between the dendrimer molecules in a monolayer on the gold surface. The effective radius concept can be used, for example, to describe the blocking effect of the interacting particles.21,23 It can be noticed that, additionally, the solid surface (in this case, the gold surface) interacts with the dendrimers molecules. The zeta potential measurements can be used to estimate the charge density on the gold surface using the Gouy−Chapman equation for a symmetric electrolyte (NaCl, in our case).24

Figure 3. Comparison of the adsorbed mass ΓMP‑SPR [ng/nm2] of G6 PAMAM dendrimer with concentration c = 5 ppm in NaCl solution at I = 1 × 10−2 M at different pH values on the gold surface using MPSPR (error estimate is ±0.06 ng/cm2). The dotted lines show the theoretical results calculated from eq 2 for coverage Θ = 0.547. The dashed curve is interpolation of experimental data.

conditions, i.e., near i.e.p., the dendrimer molecules have a tendency to form aggregates on the surface. The maximum mass of the adsorbed dendrimers ΓMP‑SPR obtained in the MP-SPR experiments is lower than the theoretical values, due to the lateral electrostatic interactions among adsorbed molecules. This effect can be taken into account by using the “effective hard particle” concept. The distance between particles (hrep), characterizing the repulsive double-layer interaction, is connected with the interaction energy between dendrimer molecules. The surface coverage obtained in the MP-SPR experiments can be used to estimate the effective radius of the dendrimers on the surface at different values of pH. In this calculation, it was assumed that the maximum adsorption amount obtained for all pH values is

σ0 =

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

(3)

where σ0 is the charge density expressed in e/cm2, ε is the dielectric constant of water, and nb is the concentration of ions. The charge density of the gold surface is sensitive to the pH of the solution and approaches the zeta potential, −32.5 mV at pH 5.5.24 Under these conditions, the charge density is equal to −0.0163 e/nm2. The gold sensor is highly charged at a high pH and weakly charged at low pH. The i.e.p. for the gold surface is

Figure 4. Comparison of the surface coverage ΘMP‑SPR of G6 PAMAM on the gold surface using MP-SPR versus (A) pH, (B) zeta potential of G6 PAMAM dendrimer ζ, and (C) effective radius aeff (●) with the electrostatic repulsion range hrep (▲). The points denote experimental results derived using MP-SPR measurement after t = 30 min adsorption of c = 5 ppm G6 PAMAM dendrimer at I = 1 × 10−2 M. The dotted lines show the theoretical results calculated from eq 2 for the G6 PAMAM monolayer (coverage ΘRSA = 0.547). The dashed curves are interpolations of experimental data. 19681

DOI: 10.1021/acs.jpcc.6b05020 J. Phys. Chem. C 2016, 120, 19678−19685

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Table 2. Electrophoretic Mobility μD, Zeta Potential ζD, Number of Uncompensated Charge ND, Charge Density σD of G6 PAMAM Dendrimers, and Surface Coverage ΘMP‑SPR on Au Surfacea μD [μm·cm·V−1·s−1]b

pH 4.1 5.0 6.0 7.2 8.2 9.0 10.0

5.6 5.1 5.0 4.4 2.8 0.8 −0.1

± ± ± ± ± ± ±

0.18 0.30 0.24 0.14 0.19 0.06 0.01

ζD [mV]c 106.3 98.7 96.0 84.6 53.0 15.3 −2.0

± ± ± ± ± ± ±

2.3 3.9 3.1 1.8 2.4 0.8 0.2

ND [e]d

σD [e·nm−2]e

ΘMP‑SPRf

21.9 20.2 19.7 17.3 10.8 3.1 −0.6

0.1273 0.1174 0.1145 0.1006 0.0628 0.0180 −0.0035

0.191 0.294 0.329 0.360 0.418 0.458 1.223

Conditions: G6 PAMAM dendrimer, I = 1 × 10−2 M, T = 298 K, η = 8.9 × 10−3 g (cm·s)−1, RH = 3.75 × 10−7 cm. bDetermined from the 6πηR 3η electrophoretic mobility.13 cCalculated from equation ζD = 2εF(κa) μe . dCalculated from equation ND = e H μe . eCalculated from equation

a

σD =

ND 4π R H 2

f

Determined from MP-SPR measurements.

