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Jul 31, 2014 - The structure and interaction in complexes of anionic Ludox HS40 silica nanoparticle, anionic bovine serum albumin (BSA) protein, and ...
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Cationic versus Anionic Surfactant in Tuning the Structure and Interaction of Nanoparticle, Protein, and Surfactant Complexes Sumit Mehan,† Vinod K. Aswal,*,† and Joachim Kohlbrecher‡ †

Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai 400 085, India Laboratory for Neutron Scattering, Paul Scherrer Institut, CH-5232 PSI Villigen, Switzerland

‡

ABSTRACT: The structure and interaction in complexes of anionic Ludox HS40 silica nanoparticle, anionic bovine serum albumin (BSA) protein, and cationic dodecyl trimethylammonium bromide (DTAB) surfactant have been studied using small-angle neutron scattering (SANS). The results are compared with similar complexes having anionic sodium dodecyl sulfate (SDS) surfactant (Mehan, S; Chinchalikar, A. J.; Kumar, S.; Aswal, V. K.; Schweins, R. Langmuir 2013, 29, 11290). In both cases (DTAB and SDS), the structure in nanoparticle−protein−surfactant complexes is predominantly determined by the interactions of the individual twocomponent systems. The nanoparticle−surfactant (mediated through protein−surfactant complex) and protein−surfactant interactions for DTAB, but nanoparticle−protein (mediated through protein−surfactant complex) and protein−surfactant interactions for SDS, are found to be responsible for the resultant structure of nanoparticle−protein−surfactant complexes. Irrespective of the charge on the surfactant, the cooperative binding of surfactant with protein leads to micellelike clusters of surfactant formed along the unfolded protein chain. The adsorption of these protein−surfactant complexes for DTAB on oppositely charged nanoparticles gives rise to the protein−surfactant complex-mediated aggregation of nanoparticles (similar to that of DTAB surfactant). It is unlike that of depletion-induced aggregation of nanoparticles with nonadsorption of protein− surfactant complexes for SDS in similarly charged nanoparticle systems (similar to that of protein alone). The modifications in nanoparticle aggregation as well as unfolding of protein in these systems as compared to the corresponding two-component systems have also been examined by selectively contrast matching the constituents.

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INTRODUCTION

There are numerous studies for the interaction of nanoparticles with protein as well as surfactant to improve the properties of their complexes.14−17 The resultant properties of complex systems depend on both nanoparticle and protein (or surfactant) as well as on various solution conditions.11,12,18−20 In all these, electrostatic interaction among the components plays an important role in controlling the structure and interaction of such complexes.10,12 For example, the interaction of silica nanoparticles with oppositely charged lysozyme protein and DTAB surfactant results in strong adsorption of protein/ micelle on the nanoparticles, which in turn leads to protein/ micelle-mediated fractal aggregation of the nanoparticles.10,12,21 The nanoparticle aggregates coexist with unaggregated nanoparticles at low protein/micelle concentrations whereas with free protein/micelle at high concentrations. In the case of similarly charged BSA protein and SDS surfactant, they show nonadsorption on silica nanoparticles which can lead to depletion force between the silica nanoparticles.22−24 This depletion interaction can result in nanoparticle aggregation

There is a lot of recent scientific and industrial interest in nanoparticles in a variety of applications from biomedical to electronic to functional materials.1−4 Many of these applications require the adsorption of amphiphilic molecules such as protein or surfactant to modify their properties for specific applications.5−9 For example, protein-coated nanoparticles have enhanced biocompatibility and have been used in diverse potential applications in drug delivery and biosensors.6,7 The adsorption of surfactant to nanoparticles gives enhanced colloid stability, which is useful in detergent and cosmetic industry.8,9 The properties of such complexes can be varied by various parameters which include charge on individual component, pH, ionic strength, etc.10−12 Nanoparticle−protein−surfactant complexes are used in food, medicine, etc., where three components in varying proportions coexist with each other.13 The protein−surfactant complex provides the advantage of effective tuning of adsorption behavior with nanoparticles by varying the concentration ratio of protein to surfactant or charge of surfactant molecules.13,14 Therefore, it is interesting to study the interactions in these complexes and tune them for various potential applications. © 2014 American Chemical Society

Received: June 20, 2014 Revised: July 24, 2014 Published: July 31, 2014 9941

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have low incoherent background and provide better contrast in neutron scattering experiments for hydrogenous components (e.g., BSA, DTAB). The mixed solvents of D2O and H2O were also used for contrast matching individual components. D2O (99.9 atom % D) and distilled deionized water from a Millipore Milli-Q unit were used as solvent. The silica nanoparticle interaction with protein and surfactant is studied at fixed concentrations of nanoparticle (1 wt %), protein (1 wt %), and surfactant (50 mM). Small-angle neutron scattering experiments were performed at the SANS-I facility, Swiss Spallation Neutron Source SINQ, Paul Scherrer Institut, Switzerland.32 The wavelength (λ) of neutron beam used was 8 Å, and the scattered neutrons from samples were detected using a two-dimensional 96 cm × 96 cm detector. Data were collected at two sample-to-detector distances of 2 and 8 m to cover a wave vector transfer (Q = 4π sin(θ/ 2)/λ, where θ is the scattering angle) range of 0.005 to 0.30 Å−1. Samples were held in standard 1 mm or 2 mm path length quartz cells during the experiments. Data were corrected for background and empty cell and normalized to absolute units of cross-section using standard procedures.

