Shell Structure of Natural Rubber Particles - American Chemical Society

Article pubs.acs.org/Langmuir

Shell Structure of Natural Rubber Particles: Evidence of Chemical Stratification by Electrokinetics and Cryo-TEM Christophe N. Rochette,†,‡ Jérôme J. Crassous,§ Markus Drechsler,∥ Fabien Gaboriaud,⊥ Marie Eloy,⊥ Benoît de Gaudemaris,⊥ and Jérôme F. L. Duval*,†,‡ †

Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC), Université de Lorraine, UMR 7360, 15 avenue du Charmois, Vandœuvre-lès-Nancy, F-54501, France ‡ Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC), CNRS, UMR 7360, 15 avenue du Charmois, Vandœuvre-lès-Nancy, F-54501, France § Physical Chemistry, Department of Chemistry, Lund University, 221 00 Lund, Sweden ∥ Laboratory for Soft Matter Electron Microscopy, Bayreuth Institute of Macromolecular Research (BIMF), University of Bayreuth, 95440 Bayreuth, Germany ⊥ Manufacture Française des Pneumatiques Michelin, 23 Place des Carmes-Déchaux, 63040 Clermont Ferrand Cedex 9, France S Supporting Information *

ABSTRACT: The interfacial structure of natural rubber (NR) colloids is investigated by means of cryogenic transmission electron microscopy (cryo-TEM) and electrokinetics over a broad range of KNO3 electrolyte concentrations (4−300 mM) and pH values (1−8). The asymptotic plateau value reached by NR electrophoretic mobility (μ) in the thin double layer limit supports the presence of a soft (ion- and water-permeable) polyelectrolytic type of layer located at the periphery of the NR particles. This property is confirmed by the analysis of the electron density profile obtained from cryo-TEM that evidences a ∼2−4 nm thick corona surrounding the NR polyisoprene core. The dependence of μ on pH and salt concentration is further marked by a dramatic decrease of the point of zero electrophoretic mobility (PZM) from 3.6 to 0.8 with increasing electrolyte concentration in the range 4−300 mM. Using a recent theory for electrohydrodynamics of soft multilayered particles, this “anomalous” dependence of the PZM on electrolyte concentration is shown to be consistent with a radial organization of anionic and cationic groups across the peripheral NR structure. The NR electrokinetic response in the pH range 1−8 is indeed found to be equivalent to that of particles surrounded by a positively charged ∼3.5 nm thick layer (mean dissociation pK ∼ 4.2) supporting a thin and negatively charged outermost layer (0.6 nm in thickness, pK ∼ 0.7). Altogether, the strong dependence of the PZM on electrolyte concentration suggests that the electrostatic properties of the outer peripheral region of the NR shell are mediated by lipidic residues protruding from a shell containing a significant amount of protein-like charges. This proposed NR shell interfacial structure questions previously reported NR representations according to which the shell consists of either a fully mixed lipid−protein layer, or a layer of phospholipids residing exclusively beneath an outer proteic film.

1. INTRODUCTION

in turn explains their strategic role in a wide range of applications. A critical analysis of the interfacial structure of NR particles is mandatory for optimizing their performance in the aforementioned applied technologies or products. In that respect, numerous studies have long been reported for deciphering the intricate relationship existing between structural details and physicochemical properties of NR colloidal particles. While a consensus has been reached on the existence of a ∼3−20 nm thick shell layer surrounding the polyisoprene core of NR

Natural rubber (NR) latex collected from Hevea brasiliensis is a major source of rubber material used in the tire industry and the hygienic and medical sectors, and it is further involved in a broad spectrum of everyday-life consumer products, e.g., shoes, cables, sport accessories, and antiadhesive surfaces.1−3 NR is generally present in the form of ∼94 wt % polydispersed polyisoprene (PI) latex particles of diameter ranging from ∼50 nm to ∼2 μm.4−6 NR particles further consist of ca. 6 wt % nonrubber components that include proteins, phospholipids, polypeptides, or fatty acids.7 This peculiar composition confers upon the particles specific physicochemical properties in terms of mechanical resistance or stability versus aggregation, which © 2013 American Chemical Society

Received: September 24, 2013 Revised: October 18, 2013 Published: October 23, 2013 14655