near pH 2.8.24 For comparison, the charge density of the dendrimer molecules (see Table 2) changes from 0.1273 e/nm2 at pH 4.0 to −0.0035 e/nm2 at pH 10.0. It should further be noted that an inverse correlation is observed between the charge of the dendrimer of the gold surface and the pH of the solution. During adsorption, two types of interactions may occur: interdendrimer interactions and dendrimer−surface interactions. Interactions between the dendrimer and the surface cause partial shielding of the net charge on the surface of the dendrimer molecules. As a result, the effective charge of the adsorbed molecules is generally less than in the bulk solution. Further, the surface causes deformation of the particles during the process of adsorption. The degree of deformation is correlated with the strength of the interaction and the properties (e.g., hydrophobicity) of the surface itself. The literature shows examples comparing the degree of deformation of dendrimers for different types of surfaces (hydrophobic or hydrophilic).25 From these data, it can be concluded that dendrimers are more deformed on hydrophilic surfaces rather than hydrophobic surfaces. This indicates that the deformation on the gold surface is not very strong because the contact angle for the gold surface is 75° (see Figure 1B). The interaction of positively charged dendrimers with oppositely charged surface results in flattening of molecules on the surface.26 Computational studies performed by Welch27 show that increasing the surface charge density maximizes the contact between molecules and the surface. Additionally, the ionic strength (κ−1 Debye length) can be used to adjust the type of contact between the surface and the dendrimer molecule during adsorption, which ultimately leads either to the deposition (capture) or escape of molecules from the surface.26,27 According to classical Deyaguin−Landau−Verwey−Overbeek theory (DLVO), the potential energy has contributions from electrostatic and van der Waals interactions. However, while the DLVO theory provides an excellent description of colloidal systems, it is not sufficient for the description of nanosized systems. This is because for nanosized systems, such as dendrimers (in our case 3.75 nm), where the size of the nanoparticles is comparable to the range of interactions, the hydrophobic interaction must also be taken into account. In this case, the local density of charge on the adsorbed surface can be crucial in determining the interaction with nanoparticles.28 Additionally, the finite size of the solvent must also be taken into account. Additionally, we attempted to interpret the dendrimer adsorption data from the MP-SPR experiments, to gain an

insight into mass transport in the MP-SPR parallel-plate channel. To enable this analysis, the physicochemical parameters of the dendrimer system must be known (the diffusion coefficient DG6 and the cross-sectional area of the molecule SG6 by AFM).13 Therefore, the geometrical PAMAM G6 coverage can be calculated from formula Θ = SG6NG6, where NG6 is the surface concentration of adsorbed dendrimers. Under the stationary convective-diffusion transport conditions, NG6 = KSPRcbt. NG6 can be calculated using the mass transport rate constant, KSPR in the MP-SPR cell, and t, the adsorption time. Then, the average surface coverage of dendrimers on the MP-SPR sensor can be estimated by the following formula Θ = SG6KSPR cbt

(4)

For the dendrimer G6, for I = 1 × 10−2 M, assuming DG6 = 6.54 × 10−7 cm2/s, KSPR = 2.08 × 10−4 cm/s, and SG6 = 44.14 × 10−14 cm2 from eq 4, the dendrimer coverage can be calculated for different concentrations and adsorption times. Thus, for c = 5 ppm and an adsorption time of 1.95 min, the average dendrimer coverage Θ = 0.547. For a ten times lower concentration, c = 0.5 ppm, an adsorption time of 19.5 min is required to obtain the same surface coverage. Figure 5 shows a comparison of experimental (using MP-SPR) and theoretical dendrimer adsorptions at pH 6.0 for three concentrations. It is evident that the theoretical and experimental dendrimer coverages are well-correlated for the three concentrations presented in Figure 5.