similar to that of oppositely charged nanoparticle−protein/ surfactant systems.25 On the other hand, the interactions in protein−surfactant complexes are significantly different, and cooperative binding of surfactant with protein leads to the unfolding of protein through micellelike clusters attached along the unfolded polypeptide chain.26,27 The cooperative binding is predominantly hydrophobic so both the anionic (SDS) and cationic (DTAB) surfactants undergo quite similar binding with anionic (BSA) protein.28,29 It is of interest to know how the different two-component interactions control the structure and interaction in three-component systems. There is also interest in knowing the role of a particular component in modifying the interaction of other two components. We have recently studied the three-component system of silica nanoparticle, BSA protein, and SDS surfactant, where all three components are anionic.14 It is shown that the interaction of individual two-component systems (nanoparticle−protein, nanoparticle−surfactant, and protein−surfactant) governs the properties of the three-component system. The nanoparticle− protein system shows depletion force-induced nanoparticle aggregates coexisting with free protein. Both components remain independent in the nanoparticle−surfactant system, whereas the surfactant interacts cooperatively with protein, leading to a micellelike cluster of the surfactants formed along the unfolded chain of protein. The structure of the threecomponent (nanoparticle−protein−surfactant) system is found to be determined by the synergetic effect of nanoparticle− protein and protein−surfactant interactions. The protein− surfactant complex controls the structure of the threecomponent system, leading to nanoparticle aggregates similar to that of the nanoparticle−protein system. The nanoparticle aggregation in the three-component system as compared to nanoparticle−protein as well as unfolding of protein in the three-component system as compared to protein−surfactant is enhanced. In the present work, a three-component system where the anionic surfactant SDS has been replaced by cationic surfactant DTAB is examined. This kind of change in the threecomponent system is expected to significantly modify the nanoparticle−surfactant and protein−surfactant interactions.21,28 The structure and interaction in these twocomponent systems and their role in the three-component system have been studied by small-angle neutron scattering (SANS). The technique of SANS is an ideal probe to study both structure and interaction of such multicomponent systems under a native environment.14,21,24,30,31 This technique is used for various kinds of nanostructure materials in a length scale of 1−100 nm. Because the neutron scattering is different for hydrogenous and deuterated components, a mixed solvent in different proportions of these components has been used in contrast variation experiments to simplify the scattering from complex systems.

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SANS ANALYSIS In SANS experiments, one measures the differential scattering cross-section per unit volume d∑/dΩ as a function of Q. For a system of monodisperse particles in a solvent, d∑/dΩ can be expressed as33,34 d∑ (Q ) = nVp2Δρ2 P(Q )S(Q ) + B dΩ

(1)

where n is number density of particles, Vp is particle volume, and Δρ2 is the scattering contrast of particles. P(Q) and S(Q) are the intraparticle and interparticle structure factors, respectively. B is the incoherent background, which predominately arises from the presence of hydrogen in the sample. P(Q) depends on the shape and size of particles.35 For spherical particles (radius R), it is given by ⎡ j (QR ) ⎤2 ⎥ Ps(Q ) = 9⎢ 1 ⎣ QR ⎦

(2)

where j1(x) = ((sin x − x cos x)/x2) is the first-order spherical Bessel function. In the case of ellipsoidal particles with its semiaxes R and εR, Pe(Q) can be written as35 Pe(Q ) =

∫0

π /2

Ps(Q , r )sin β dβ

r = R sin 2 β + ε 2 cos2 β

(3) (4)

where ε < 1 for oblate ellipsoidal and ε > 1 for prolate ellipsoidal shape of particles. β is the angle between the semimajor axis and wave vector transfer. For a polydisperse system, d∑/dΩ is modified by the size distribution of particles as36

EXPERIMENTAL SECTION

Electrostatically stabilized colloidal suspension of Ludox HS40 silica nanoparticles (catalogue no. 420816), BSA protein (catalogue no. A2153), and dodecyl trimethylammonium bromide surfactant (catalogue no: D5047) were purchased from Sigma-Aldrich. The stock solutions of each component were prepared by dissolving the required weighted amounts of components in 20 mM phosphate buffer (pH = 7) in the presence of 0.2 M NaCl. The salt is used to reduce the electrostatic interaction between different components. The neutron scattering experiments provide a different contrast for the particles in H2O and D2O. The samples were prepared in D2O which

d∑ (Q ) = dΩ

∫

d∑ (Q , R )fs (R )dR + B dΩ

(5)

where fs(R) is the size distribution function. It is usually accounted for by log-normal distribution as given by f (R ) = 9942

⎡ (ln R /R )2 ⎤ med ⎥ exp⎢ − 2 Rσ 2π 2σ ⎦ ⎣ 1

(6)