dx.doi.org/10.1021/la4036858 | Langmuir 2013, 29, 14655−14665

Langmuir

Article

particles,7−9 the arrangement of that shell layer in terms of nonrubber components (e.g., lipids and proteins) still remains controversial. Similarly to the nomenclature used in ref 9, we employ in the following the term “lipids” in a generic sense, including therein lipids, phospholipids, free fatty acids, or fatty acids associated with phospholipids. In detail, two NR interfacial structure formulations have emerged so far. According to one of these, denoted hereafter as formulation (i), the shell consists of a mixed layer of proteins and lipids without any spatial separation between these two types of components within the particle shell.7−9 In contrast, a second formulation (ii) proposes that the NR interface consists of a lipid monolayer surrounding the polyisoprene core beneath a peripheral protein film facing the outer aqueous medium.10 While formulation (ii) is hypothesized on the basis of indirect evidence in very few studies only,10 there is an abundance of experimental work underpinning a mixed lipid− protein NR surface structure.7−9 In particular, recent nanoindentation experiments by atomic force microscopy (AFM) on NR particles9 were claimed to support formulation (i) for the NR interface. The authors argue that the observed “single distortion on the [NR] indentation curve [corresponds] to a single-layer structure instead of a stepwise penetration [of the AFM tip] for a double layer morphology.” This statement is, however, questionable in view of the AFM nanoindentation analysis by Francius et al.11 on both nude soft bacterial membranes and genetically modified bacterial cells that selectively exhibit protruding soft surface appendages anchored at the membrane. The results reported in ref 11 indeed evidence that the presence of a single nonlinear compression domain in the force versus indentation curve is fully consistent with the existence of a structured (or multilayered) membrane/ soft appendage biological interphase. In line with this, Gaboriaud et al.12 showed that the deformation/indentation of a bacterial membrane upon compression by an AFM tip may be concomitant with that of the cytoplasmic cell component beneath the membrane, thus resulting in a monotonic indentation regime that reflects both membrane elasticity and cytoplasm deformation. It is further emphasized that the AFM phase contrast imaging procedure followed in ref 9, together with the rhodamine labeling of amine functional groups from proteins and phospholipids, solely supports the existence of a shell at the NR particle surface but remains inappropriate for revealing univocally a possible in-depth organization of proteins and phospholipids. Another experimental work based on the measurements of NR particle electrophoretic mobility points out a mixed protein−lipid structure NR interphase.7 In view of the recent developments done for the theoretical modeling of core−shell particle electrokinetics,13−17 the quantitative interpretation of the reported NR mobility data may, however, be criticized. First, the modeling of electrokinetics is often based on the use of the conventional zeta (ζ)-potential concept for deriving electrostatic and structural features of NR particles. This theoretical strategy is unfortunately physically inappropriate because it strictly holds for hard (impermeable) colloidal systems and it is meaningless for core−shell colloidal systems such as NR particles.17 A more appropriate interpretation requires the explicit account of particle shell permeability (to ions and solvent), as done by Ho et al.18 using Ohshima’s analytical equation for the electrophoretic mobility of soft rubber particles (i.e., ion and water permeable). However, the range of accuracy of that expression remains severely limited as

it tacitly presumes a homogeneous shell surrounding the particle core and ignores the possible presence of layers different in nature and with distinct electrohydrodynamic properties in the particulate shell component.19 These shortcomings then render impossible the detection of any spatial organization of proteins−lipids across the NR shell layer. In addition, Ohshima’s model becomes inexact at sufficiently low electrolyte concentrations and/or large shell potentials where polarization of the electric double layer by the applied electric field is significant.14−16 It is further inappropriate for a sufficiently small core particle radius and/or thin shell layer thickness, cases where both potential and hydrodynamic flow field distributions at the particle/solution interphase strongly depend on particle dimensions.13−17 In this work, we provide for the first time a quantitative methodology for evidencing unambiguously a bilayer structure organization of the shell surrounding the polyisoprene core of NR particles (∼50 nm in radius) issued from Hevea brasiliensis tree species. Electron density profiles derived from cryogenic transmission electron microscopic (cryo-TEM) analysis first highlight the presence of a 2−4 nm thick shell layer. The electrokinetic properties of NR particles are further measured over a wide range of electrolyte concentration (4−300 mM). Data firmly invalidate the “hard” representation of the NR shell often reported in the literature, and they support that the NR shell layer is permeable to ions and fluid flow; i.e., NR particles are soft from an electrokinetic point of view. In addition, at a fixed pH value below 3, the electrophoretic mobility of NR particles changes sign with varying electrolyte concentration. We show that this peculiar pH and electrolyte concentration dependence of the NR electrophoretic mobility is symptomatic of a chemical stratification of the shell in the form of two successive shell layers, each characterized by distinct properties in terms of charge dissociation constants and thickness. Using a recently developed electrohydrodynamic formalism for heterogeneous soft multilayered particles,19 the complete set of electrokinetic data collected over 7 pH units and ∼2 orders of magnitude in electrolyte concentration is quantitatively reconstructed. The results suggest that the outer region of the NR shell component is analogous to that of a bilayer interphase consisting of a proteinaceous layer anchored at the NR polyisoprene core surrounded by negatively charged protruding lipidic residues facing bulk electrolyte solution.

2. MATERIALS AND METHODS 2.1. Natural Rubber Particle Suspensions. Concentrated latex high ammonia (60 wt %, HA) was purchased from Trang Latex Co. Ltd., Thailand. We used the latex HA to avoid contamination by nonrubber particles in the field latex. Prior to use, NR suspensions were stirred for ∼1 min and subsequently left to equilibrate for a period of 10 min (unless otherwise specified). A concentration of 2 × 10−4 (weight percent) was adopted for the electrokinetic and size measurements, while a 7% concentration was used for cryo-TEM measurements. Conditions for size and electrokinetic experiments are in line with the absence of NR aggregates (micrometers in size or even larger). 2.2. Cryogenic Transmission Electron Microscopy (CryoTEM). Cryo-TEM makes it possible to observe colloidal particles mummified in an aqueous solvent following thermal shock fixation conditions. It does not suffer from impairing dehydrating steps likely to alter the searched colloidal interfacial structure, a situation recurrently encountered when using other microscopy preparations such as drying or embedding for conventional transmission electron microscopy (TEM). Instead, the cryogenic method permits an instantaneous vitrification of the solvent and thus allows for exploring 14656