Figure 5. Time dependence of the adsorbed mass (ΓMP‑SPR) on the Au SPR sensor after adsorption of G6 PAMAM at I = 1 × 10−2 M at pH 6.0 for three different concentrations: (1) c = 0.05 ppm, (2) c = 0.1 ppm, (3) c = 5 ppm. The dashed lines show the theoretical results calculated from eq 4 and the solid lines show experimental data. 19682

DOI: 10.1021/acs.jpcc.6b05020 J. Phys. Chem. C 2016, 120, 19678−19685

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Figure 6. (A) Time dependence of the frequency shift (Δf), (B) the dissipation energy (ΔD), and (C) the adsorbed mass (ΓQCM‑D) on the Au QCM sensor after adsorption of c = 5 ppm G6 PAMAM at I = 1 × 10−2 M and various pH ranging from 4.0 to 10.0.

Adsorption of G6 PAMAM Followed by QCM-D Method. The efficiency of adsorption of dendrimers was also measured using a crystal microbalance with dissipation energy monitoring. Two system parameters are recorded in the QCMD method: the change in frequency, Δf, and the change in dissipation energy, ΔD. The procedure for all QCM-D experiments was identical to that of MP-SPR. Using the Sauerbrey model, the frequency signal was used to calculate adsorbed mass on the sensor surface. We chose the Sauerbrey model because of the low levels of dissipation energy for all measurements. In the pH range of 4.0−7.0, the dissipation energies are in the range 0.5−0.75. For pH > 7, the dissipation energy increases monotonically with pH, and a value of 1.5 is obtained at pH 10.0. These data suggest that the adsorbed films are rigid. The dependence of Δf (Figure 6A), ΔD (Figure 6B). and ΓQCM‑D (Figure 6C) on the adsorption time is shown for various kinetic runs acquired by QCM-D. The signal rises steeply and linearly with time for t < 5 min, after which it slowly starts approaching the saturation value. The occurrence of the linear regime for the initial time indicates that the adsorption of PAMAM dendrimers is controlled by the bulk transport. After 30 min adsorption, the film was rinsed with the supporting electrolyte solution for the next 30 min. The adsorption of dendrimers on the gold surface sensor is irreversible, as indicated by the low degree of desorption. The highest desorption is observed at pH 4.0 when the gold surface is least charged. In the pH range of 6−10, the degree of desorption remains constant at approximately 2.5−5%. In this regime, the surface charge of gold has its highest value. Figure 7 shows a comparison of results for all tested pH values. These measurements indicate the mass of dendrimers irreversibly adsorbed on the gold surface. A value of 100 ng/ cm2 is obtained at pH 4.0, and the values increase considerably with increasing pH. The maximum mass is adsorbed at pH 10.0. The maximum mass of 700 ± 0.3 ng/cm2 is six times

Figure 7. Comparison of the adsorbed mass, ΓQCM‑D [ng/cm2] of dendrimers, obtained from QCM-D (error estimate is ±0.3 ng/cm2). The G6 PAMAM concentration c = 5 ppm in NaCl solution at ionic strength I = 1 × 10−2 M for various pH conditions. The dotted lines show the theoretical results calculated from eq 2 for the G6 PAMAM monolayer (coverage Θ = 0.547) and G6 PAMAM monolayer +80% H2O. The dashed curves are interpolation of experimental data.

higher than the mass at the lowest tested pH. For any pH, the adsorbed mass of dendrimers on the QCM sensor is much higher than theoretical value ΓAd = 123 ng/cm2. This discrepancy may be explained by two reasons. The first explanation is the possible formation of multilayers due to surface aggregation of the uncharged dendrimers (at pH 10 i.e.p.). Additionally, it must be noted that the QCM-D method senses the total mass adsorbed at the sensor surface. This means that the measured mass is the sum of the mass of dendrimers adsorbed on the sensor plus the mass of the water bound to the adsorbed film. The thickness of the films adsorbed on the gold sensor was calculated from the QCM-D data. The dendrimer film thickness varies in the range from 2.1 to 6.6 nm. For pH 4.0, the film thickness is 2.1 nm and gradually increases to 6.6 nm for pH 10. Because it is known that the hydrodynamic radius of 19683