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where Rmed and σ are the median value and standard deviation, respectively. The mean (Rm) and median values are related as Rm= Rmed exp(σ2/2). In the case of interaction leading to the adsorption of micelles or proteins on nanoparticles, d∑/dΩ for such a core− shell structure can be expressed as35

throughout the data analysis.40 The modeled scattering profiles were smeared by the appropriate resolution function to compare with the measured data. The corrections for incoherent background were taken in the model calculations as a fitting parameter. Because the scattering in the high Q region is fully dominated by incoherent scattering, no attempts were made to make much use of high Q data in the modeling. The fitted parameters in the analysis were optimized by means of a nonlinear least-squares fitting program.41 The fitted data are represented by the solid lines to the experimental data points.

d∑ (Q ) = dΩ 2 ⎡ ⎧ 3j (Qr1) ⎫ ⎧ 3j (Qr2) ⎫⎤ 1 1 ⎢ ⎥ ⎨ ⎬ ⎨ ⎬ S(Q ) + (ρshell − ρs )V2 n (ρc − ρshell )V1 ⎢⎣ ⎩ Qr1 ⎭ ⎩ Qr2 ⎭⎥⎦ ⎪

⎪

⎪

■

⎪

(7)

+B

RESULTS AND DISCUSSION We first characterize and compare the SANS data from pure components of nanoparticle, protein, and surfactant systems. The data from pure 1 wt % HS40 silica nanoparticle, 1 wt % BSA protein, and 50 mM (1.54 wt %) DTAB surfactant systems are shown in Figure 1. Nanoparticles and proteins show typical

where ρc, ρshell, and ρs are, respectively, the scattering length densities of the core, shell, and solvent. The dimensions r1, r2, V1, and V2 are the inner radius, outer radius, volume of core, and volume of the core along with the shell, respectively. S(Q) is decided by the interparticle interaction, and for repulsive particles it has been calculated as derived by Hansen and Hayter from the Ornstein−Zernike equation under the rescaled mean spherical approximation.37 The particles are approximated by the equivalent sphere interacting through a screened Coulomb potential, which is given by u(r ) = u0σ

exp[−κ(r − σ )] , r

r>σ

(8)

where σ is the particle diameter, κ is the Debye−Hückel inverse screening length, and u0 is the contact potential. The strong interaction between oppositely charged micelles or proteins with nanoparticles is known to result in aggregation of nanoparticles. The aggregates are characterized by a mass fractal structure for which S(Q) is given by38 Sf (Q ) = 1 +

D Γ(D − 1) 1 sin{(D − 1) D (D − 1)/2 (Qr0) ⎡ 1 ⎤ 1 + ⎥ ⎣⎢ (Qξ)2 ⎦

× tan−1(Qξ)}

(9)

Figure 1. SANS data of 1 wt % HS40, 1 wt % BSA, and 50 mM DTAB in the presence of salt (0.2 M NaCl) in D2O. Inset shows the comparison of scattering profile of 50 mM DTAB (cationic) and 50 mM SDS (anionic) surfactants. The solid curves are theoretical fits to the experimental data.

where D is fractal dimension, ξ is the overall size of a fractal aggregate and r0 is characteristic dimension of individual particles. The cooperative binding of surfactant with protein is known to result in micellelike clusters of surfactant formed along the unfolded polypeptide chain. The cross-section for this bead− necklace model of a protein−surfactant complex can be written as39 d∑ N2 (Q ) = 1 (bm − Vmρs )2 P(Q )Sf (Q ) + B dΩ NpN

scattering profiles determined by the intraparticle structure factor P(Q), where interparticle structure factor (S(Q) ∼ 1) contribution can be neglected.14,21 On the other hand, a significant presence of interparticle structure factor is observed in the case of the DTAB surfactant system. The features of the DTAB surfactant are very much similar to that of SDS surfactant (inset of Figure 1). The fitted parameters of all the components are given in Table 1. The HS40 silica nanoparticles are spherical with a mean radius of 87.1 Å and polydispersity of 0.22.21,25 The BSA proteins have oblate ellipsoidal shape with semiminor and semimajor axes of 13.6 and 42.3 Å, respectively.14,21,42 The DTAB micelles form prolate ellipsoidal core−shell structure with the semiminor axis, semimajor axes, and thickness as 16.7 Å, 21.0 Å, and 5.8 Å, respectively.43,44 The aggregation number of micelles is found to be 70. The structure of SDS micelle is similar to that of DTAB micelles except they are oppositely charged. S(Q) was calculated using screened Coulomb potential between micelles (eq 8) under the rescaled mean spherical approximation.37 The effective charges on the DTAB and SDS micelles are found to be around +21 and −25

(10)

where N1, Np, and N are, respectively, the number density of the total surfactant molecules, number density of protein molecules, and number of such micelles attached to the polypeptide chain. bm, ρs, and Vm represent the scattering length of the surfactant molecule, scattering length density of solvent, and volume of the surfactant molecule, respectively. P(Q) for spherical micellelike clusters is given by eq 2, and Sf(Q) of mass fractal structure (eq 9) can be used for modeling the bead−necklace structure of adsorbed micelles along the unfolded protein chain. The data have been analyzed by comparing the scattering from the combination of different models to the experimental data. The corrections for instrumental smearing were made 9943