Langmuir

Article

particle structure in the native state. Samples were prepared by diluting 10 times NR stock suspensions (70 wt %). A drop of this sample suspension was then vitrified on a lacey carbon film covered copper grid by liquid ethane at its freezing point (90 K) by means of a cryobox (Carl Zeiss Microscopy, Germany). Observation of the cryomicrographs and derivation of the corresponding electron density profiles were then performed at 90 K with a LEO EM922Omega EFTEM instrument (Carl Zeiss Microscopy, Germany) using an aperture of 1.25 mrad and an acceleration voltage of 120 kV. In this work, the analysis of electron density profile was carried out on ca. 60 NR particles. 2.3. Electrokinetics (Electrophoresis) and Dynamic Light Scattering (DLS). Size and electrokinetic analyses of NR particles were performed over a wide range of KNO3 electrolyte concentrations (4−300 mM) whose pH was varied in the range 1−8. All electrolytes used in this study to fix the salinity and pH of the suspensions have a purity larger than 99% (KNO3 from Roth, Karlsruhe, 0.1 mol/L KOH from VWR International, Leuven, and 1 mol/L HNO3 from Fluka). The particle concentration adopted for the experiments was 2 × 10−4 (weight percent). The size distribution of NR particles was obtained from dynamic light scattering (DLS) using a Zetasizer Nano ZS instrument (He−Ne red laser, 633 nm, Malvern Instruments) at 25 ± 0.1 °C. This apparatus is equipped with an automatic laser attenuator and an avalanche photodiode detector. The position of the latter is located 173° relative to the laser source so that backscattering detection is ensured. Experiments were driven by the Dispersion Technology Software provided by Malvern Instruments. The suspensions were preequilibrated for a period of ∼10 min prior to measurement. It was systematically verified that shorter and longer pre-equilibration steps (up to 4 h) did not lead to significant changes in size distribution (Supporting Information, Figure S1). As a first approximation, measured diffusion coefficients were converted into particle size using the Stokes−Einstein relationship. The measurements were repeated at least three (up to nine) times using at least two different and freshly prepared NR suspensions. DLS data were further compared to the particle size obtained from the cryo-TEM observation of ca. 860 particles (section 4.1). The electrophoretic mobility of NR particles was measured by laser Doppler electrophoresis, also known as phase analysis light scattering (PALS). The rate of change of the phase shift between the scattered light and a reference beam is correlated to the particle velocity and thus allows for evaluating the particle electrophoretic mobility. For the measurements, a constant direct-current electric field of 40 V/cm was applied across the cell. The reproducibility of the results was addressed by measuring three times the electrophoretic mobility of NR particles under fixed pH and ionic strength conditions and by iterating the measurements with three different NR suspensions. The pH, particle concentration, and electrolyte concentration conditions adopted in the electrokinetic and size measurements correspond to situations where NR suspensions are stable against aggregation, as verified from DLS data (Supporting Information, Figures S2−S4). Measurements were performed after a NR suspension equilibration time of 10 min.

Figure 1. Schematic representation of cryo-TEM evaluation of the gray value profile G(r) of a core−shell particle embedded in a thin film of hyperquenched glassy water (HGW). The radius of the particle core is denoted as RC, and RS pertains to the radius of the whole particle including the shell layer thickness. r is the radial coordinate with the origin taken at the center of the particle core. G0 is the gray value obtained from scattered electrons crossing the HGW.

may be neglected.20 Then only the amplitude contrast brought about by elastic and inelastic scattering processes needs to be taken into account in the analysis. Depending on the objective aperture α0, the total electron scattering cross section of a given atom i, hereafter denoted as σT,i(α0), is expressed according to21−23 σT, i(α0) = σel, i(α0) + σin, i

(1)

where the indexes “el” and “in” refer to the elastic and inelastic scattering processes, respectively. The inelastic scattered electrons are mainly transmitted through the objective aperture, and the corresponding cross section term σin,i may be evaluated following Wall et al.24 σin, i = 1.5 × 10−6Z1/2β −2 ln(2/ϑe)

(2)

where Z is the atomic number of the ith scattering element and β is the ratio between the speed of the electrons and that of the light; i.e., β2 = 1 − [E0/(E + E0)2] with E the acceleration voltage, E0 the rest energy (E0 = m0c2), m0 the electron mass, and c the speed of light. The quantity ϑe in eq 2 is defined by ϑe = El/[β2/(E + E0)] and El is the average energy loss set to 20 eV following the evaluation by Wall et al. for organic materials.24 Equation 2 is not valid for hydrogen,23 and the scattering cross section for that element is set to 12 pm2 at 120 keV.20,25,26 Following Langmore and Smith,21,22 the elastic cross section σel,i(α0) of the ith element (eq 1) may be derived at small angles via the relationship

3. THEORY 3.1. Evaluation of Particle Size Distribution and Shell Layer Thickness of NR Particles by Cryogenic Transmission Electron Microscopy (Cryo-TEM). The pixels constituting cryogenic transmission electron micrographs exhibit a contrast (gray value) whose magnitude depends on the extent the incident intensity I is lowered when crossing the sample (Figure 1). The higher the concentration and/or atomic weight (Z) of the sample atoms, the darker are the corresponding pixels. Gray values were evaluated on the basis of the Beer−Lambert law. For that purpose, it is necessary to first describe the scattered electrons in terms of the first order of amplitude and phase contrast, as detailed elsewhere.20 Under the condition of focused specimen met here, the phase contrast