DOI: 10.1021/acs.jpcc.6b05020 J. Phys. Chem. C 2016, 120, 19678−19685

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CONCLUSIONS The combination of MP-SPR and QCM-D measurements allows the investigation of factors that govern the immobilization of PAMAM dendrimers on an Au sensor surface. Changes in pH have a strong effect on the binding affinity of the polymer to the surface and additionally improve the self-assembling behavior of PAMAM dendrimers. The trends in the binding affinity and the surface saturation amount correspond well with the degree of change of protonation of the adsorbed molecules. The highest amount of adsorption was obtained at pH = 10.0 (close to the i.e.p. of G6 PAMAM) and the lowest at pH 4.0, by both methods. For all measurements, the mass obtained by the QCM method was six times higher than that obtained by MPSPR. This comparison allowed the estimation of water molecules associated with the dendrimer structure and determination of how the degree of hydration changes with pH. These data very clearly indicate how the structure of the dendrimer can influence the properties of the polymer film formed on the gold surface. PAMAM films on gold surfaces are composed of 80% of water. This is a particularly large value compared to the amount of water associated with the dendrimer molecules during the swelling process, which was estimated as 25−30%. This is one of the additional advantages of dendrimeric systems.

the dendrimer is 3.75 nm, the surface is expected to be a monolayer rather than a multilayer. Assuming a high degree of hydration in the monolayer of the PAMAM film, e.g., at a level of 80%, the mass adsorbed on the surface can be 615 ng/cm2. This value is approached at pH 9.0. At a pH in the range of 8.0−4.0, the mass adsorbed is much lower. Under these conditions, the PAMAM dendrimer does not form a full monolayer on the gold surface. Comparison of MP-SPR and QCM-D Results. MP-SPR and QCM-D are well-established techniques for studying polymer adsorption.14,15 The comparison of this methods is important for biological samples or swelling systems. A comparison of the dendrimer mass adsorbed on the gold surface shows that both methods yield similar trends. In the entire pH range, the adsorbed amount obtained by QCM-D is much higher than that by MP-SPR. This discrepancy is caused by the additional water mass sensed by the QCM-D method. From the combination of the QCM-D and MP-SPR data, with the assumption that the excess mass measured in QCM-D compared to the MP-SPR mass is due to trapped water molecules, we estimated the fraction of water in the dendrimers film according to eq 5. Γ H2O[%] =

ΓQCM ‐ D − ΓMP ‐ SPR ΓQCM ‐ D

Article

× 100

■

(5)

where ΓMP‑SPR is the adsorbed mass from MP-SPR method, ΓQCM‑D is the adsorbed mass from QCM-D method, and ΓH2O [%] is fraction of water in the dendrimer film. The structure of the resulting films is strongly dependent on the deposition conditions. In the pH range of 4.0−9.0, the dendrimer films show 83% hydration for pH 4.0, 78% for pH 7.0, and 83% for pH 9.0. Finally, at pH 10.0, the degree of hydration slowly decreases to 64%. These results indicate that sixth generation PAMAM dendrimers form very hydrated films on gold surfaces (Figure 8).

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +48 126395125. Fax: +48 124251923. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grant NCN OPUS 2012/07/B/ ST5/00767. The authors thank the Bionavis Company for the cooperation and support.

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REFERENCES

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Figure 8. Comparison of the adsorbed amount of the G6 PAMAM dendrimers mass Γ [ng/cm2] on an Au sensor obtained using MP-SPR and QCM-D techniques (error estimates are ±0.06 and ±0.3 ng/cm2, respectively). The dashed curves are interpolations of experimental data.

Please note that the molecules of dendrimers undergo reversible swelling.29 For an ionic strength of I = 1 × 10−2 M, the difference in hydration between pH 4.0 and 8.0 is 25%.13 Three types of water may be distinguished in the film structure: waters that are a part of the internal structure of dendrimers, those forming the outer shell of hydration, and those that are loosely bound or associated with the dendrimer film. Similar levels of dendrimer hydration were observed on a silica surface.30 19684

DOI: 10.1021/acs.jpcc.6b05020 J. Phys. Chem. C 2016, 120, 19678−19685

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DOI: 10.1021/acs.jpcc.6b05020 J. Phys. Chem. C 2016, 120, 19678−19685