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Table 1. Structural Parameters of 1 wt % HS40 Silica Nanoparticles, 1 wt % BSA Protein, and 50 mM DTAB Surfactant Solutions in the Presence of 0.2 M NaCl in D2O system

shape

silica nanoparticles BSA protein DTAB micelles

polydisperse spherical oblate ellipsoidal (ε < 1) prolate core−shell ellipsoidal (ε > 1)

structural dimensions radius Rm = 87.1 ± 1.0 Å semimajor axis R = 42.3 ± 0.5 Å semiminor axis R = 16.7 ± 0.2 Å aggregation number Na = 70 ± 4

e.u., respectively.43,44 The value of Debye screening length (1/ κ) of the solution (20 mM phosphate buffer with 0.2 M NaCl) is about 6.0 Å. The structural parameters of all the components (silica nanoparticles, protein, and micelles) are found to be in agreement with those reported in the literature.21,42,43 The differences in microstructure of nanoparticle−protein−surfactant for DTAB vs SDS are expected to be due to differences in the interaction of nanoparticle−surfactant and protein− surfactant complexes (anionic−cationic vs anionic−anionic) in these systems, as has been examined. SANS data of the mixture of a three-component system of silica nanoparticle−BSA protein−DTAB surfactant is shown in Figure 2. The SANS data as measured are also compared with

polydispersity σ = 0.22 ± 0.02 semiminor axis εR = 13.6 ± 0.2 Å semimajor axis εR = 21.0 ± 0.4 Å charge Z = 21 ± 2 e.u.

shell thickness t = 5.8 Å

nanoparticle−BSA protein−DTAB surfactant are also found to be significantly different from those of the silica nanoparticle− BSA protein−SDS surfactant system (inset of Figure 2), which in turn indicates that the interaction of the two components as well as type of surfactant plays an important role in tuning the properties of the three-component system.14 We have therefore examined two-component systems of silica nanoparticle−BSA protein, silica nanoparticle−DTAB surfactant, and BSA protein−DTAB surfactant for their role in the three-component system. Comparisons are also made for DTAB surfactant with respect to SDS surfactant in these systems. Among different possible two-component systems in the three-component system of silica nanoparticle−BSA protein− DTAB surfactant, we first examine interaction of the nanoparticle−protein system where both components are similarly (negative) charged. SANS data of 1 wt % HS40 with 1 wt % BSA are shown in Figure 3. The data, unlike those of the pure

Figure 2. SANS data of 1 wt % HS40 silica nanoparticles + 1 wt % BSA protein + 50 mM DTAB surfactant compared with the sum of scattering from individual components (1 wt % HS40 silica nanoparticles, 1 wt % BSA protein, and 50 mM DTAB surfactant) in the presence of salt (0.2 M NaCl) in D2O. Inset shows the comparison of scattering profiles of 1 wt % HS40 silica nanoparticles + 1 wt % BSA protein + 50 mM DTAB (cationic) surfactant with 1 wt % HS40 silica nanoparticles + 1 wt % BSA protein + 50 mM SDS (anionic) surfactant.

Figure 3. SANS data of 1 wt % HS40 silica nanoparticles + 1 wt % BSA protein compared with the sum of scattering from individual components (1 wt % HS40 silica nanoparticles and 1 wt % BSA protein) in the presence of salt (0.2 M NaCl) in D2O. Inset shows the scattering profile of 1 wt % HS40 silica nanoparticles + 1 wt % BSA protein along with 1 wt % HS40 silica nanoparticles and 1 wt % BSA protein in the presence of salt (0.2 M NaCl) in H2O. The solid curves are theoretical fits to the experimental data.

the simple addition of scattering from three individual components. The scattering profile over the whole Q range can be divided into three regions. The scattering from region III in high Q range almost overlaps with the sum of scattering from individual components. There are significant differences in scattering from the two profiles in the intermediate Q range (region II) and dramatic differences in the low Q range (region I). The observed differences in scattering profiles (regions I and II) suggest strong interactions among the components. In particular, the strong buildup of scattering and linearity on log− log scale indicates the formation of large size fractal aggregates in solution.18,25 The presence of these aggregates is also evident from the observed solution turbidity. The SANS data of silica

components (Figure 1), show a buildup of scattering in the low Q range. This cannot be explained on the basis of scattering as the sum of the two repulsive components. The buildup of scattering in the low Q range may arise from the protein adsorption on the nanoparticle17,21,45 (core−shell structure) or by the attractive depletion interaction between nanoparticles set up by the nonadsorption of protein.22,23,25 The proteins are known to have both the negative and positive patches, which can result in site-specific adsorption of protein on the nanoparticle even though they are overall similarly charged.46,47 9944