σel, i(α0) = σel,̅ i[1 − 5−1λ−1 sin(α0/2)]

(3)

−3

where λ = 2.5 × 10 nm is the electron wavelength and σ̅el,i is the total elastic cross section of an element i evaluated from Dirac partial-wave analysis by means of the algorithm described by Mayol and Salvat27 using the NIST electron elasticscattering cross section database (SRD 64, version 3.2) for an energy of 120 keV. The relative decrease dI/I of the intensity passing through a sample material j along a distance dl is then given by 14657


Langmuir

Article

Table 1. Evaluation of the Distances lC and lS Covered by the Electrons at Position r Across the Core and Across the Shell Component of the Particle Depicted in Figure 1a r

lC

r < −RS and r > RS −RS < r < −RC and RC < r < RS −RC < r < RC a

2(RC2

lS

0 0 − r2)1/2

0 2(RS2 − r2)1/2 2[(RS2 − r2)1/2 − (RC2 − r2)1/2]

The radial coordinate system adopted is specified in Figure 1. n

i Nν dI = −md, j∑ A i σT, i(α0) dl I Mi i=1

is not met in the case of NR particles whose particle shell is composed of two successive layers: a thick layer that is detectable by cryo-TEM and a second layer (evidenced by electrokinetics (section 4.2)) whose typical thickness does not exceed the characteristic pixel size in cryo micrographs. 3.2. Electrokinetics of Multilayered Soft Particles. We consider the spherical, core−shell particle with core radius RC that moves with a velocity U⃗ in an electrolyte under the action of an applied dc electric field E⃗ . The origin of the spherical coordinate system (r,θ,φ) is placed at the center of the particle, and the polar axis (θ = 0) is set parallel to E⃗ , a nomenclature similar to that adopted in previous studies.13,14 The electrolyte is composed of N types of ionic, mobile species with valences zi, bulk concentrations c∞ i , and limiting ionic conductivities λi° (i = 1, ..., N). In line with the colloidal system investigated here, we further consider that the particle shell consists of j = 1, ..., M successive layers of thickness δj, where j = 1 and j = M correspond to the layer supported by the particle core and to the peripheral layer facing the outer solution, respectively. The charges located within each layer j originate either from the deprotonation of basic groups denoted ≡RHj+ or from that of acid groups ≡RHj according to

(4)

where σT,i(α0) is defined by eqs 1−3, md,j is the mass density of material j, ni and Mi are the number of elements i in material j and the molar mass of element i, respectively, νi is the stoichiometric coefficient of the ith element, and NA is the Avogadro number. The contrast parameter of the jth material, denoted as kj(α0), can be expressed as ni

1/kj(α0) =

∑ NAνσi T,i(α0)/Mi

(5)

i=1

After integrating eq 4 and combining with eq 5, we obtain I /I0 = exp[−md, jl /kj(α0)]

(6)

where I0 is the intensity of the incident beam. The decrease of the intensity is then directly related to kj(α0)/md,j that corresponds to the total mean free path length for material j, a quantity that may be evaluated as detailed elsewhere.20 The searched gray values G(r) evaluated at a given position r in the cryo micrographs (Figure 1) are then directly proportional to the corresponding intensity I(r) pending appropriate definition of the distance l covered by the electrons across material j at position r. Introducing G0 ∝ I0 exp[−md,wlT/kw(α0)], the contribution by the hyperquenched glassy water, and lT = lw + ∑Pj=1j lj the total thickness of the sample with lw that of the vitrified water and pj the total number of material types within the analyzed particle, we obtain

≡RHj ⇄ ≡R j− + H+

and ≡RHj+ ⇄ ≡R j + H+

∑ md,jlj/kj(α0)] j=1

(10)

where ≡R refers to the polymeric material within layer j that supports the protons. The charging mechanism of layer j is specified by setting εj = −1 when eq 9 applies, and εj = +1 for the other case where eq 10 adequately depicts the origin of the charge. The acidity constant associated with the relevant acid− base reaction taking place in the jth layer is then denoted as Ka,j = 10−pKa,j. The total concentration of ionizable groups ≡Rj within the jth layer is called nj. Within this representation of the particulate shell, the profile for the concentration of ionizable groups, n(r), is a succession of discontinuous square functions of amplitude nj. The possibility of smooth (or diffuse) transition for n(r) across the shell layer is introduced with adopting the following expression of n(r)28,29

pj

G(r ) ∝ I0 exp[−md,w l w /k w(α0) −

(9)

(7)

The ratio G(r)/G0 may be written in the final form pj

G(r )/G0 = exp[−∑ l j(r )(md, j /kj(α0) − md,w /k w(α0))] j=1

(8)

where we have explicitly written the r dependence of the distances lj. The quantity md,j/kj(α0) − md,w/kw(α0) corresponds to the contrast for material j as evaluated for a given objective aperture α0. For the situation of a soft particle composed of an homogeneous core surrounded by an outer homogeneous shell layer (Figure 1), the distances lC and lS pertaining to the core and the shell component of the particle, respectively, depend on r, RC, and RS via the expressions collected in Table 1. The theory may be straightforwardly extended to cases where the particle shell consists of different layers, each characterized by a given kj(α0)/md,j. The latter theoretical extension is meaningful providing the accuracy of the experimental data is sufficient for discriminating electrons scattered by the various shell constituents. As later argued (section 4.1), this condition