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To confirm this, SANS experiments were carried out using a solvent (H2O) where the scattering from the protein is minimized (inset of Figure 3). The scattering data of 1 wt % HS40 with 1 wt % BSA in H2O still show features similar to those in D2O. This clearly shows that SANS data in Figure 3 do not support the core−shell structure of protein adsorption on nanoparticles, which otherwise should have given data (1 wt % HS40 with 1 wt % BSA in H2O) similar to those of the pure nanoparticle (1 wt % HS40 in H 2 O) system. These observations therefore confirm that the buildup of scattering in the low Q range arises from the formation of nanoparticle aggregates due to nonadsorption of protein, leading to depletion interaction between the nanoparticles.15,25 Further, the high turbidity suggests that the system is much beyond the onset of nanoparticles experiencing depletion interaction; instead, they have achieved complete aggregation. The formation of aggregates (unlike attractive interaction) was also confirmed by dynamic light scattering measurements where a very small diffusion coefficient (large hydrodynamic size) was obtained even when the sample was diluted 10 times. Upon dilution, the increase in the average distance between the nanoparticles is expected to suppress the interaction. The linearity in the scattering profile of Figure 3 in the low Q region on log−log scale indicates that the aggregates follow fractal morphology.18,38 The fractal structures are usually determined by the three parameters comprising overall size of fractal (correlation length), building block radius, and fractal dimension. These parameters can be determined from the low Q cutoff, high Q cutoff, and slope of the data, respectively.18,25,38 The absence of low Q cutoff implies that the overall size of fractal aggregates are much larger than 2π/ Qmin ∼ 1000 Å, which can be measured from the Qmin value of the present measurement. The high Q cutoff value, as expected, corresponds to the diameter of the nanoparticles. The data are fitted with fractal aggregates of nanoparticles coexisting with free protein. The fractal dimension is found to be 2.55, indicating the diffusion-limited aggregation type fractal morphology of nanoparticle aggregates.48 The features of nanoparticle aggregation are also observed in the two-component silica nanoparticle−DTAB surfactant system. In this case, unlike that of silica nanoparticle−BSA protein, the electrostatic interaction mediated by oppositely charged surfactant between nanoparticles is expected to be responsible for the nanoparticle aggregation.21 A very small amount of DTAB is known to cause the nanoparticle aggregation, which rules out the possibility of aggregation through the process of charge neutralization on the nanoparticles. Figure 4 shows the SANS data of 1 wt % HS40 + 50 mM DTAB and also compared with 1 wt % HS40 + 1 wt % BSA. The scattering profile of 1 wt % HS40 + 50 mM DTAB shows linearity in the low Q range from the fractals of nanoparticle aggregates.21,38 The scattering in the intermediate to high Q range is dominated by DTAB micelles. The difference in microstructures of nanoparticle aggregates in the silica nanoparticle−DTAB surfactant system and silica nanoparticle−BSA protein system is also evident from the difference in scattering in the low Q range. The nanoparticle aggregates in the silica nanoparticle−DTAB surfactant system have a higher slope in the low Q region than that in the silica nanoparticle− BSA protein system. The higher slope (fractal dimension) means higher packing fraction of nanoparticles in these aggregates, which is expected because of the strong electrostatic binding mediated by surfactant micelles among the nano-

Figure 4. SANS data of 1 wt % HS40 silica nanoparticles + 50 mM DTAB surfactant along with 1 wt % HS40 silica nanoparticles + 1 wt % BSA protein in the presence of salt (0.2 M NaCl) in D2O. Inset shows the comparison of scattering profile of 1 wt % HS40 silica nanoparticles + 50 mM DTAB (cationic) surfactant with 1 wt % HS40 silica nanoparticles + 50 mM SDS (anionic) surfactant.

particles.10,12,38 The difference in scattering in the intermediate and high Q range is from the different scattering contrast and volume fractions of proteins and micelles in solution. The SANS data of silica nanoparticles with DTAB and SDS are compared in the inset of Figure 4. It had been found for SDS (very different from DTAB) that it is nonadsorbing as well as nondepleting even up to very high concentration.14,21 We believe that nanoparticle−surfactant interaction is the most important factor in deciding the differences in the structures of a three-component system having surfactant as DTAB or SDS. Further, the role of surfactant micelles in the nanoparticle− surfactant aggregates has been examined by contrast matching the individual components systematically. Figure 5 shows SANS data of the HS40 nanoparticle−DTAB surfactant system where (a) surfactant is contrast matched and (b) nanoparticles are contrast matched. The scattering of the nanoparticle−surfactant system as compared to the onlynanoparticle system under the surfactant contrast matched condition (2 vol % D2O in mixed D2O/H2O solvent) has very different behavior (Figure 5a). There are clearly two distinct features observed in the low and intermediate Q regions compared to that of the nanoparticle−surfactant system. This shows the formation of a new structure of nanoparticles in the presence of surfactant. The large scattering with linear dependence in the low Q region confirms the new structure as nanoparticle aggregates which are characterized by a mass fractal.21,48 The fractal dimension for the nanoparticle− surfactant system (D = 2.72) is found to be significantly larger than diffusion-limited aggregation of nanoparticles in the presence of protein.48,49 The lower scattering for the nanoparticle−surfactant system compared with the nanoparticle-only system in the intermediate Q range indicates that the building block size in the aggregate structure is significantly higher than the nanoparticle size, which possibly arises from surfactant micelle mediation of the nanoparticle aggregates. This point is made clear in Figure 5b where the nanoparticles in the nanoparticle−surfactant system are contrast matched (60 vol % D2O in mixed D2O/H2O solvent). 9945

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single micelle to favor attractive electrostatic interaction between them for their aggregation.18,21 We have observed that nanoparticles undergo aggregation in the presence of both the protein and surfactant despite their very different nature of interaction with the nanoparticles. Therefore, the interaction of protein and surfactant is going to play an important role when both components are present in the nanoparticle solution. The surfactant molecules are known to strongly interact with the protein through electrostatic as well as hydrophobic interactions.51−53 SANS data of 1 wt % BSA protein with 50 mM DTAB surfactant are shown in Figure 6. The figure also shows for comparison the data of the

Figure 6. SANS data of 1 wt % BSA protein + 50 mM DTAB surfactant compared with the sum of scattering from individual components (1 wt % BSA protein and 50 mM DTAB surfactant) in the presence of salt (0.2 M NaCl) in D2O. Inset shows the scattering profile of 1 wt % BSA protein + 50 mM DTAB (cationic) surfactant compared with 1 wt % BSA protein + 50 mM SDS (anionic) surfactant. The solid curves are theoretical fits to the experimental data.

Figure 5. SANS data of 1 wt % HS40 silica nanoparticles + 50 mM DTAB surfactant in the presence of salt (0.2 M NaCl) with (a) micelles are contrast matched compared with 1 wt % HS40 silica nanoparticles and (b) nanoparticles are contrast matched compared with 50 mM DTAB surfactant.

calculated sum of scattering from individual 1 wt % BSA protein and 50 mM DTAB surfactant systems. The measured scattering profile of BSA protein−DTAB surfactant (unlike the sum of scattering of individual components) shows linearity in the intermediate Q range, representing a fractal kind of structure. This fractal structure is interpreted in terms of micellelike clusters of surfactant formed along the unfolded polypeptide chain of protein in accordance with the bead− necklace model as known in the literature.29,39,54,55 The data are fitted using the structure factor of mass fractal S(Q) (eq 9) and form factor P(Q) of spherical micelles (eq 2). The analysis (Table 2c) provides the size of micellelike clusters as building blocks (16.8 Å), the overall size of the complex as correlation length (61.8 Å), and the packing of micelles as the fractal dimension (1.59). The inset in Figure 6 shows the comparison of SANS data from the protein−surfactant system for cationic (DTAB) with anionic (SDS) surfactant. The scattering profiles from these systems show quite similar features. The size of the micellelike clusters is found to be smaller (16.8 Å for BSA− DTAB as compared to 18.6 Å for BSA−SDS) and the overall size of the complex larger (61.8 Å for BSA−DTAB as compared to 39.8 Å for BSA−SDS) in the case of DTAB compared with SDS.14 This suggests that cationic DTAB micelles are relatively

The scattering from surfactant micelles in the nanoparticle− surfactant system is observed to be very different from that of pure surfactant solution. Similar to Figure 5a, there are again two distinct features observed in Figure 5b in the low and intermediate Q regions compared to that of the nanoparticle− surfactant system. The large scattering in the low Q region followed by a hump in the intermediate Q value arises from aggregates of the core−shell structure of surfactant micelles surrounding the nonvisible nanoparticles.21,31,50 The data are fitted using mass fractal distribution of shells of micelles [d∑/ dΩ(Q) ∼ P(Q)S(Q)] for S(Q) as given by eq 9 and P(Q) by eq 7. The fractal dimension of 2.72 of nanoparticle aggregates is found to be similar to when the micelles are contrast matched, and the thickness of micelles adsorbed around the nanoparticles has a value about the size (diameter 41 Å) of the micelles. Interestingly, the building block size (radius 107 Å) of the fractal aggregates is significantly less than that of the sum of the nanoparticle size (radius 87 Å) and micelle size (diameter 41 Å). This is an indication that the nanoparticles are bridged by a 9946

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Table 2. Fitting Parameters of Two-Component Systems at pH = 7 and 0.2 M NaCl Salt (a) Nanoparticle−Protein System Characterized by Protein Depletion-Induced Fractal Aggregates of Nanoparticles particle radius R (Å)

system

building block radius r (Å)

fractal dimension D

1 wt % HS40 + 1 wt % BSA 87.1 ± 1.0 87.1 ± 1.0 2.55 ± 0.20 (b) Nanoparticle−Surfactant System Characterized by Surfactant Micelle-Mediated Fractal Aggregates of Nanoparticles system

particle radius R (Å)

shell thickness t (Å)

building block radius r (Å)

fractal dimension D adsorbed micelles per particle N

1 wt % HS40 + 50 mM DTAB 87.1 ± 1.0 41.0 ± 0.5 107.0 2.72 ± 0.20 50 (c) Protein−Surfactant System Characterized by Fractal Structure of Micellelike Clusters Formed along the Unfolded Protein Chain system

micelle radius r (Å)

correlation length ξ (Å)