M−1

n(r ) =

∑ {nj − nj + 1}f j (r) + nM fM (r) j=1

(11)

where j

f j = 1,..., M (r ) = {1 − tanh[(r − R C −

∑ δk)/αj]}/2 k=1

(12)

The gradual variation of n(r) between two adjacent layers j and j + 1 is then governed by the quantities αj, as detailed elsewhere.28 αj defines the sharpness of the interface between layers j and j + 1 and, in particular, eq 11 reduces to a succession of step functions in the limit αj → 0.28 The local 14658


Langmuir

Article

Figure 2. Left: Typical cryo-TEM micrograph of a 7 wt % NR colloidal particle suspension. Right: The circles correspond to the size distribution of NR particles as obtained from DLS measurements, and the histograms correspond to that estimated from cryo-TEM analysis on 863 particles. The dotted and full lines are Gaussian distributions with mean particle diameters of 82 ± 15 nm and 90 ± 25 nm, respectively. Experimental conditions: natural pH (5.6−5.8), aqueous solution with no KNO3 salt added, and a particle concentration of 2 × 10−4 wt % (DLS) and 7 wt % (cryo-TEM).

in the system and the steady incompressible flow. For the sake of simplicity and for model constraining purposes, we consider that the flow penetration length 1/λo is similar for each shell layer. This allows expressing in the Navier−Brinkman equation the radial distribution of the friction coefficient, k(r), via k(r) = kM f M(r),19 where kM = ηλo2 is the friction coefficient pertaining to layer M and η is the dynamic viscosity of water. The theory applies without approximation of particle size and particle charge and further accounts for double layer polarization processes that possibly come into play at sufficiently low electrolyte concentration and high particle charge. The reader is referred to ref 19 for further details.

density of fixed charges at a given position r, denoted as ρfix(r), depends on the corresponding local concentration of ionizable sites, n(r) (eqs 11 and 12), on the local dimensionless electrostatic potential y(r) (y(r) = Fψ(r)/RT with T the absolute temperature, R the gas constant, ψ the potential, and F the Faraday number), on solution pH, and on the nature of the acid−base equilibrium that applies at the position r. For the acid−base reactions given in eqs 9 and 10, we obtain28,29 M−1

ρfix (r ) = ρM { ∑ {γj , Mμj (y(r )) − γj + 1, Mμj + 1(y(r ))}f j (r ) j=1

+ μM (y(r ))fM (r )}

(13)

4. RESULTS AND DISCUSSION 4.1. Particle Size Distribution and Evaluation of NR Shell Layer Thickness. We report in Figure 2 typical images of NR particle suspensions as obtained from cryogenic transmission electron microscopy. The gray background corresponds to the low-density hyperquenched glassy water, while darker spheres are the NR particles. A majority of the particles (70%) with a diameter below 140 nm was observed, and the presence of larger pear-shaped particles (up to 500 nm in equivalent hard-sphere diameter) could be detected, in line with conclusions of previous work.4,5,9 A quantitative analysis carried out on ca. 860 particles by means of Digital Micrograph Software (Gatan, version 3.6.5) reveals that the mean diameter of the most abundant particles is 90 ± 25 nm (Figure 2). This value is in good agreement with that obtained from DLS analysis (82 ± 15 nm). Both types of size measurements further confirm the intrinsic polydisperse nature of the particle suspensions analyzed with, in particular, the existence of a minor population of large particles with diameter in the range 150−350 nm. In the rest of the study, the only population corresponding to the most abundant particles (diameter in the range 50−140 nm, Figure 2) was considered for cryo-TEM analysis of NR interfacial structure using eq 8. The r-dependent gray contrast (or electron density profile) at the NR/solution interface was extracted for 60 NR particles. In order to obtain statistically

where the dimensionless γj,M are defined by γj,M = ρj/ρM and the quantities ρj = Fεjnj correspond to the maximum charge density in layer j in the limits where ionizable groups are therein uniformly distributed and completely dissociated. In eq 13, the dissociation functions μj(y(r)) for the ionizable groups located at the position r within the jth layer are defined by μj (y(r )) = 1/[1 + 10−εj(pKa,j − pH) exp(εjy(r ))]

(14)

In the limit εj{pKa,j − pH} ≫ 1, complete dissociation of the ionizable groups in layer j is reached and μj(y(r)) → 1. The set of coupled electrohydrodynamic equations governing the electrophoretic mobility μ of the above particle has been derived by Langlet et al.,19 who extended the theory by Duval and Ohshima14 applicable to diffuse soft particles with a monolayered shell compartment. Briefly, under given conditions of pH and solution ionic strength, μ is obtained by resolving the following:19,30 (i) y(r), as determined by the nonlinear Poisson−Boltzmann equation that includes the radial distribution for the density of fixed charges across the shell (eqs 13 and 14), (ii) the Navier−Brinkman equation that involves the friction force exerted by the particle on the electroosmotic flow, having in mind that this friction term depends on the radial distribution of polymer segments (eq 12) and on the nominal flow penetration length 1/λo for the shell as a whole, and (iii) the continuity equations for the N mobile ions present 14659


Langmuir

Article

Table 2. Mass Density md,j, Reciprocal of the Mean Free Path Length md,j/kj(α0), and Cryo-TEM contrast md,j/kj(α0) − md,w/kw(α0) for Different Materials ja

relevant data for a given selected particle, electron density profiles were further averaged from those collected along three lines separated by ca. 60°, as schematically illustrated in Figure 3. Because the background contribution is not identical all over

material j NR particle core NR particle shell HGW polyisoprene hevamine hevein REF SR α-lecithin

md,j (g/cm3)

md,j/kj(α0) (nm−1)

md,j/kj(α0) − md,w/kw(α0) (nm−1)

−

−

(1.182 ± 0.047) × 10−3

−

−

(3.009 ± 0.622) × 10−3

0.920 0.910 1.350 1.350 1.350 1.350 1.058

7.047 8.189 1.113 1.112 1.117 1.113 9.310

× × × × × × ×

10−3 10−3 10−2 10−2 10−2 10−2 10−3

0 1.142 4.083 4.070 4.125 4.081 2.263

× × × × × ×

10−3 10−3 10−3 10−3 10−3 10−3

a

The values of the contrast derived for the NR particle core and shell are indicated [values obtained from reconstruction of G(r)/G0 profiles (see example in Figure 3) collected on 60 NR particles]. For the sake of comparison, the contrasts for polyisoprene, hevamine, hevein, rubber elongation factor (REF) protein, small rubber protein (SR), and α-lecithin were evaluated for an acceleration voltage of 120 kV and an aperture of 1.25 mrad, with filtering of the energy stemming from the inelastic scattering contribution. Further details in the text.

Figure 3. Typical electron density profiles G(r) normalized to G0 as obtained from cryo-TEM for the particle displayed in the inset (symbols). The full line corresponds to data reconstruction from eq 8 on the basis of a core−shell particle representation (RC = 45.7 nm, RS = 49 nm, md,core/kj(α0) − md,w/kw(α0) = 1.195 × 10−3 nm−1 and md,shell/kshell(α0) − md,w/kw(α0) = 2.960 × 10−3 nm−1), while the dotted line is a prediction from eq 8 ignoring the contribution stemming from the particle shell component (RC = RS = 49 nm and md,core/kcore(α0) − md,w/k(α0) = 1.33 × 10−3 nm−1). Error bars pertain to deviations evaluated from the electron density profiles collected along the three lines shown in the inset.

voltage of 120 kV, objective aperture of 1.25 mrad). Within the experimental error bar, the contrast derived for the NR particle core is in good agreement with that expected for polyisoprene material. In addition, the contrast obtained for the shell layer is in between that expected for hevamine and α-lecithin (Table 2). Evaluation for other constitutive proteins of the shell such as hevein, rubber elongation factor protein (denoted as REF32), and small rubber proteins33−35 leads to similar contrast values compared to that derived for hevamine. Any further quantitative identification of the chemical composition of the NR shell component remains speculative because the range of values obtained for the NR shell contrast covers that of the proteins and phospholipids constituting the NR shell component. Obviously, additional data are then required to derive further information on the possible organization of proteins and lipids within the particle shell component. In section 4.2, we show that an advanced analysis of the electrophoretic response of NR particles over a broad range of pH and electrolyte concentration conditions sheds light on the structural organization of the NR shell component. 4.2. Electrokinetics. The dependence of the NR electrophoretic mobility μ on solution ionic strength at neutral pH is given in Figure 4. The results quantitatively agree with the electrokinetic data previously reported by Ho et al.18 under such pH and electrolyte concentration conditions. In detail, the mobility is systematically negative over the whole range of salinity levels tested. This is in line with the presence of, e.g., deprotonated carboxylic groups from shell proteins. Upon increasing suspension ionic strength, μ decreases in magnitude due to the screening of the shell charge by the ions present in the electrolyte medium. For sufficiently large ionic strengths, μ tends asymptotically to a finite nonzero plateau value, which is clear from the inspection of the curve μ vs ionic strength in linear scale (not shown). This feature is the typical signature for the presence of an ion- and water-permeable shell layer supported by the particle core surface,13−17 thereby confirming the cryo-TEM analysis displayed in Figure 3. The resulting soft (core−shell) nature of the NR particles dismisses de facto the

the sample surface area due to nonuniform hyperquenched glassy water film thickness (see Figure 2), G0 was estimated upon third-order polynomial extrapolation of four contrast values selected outside the particle along the line where electron density profiles were measured. As a last step, the obtained data displayed in Figure 3 were analyzed on the basis of eq 8. For that purpose, the radius of the particle core was derived from the cryo-TEM image (as done for generating the size distribution given in Figure 2); the thickness of the particle shell layer together with the core and shell contrasts md,j/kj(α0) − md,w/kw(α0) were evaluated by least-mean-square data analysis. We obtained a shell thickness of 3.2 ± 1.2 nm, a value that is 5 times larger than the pixel size (0.6 nm), thus confirming the suitability of the cryo-TEM technique for deriving the core−shell nature of NR particles. Figure 3 clearly shows that an appropriate reconstruction of the electron density profiles necessarily requires the introduction of a shell particle component in the modeling of G(r). Ignoring this component leads to electron density values that smoothly decrease when entering the particles, which does not conform to the steep decrease of G(r) with decreasing r from 50 nm to ca. 46 nm and to the local minimum of G(r) at r ≈ 46 nm. As detailed in section 4.2, the shell layer thickness derived from cryo-TEM helps in constraining the theoretical recovery of NR electrophoretic mobility data. Quantitatively, the cryo-TEM contrasts md,j/kj(α0) − md,w/ kw(α0) obtained for the particle core and particle shell are (1.182 ± 0.047) × 10−3 and (3.009 ± 0.622) × 10−3 nm−1, respectively (values averaged over the results obtained for the 60 NR particles analyzed). For the sake of comparison, the theoretical contrasts for cis-polyisoprene (constituent of NR particle core), for one of the proteins that mainly constitute the NR particle surface (i.e., hevamine31), and for α-lecithin (main phospholipid in NR according to ref 7) are reported in Table 2 under the conditions of interest in this study (acceleration 14660