fractal dimension D

number of micelles n

aggregation number N

1 wt % BSA + 50 mM DTAB

16.8 ± 0.2

61.8 ± 4.0

1.59 ± 0.10

10

35

protein−SDS surfactant in the high Q region.14 In this case, the system consists of protein−surfactant complexes coexisting with their depletion-induced aggregates of nanoparticles. This in turn suggests that irrespective of the nature of surfactant (DTAB or SDS), similar kinds of protein−surfactant complexes are formed. The interaction of these protein−surfactant complexes with nanoparticles governs the resultant structure of nanoparticle−protein−surfactant systems. This is perhaps expected among the three components (nanoparticle, protein, and surfactant); protein and surfactant are much smaller in size and therefore have a higher possibility of interaction because of higher mobility in solution, hence controlling the resultant structure. The details of these systems have been investigated by selectively contrast matching different components as shown in Figure 8. Figure 8 shows SANS data of the 1 wt % HS40 silica nanoparticle−1 wt % BSA protein−50 mM DTAB surfactant system for the solution conditions: (a) surfactant and (b) nanoparticles are contrast matched. Figure 8a compares data of the three-component nanoparticle−protein−surfactant system with the two-component nanoparticle−protein and nanoparticle−surfactant systems under the conditions of surfactant contrast matching. All these systems, as discussed previously, show fractal aggregates of nanoparticles. However, there are significant differences in the features of data of these systems, which may correspond to different structures formed through different mechanisms of nanoparticle aggregation.10,12 The lower scattering intensity in the case of nanoparticle−protein− surfactant and nanoparticle−surfactant systems compared with the nanoparticle−protein system corresponds to higher building block size, which is an indication of nanoparticle aggregation mediated by surfactant micelles or the protein− surfactant complex for the nanoparticle−protein−surfactant system similar to that of surfactant micelles for nanoparticle− surfactant system.21 In the case where nanoparticle aggregation is also mediated by only surfactant micelles for the nanoparticle−protein−surfactant system, the fraction of free micelles available for the protein will decrease, which in turn should lead to scattering significantly different from that of the protein− surfactant system in the absence of nanoparticles. However, we have observed that the scattering of the nanoparticle−protein− surfactant system almost matches that of the protein−surfactant system in the intermediate to high Q region (>0.05 Å−1) (Figures 7 and 8b). The rearrangement of micelles in the protein−surfactant complex could be the reason for the almost same value of building block size of nanoparticle−protein− surfactant and nanoparticle−surfactant aggregates. The fitted parameters are given in Table 3. The fractal dimension of nanoparticle−protein−surfactant aggregates is interestingly found to be quite similar to that of nanoparticle−protein

more effective in protein unfolding than anionic SDS micelles.28,56 However, it is expected that the different overall charge on the protein−surfactant complex for the two surfactants (DTAB and SDS) will lead to their different interaction with nanoparticles. We now examine the structure of the three-component nanoparticle−protein−surfactant system in Figure 7 in terms of

Figure 7. SANS data of 1 wt % HS40 silica nanoparticles + 1 wt % BSA protein + 50 mM DTAB surfactant along with scattering data of 1 wt % HS40 silica nanoparticles + 50 mM DTAB surfactant and 1 wt % BSA protein + 50 mM DTAB surfactant in the presence of salt (0.2 M NaCl) in D2O. Inset shows the scattering profile of 1 wt % HS40 silica nanoparticles + 1 wt % BSA protein + 50 mM SDS (anionic) surfactant along with 1 wt % HS40 silica nanoparticles + 1 wt % BSA protein and 1 wt % BSA protein + 50 mM SDS surfactant.

behavior of two-component systems as observed in Figures 3−6. In the three-component nanoparticle−protein−surfactant system, the possible two-component interactions which could play a role in deciding the interaction and resultant structure are (i) nanoparticle−protein, (ii) nanoparticle−surfactant, and (iii) protein−surfactant. However, the SANS data in Figure 7 show that the present system is represented by the features of silica nanoparticle−DTAB surfactant in the low Q region and BSA protein−DTAB surfactant in the high Q region. This indicates that the silica nanoparticle−BSA protein−DTAB surfactant consists of protein−surfactant complexes coexisting with surfactant or protein−surfactant complex-mediated aggregates of nanoparticles. These results are clearly different from the SANS data of silica nanoparticle−BSA protein−SDS surfactant (inset of Figure 7), which are represented by silica nanoparticle−BSA protein in the low Q region and BSA 9947