Langmuir

Article

Figure 4. Left: Electrophoretic mobility of NR particles measured at neutral pH as a function of solution ionic strength. Right: Experimental electrophoretic mobility of NR particles as a function of pH at 4, 30, 50, and 100 mM electrolyte concentration (indicated), values given at pH 5.8. In both panels, the symbols correspond to measurements and full lines correspond to mobility evaluation from the theory outlined in section 3.2 for a core particle of radius RC = 45 nm (Figure 2) surrounded by a bilayered shell (M = 2). Model parameters for the first cationic layer surrounding the particle core: pKa,1 = 4.2 ± 0.1, δ1 = 3.5 ± 1.3 nm, α1/δ1 = 0.12 ± 0.05, ε1 = +1, and n1 = 400 ± 10 mM. Model parameters for the second anionic layer at the periphery of the shell: pKa,2 = 0.7 ± 0.1, δ2 = 0.6 ± 0.02 nm, α2/δ2 = 0.33 ± 0.1, ε2 = −1, and n2 = 575 ± 10 mM. Other model parameter: 1/λo = 0.5 ± 0.03 nm.

electrokinetic analyses carried out, e.g., in ref 7, where the concept of ζ-potential is adopted. We further report in Figure 4 the variation of μ with changing pH in the range 1−8 at fixed KNO3 electrolyte concentrations c∞ (4−100 mM). For a given c∞, μ remains constant and negative with decreasing pH from 8 to ca. 6. In this pH window, e.g., carboxylic groups present in the shell are fully dissociated and μ basically remains independent of pH. With further decreasing pH, μ decreases in magnitude and changes sign. This sign inversion stems from the presence of positively charged (protonated) amino groups in the shell. At sufficiently low pH and low c∞, the contribution of the protons to the overall ionic strength of the solution may lead to a significant compression of the electric double layer and therefore to a reduction in μ, which is reflected by a mobility maximum at low pH and c∞ = 4, 30, and 50 mM. At fixed pH value, an increase in c∞ leads to a strong screening of the charges carried by the NR shell and a reduction in |μ|. In the following, we define the point of zero mobility (denoted as PZM) as the pH value that corresponds to μ = 0. In the case of soft particles, this latter definition is more adequate than the commonly adopted isoelectric point (IEP) defined for hard particles by the pH value where the ζ-potential is zero, recalling that the ζ-potential has no physical basis for ion- and waterpermeable particles.13−17 The striking feature in Figure 4 is the strong dependence of the PZM on c∞. The PZM decreases indeed from ∼3.6 to 1 with increasing c∞ from 4 to 100 mM. Additional measurements of the NR electrophoretic mobility in the range 4−300 mM further indicate that the decrease in PZM with c∞ follows a sigmoidal dependence (Figure 5). In detail, PZM remains quasi-constant for c∞ ≤ 15 mM (PZM ∼ 3.6), it further decreases dramatically from 3.6 to 0.8 with increasing c∞ from 15 to 200 mM, and it essentially remains constant for c∞ ≥ 200 mM. The existence of specific adsorption of anions (NO3− in our case) at the NR particle surface might qualitatively explain the sign inversion of the mobility with c∞ at fixed pH. However, such superequivalent anion adsorption36 is very unlikely here because the electrokinetic response of NR particles measured as a function of NaCl concentration18 was found similar to that obtained with KNO3; i.e., it was independent of the nature of the ions used. In addition, the 3 pH unit magnitude in the variation of the PZM with changing c∞ (Figure 5) is not

Figure 5. Dependence of the point of zero electrophoretic mobility (PZM) as a function of solution ionic strength. Symbols, measurements; solid line, theory (section 3.2) with the parameters specified in Figure 4.

compatible with the specific adsorption of relatively indifferent anions such as NO3−. Instead, the above peculiar electrokinetic features of NR particles are compatible with a stratification of charges defined by distinct protolytic properties (i.e., different dissociation pK values) and distributed within the soft permeable shell layer surrounding the particle hard core.19 As detailed below, this explanation is supported by the electrokinetic theory outlined in section 3.2 and developed by Langlet et al.19 According to these authors, providing that the thickness of the outermost layer is comparable to the flow penetration length 1/λo and/or the Debye length 1/κ, the measured electrophoretic mobility reflects the electrostatic and hydrodynamic (friction) contributions from both the outer surface layer and the innermost layers located underneath. As a result, the PZM dependsamong other parameterson the electrolyte concentration that fixes the extension of the electric double layer within the soft stratified system analyzed. This conclusion was first given by Shinagawa et al.37 on the basis of their evaluation of electrostatic potential profiles across ion-penetrable membranes defined by nonuniform distribution of acidic and basic groups. The latter study, however, ignored the importance of flow penetration within the membranes under electrokinetic conditions. This hydrodynamic correction was included by 14661