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through the protein−surfactant complex due to a decrease in the charge on the micelle may not be as effective as with the surfactant micelles alone.10,12,21 Figure 8b shows the SANS data of the three-component nanoparticle−protein−surfactant system compared with that of the two-component protein− surfactant and nanoparticle−surfactant with nanoparticles that are contrast matched. The scattering profile of the nanoparticle−protein−surfactant system clearly shows a profile very different from that of the protein−surfactant system. The buildup of scattering in the low Q region is due to the shell formation of micellelike clusters of the protein−surfactant complex around nonvisible nanoparticles in these aggregates.17,21,57 The scattering profile of the nanoparticle− protein−surfactant system is also compared with the nanoparticle−surfactant system (nanoparticles contrast matched) in the inset of Figure 8b. The scattering profile of the nanoparticle−protein−surfactant system is considerably different from that of the nanoparticle−surfactant system. The data are different in the low Q region because of differences in the fractal dimension of the two systems (Table 3), whereas the differences in the intermediate Q range arise because of different structures (micelles versus protein−surfactant complex) coexisting with nanoparticle aggregates. The data are fitted with a shell of protein−surfactant complex around the nonvisible nanoparticles in their aggregates coexisting with free protein−surfactant complexes. The shell thickness is found to be around 43 Å. The similar values of shell thicknesses for nanoparticle−protein−surfactant and nanoparticle-surfactant systems (Table 2b) is in accordance with the rearrangement of micelles in the adsorbed protein−surfactant complex on nanoparticles in leading to their (nanoparticle) aggregation in the nanoparticle−protein−surfactant system. Moreover, the unfolding of protein in the free protein−surfactant complex in the three-component system is found to be enhanced by the presence of nanoparticles. This in turn suggests that the structure in the nanoparticle−protein−surfactant complexes is determined by the modified interactions of individual twocomponent systems.

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Figure 8. SANS data of (a) 1 wt % HS40 silica nanoparticles + 1 wt % BSA protein + 50 mM DTAB surfactant compared with 1 wt % HS40 silica nanoparticles + 1 wt % BSA protein and 1 wt % HS40 silica nanoparticles + 50 mM DTAB surfactant in the presence of salt (0.2 M NaCl) with micelles contrast matched and (b) 1 wt % HS40 silica nanoparticles + 1 wt % BSA protein + 50 mM DTAB surfactant compared with 1 wt % BSA protein + 50 mM DTAB surfactant in the presence of salt (0.2 M NaCl) with nanoparticles contrast matched. Inset shows the scattering profile of 1 wt % HS40 silica nanoparticles + 1 wt % BSA protein + 50 mM DTAB surfactant along with 1 wt % HS40 silica nanoparticles + 50 mM DTAB surfactant with nanoparticles contrast matched.

CONCLUSIONS Small-angle neutron-scattering measurements have been carried out to study the structure and interaction in three-component systems of anionic Ludox HS40 silica nanoparticles, anionic bovine serum albumin (BSA) protein, and cationic dodecyl trimethylammonium bromide (DTAB) surfactant. The results are compared with similar complexes in which DTAB surfactant has been interchanged with anionic sodium dodecyl sulfate (SDS) surfactant. The structure in these three-component systems is interpreted in terms of the interactions of individual two-component systems. The nonadsorption of BSA leads to depletion force-induced aggregation of nanoparticles. The

aggregates but less than that of nanoparticle−surfactant aggregates. This is possible because bridging of nanoparticles

Table 3. Fitting Parameters of the Three-Component Nanoparticle−Protein−Surfactant System at pH = 7 and 0.2 M NaCl salt (a) Surfactant is Contrast Matched particle radius R (Å)

system 1 wt % HS40 + 1 wt % BSA + 50 mM DTAB 1 wt % HS40 + 50 mM DTAB

building block radius r (Å)

87.1 ± 1.0 87.1 ± 1.0 (b) Nanoparticle is Contrast Matched

fractal dimension D 2.55 ± 0.2 2.72 ± 0.2

108.0 107.0

system

micelle radius r (Å)

fractal dimension D

correlation length ξ (Å)

number of micelles n

aggregation number N

BSA−DTAB HS40−BSA−DTAB

16.8 ± 0.2 16.8 ± 0.2

1.59 ± 0.10 1.30 ± 0.12

61.8 ± 4.0 70.0 ± 4.4

9 11

35 29

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micelle-mediated aggregation of nanoparticles is also observed in the oppositely charged nanoparticle−surfactant system. These nanoparticle aggregates are characterized by mass fractals. In the case of the protein−surfactant system, the surfactant molecules interact cooperatively, leading to the bead−necklace structure of micellelike clusters adsorbed along the unfolded protein chain. The nanoparticle−surfactant (mediated through the protein−surfactant complex) and protein−surfactant interactions for DTAB govern the resultant structure of nanoparticle−protein−surfactant complexes. The fractal dimension of nanoparticle−protein−surfactant aggregates is found to be quite similar to that of nanoparticle− protein aggregates but less than that of nanoparticle−surfactant aggregates. There is rearrangement of micelles in the adsorbed protein−surfactant complex on nanoparticles in leading to their (nanoparticle) aggregation in the nanoparticle−protein− surfactant system. On the other hand, the unfolding of protein in free protein−surfactant complex is found to be significantly enhanced with respect to that without the presence of nanoparticles. The roles of DTAB and SDS are found to be interestingly different, where nanoparticle−protein (mediated through protein−surfactant complex) and protein−surfactant for SDS decide their resultant structure. The present results thus show that the structure and interaction of the threecomponent nanoparticle−protein−surfactant systems can simply be modified by the change in the charge state of the surfactant.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +91 22 25594642. Fax: +91 22 25505150. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is based on SANS experiments performed at the Swiss Spallation Neutron Source SINQ, Paul Scherrer Institut, Villigen, Switzerland.

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