Langmuir

Article

Figure 6. (A) Dependence of the electrophoretic mobility of a core−shell particle on pH and electrolyte concentration (indicated). Data are evaluated from the theory of section 3.2 applied to the limiting situation where anionic and cationic charges are homogeneously distributed within a step-function-like shell layer, as illustrated by the scheme. Model parameters: RC = 50 nm, 1/λo = 1 nm; eq 13 is replaced here by ρfix(r) = [ρ1μ1(y(r)) + ρ2μ2(y(r))]f(r) with f(r) = {1− tanh [(r − RC−δ)/α]}/2 (α → 0, δ = 13.5 nm the shell thickness) and the indices 1 and 2 pertain to the cationic (ε1 = +1) and anionic (ε2 = −1) types of charges in the shell with pKa,1 = 6, pKa,2 = 3, n1 = n2 = 100 mM (solid lines), and n1 = 300 mM/ n2 = 100 mM (dotted lines). (B) Electrophoretic mobility of a core/chemically stratified shell particle (see scheme) evaluated from the theory in section 3.2 as a function of pH and electrolyte concentration (indicated). Inset: dependence of the corresponding PZM on solution ionic strength. Model parameters in panel B: as in panel A except that M = 2, n1 = 300 mM, n2 = 100 mM, δ1= 10 nm, α1/δ1 = 0.2, δ2 = 3.5 nm, and α2/δ2 = 0.3. Arrows in panels A and B indicate the position of the point of zero mobility (PZM).

In Figure 6, we evaluate as a function of pH and c∞ the electrophoretic mobility of a core particle surrounded by (i) a shell layer where positively and negatively charged groups (eqs 10 and 9, respectively) are uniformly distributed (Figure 6A), and (ii) a shell layer where these groups are distributed in separated inner and outer shell layers, respectively (M = 2 in eqs 11−14) (Figure 6B). The other model parameters, in particular the thickness δj=1,2 and the charge density ρj=1,2 of layers 1 and 2, are specified in the caption of Figure 6. Conformably to expectation,19 the PZM in the situation (i) above does not depends on c∞ and it is given by the classical expression PZM = (pKa,1 + pKa,2)/2 under the condition where the concentrations of positive and negative charges are equal (i.e., n1 = n2). In situation (ii), the spatial separation of the two types of charges in the particulate shell leads to a decrease of the PZM with increasing c∞, in agreement with the theoretical conclusions given in ref 19 (Figure 6B). In line with Figure 5, one may further identify two asymptotes at c∞ → 0 and c∞ → ∞ for the dependence of the PZM on c∞. At c∞ → 0 or equivalent δ2 ≪ 1/κ, the outer layer is immaterial in defining the potential distribution across the particle core/shell/solution interphases. The mobility μ and PZM then essentially reflect the electrostatic properties of the innermost layer, in particular the magnitude and sign of the charge it carries, to an extent that depends on the ability of the flow to penetrate within the shell. In the other extreme c∞ → ∞ or δ2 ≫ 1/κ, μ and PZM are predominantly governed by the electrohydrodynamic features

Langlet et al.19 in their electrokinetic formalism. Later, Duval et al.29 demonstrated the strong analogy existing between the electrophoretic properties of soft multilayered particles and the electrokinetic features of soft planar polyanionic/polycationic multilayers (or polyelectrolyte multilayers, PEM) obtained via streaming current/streaming potential measurements. Similarly to the PZM of stratified particulate systems, the point of zero streaming current (hereafter denoted as PZSC) of PEM may indeed depend on electrolyte concentration under the condition where the thickness of the outermost layer is comparable to 1/λo and/or 1/κ.29 On a qualitative level, the strong dependence of PZM and PZSC on c∞ has been detected for guinea-pig polymorphonuclear leukocytes,38 for positively charged poly(ethyleneimine) (PEI)/negatively charged poly(acrylic acid) (PAA) bilayers,29 and for PEI-cushioned lipidic membranes.29 Due to the complexity of these systems and, e.g., the difficulty of measuring accurately the thickness of the polymeric surface layer of core− shell particles, there has been so far no attempt to reconstruct electrokinetic data collected on soft chemically stratified systems with adequate theory. In the following, we successfully report the consistent modeling of the NR electrophoretic properties reported in Figures 4 and 5 on the basis of the formalism outlined in section 3.2. For the sake of demonstration, we further detail beforehand how the chemical stratification of a particle shell component dramatically impacts the electrokinetic properties of the particle as a whole. 14662


Langmuir

Article

of the only outer layer. For intermediate values of c∞, the electrokinetic properties of the core−shell particle are determined by the physicochemical characteristics of both layers whose contributions depend on the respective length scales δ1, δ2, 1/κ, and 1/λo. As previously detailed,19 the amplitude and direction of the shift in PZM with c∞ further depend on ΔpKa,j=1,2 = pKa,2 − pKa,1, on the respective magnitudes of the charge densities ρj=1,2 = Fεj=1,2nj=1,2, and on the quantities α1,2. Upon introducing some diffuseness for the transition of the segment density profile from layer 1 to layer 2 and/or from layer 2 to the outer solution, the corresponding parameters α1,2 ≠ 0depending on their magnitude as compared to 1/κ, 1/λo, and δ1,2may indeed lead to significant modifications of the electroosmotic flow distribution and the potential profiles across the shell as compared to the situation of step-function-like repartition of layers. In turn, this modulates the respective c∞-dependent contributions of layers 1 and 2 to the particle electrokinetic response. The reader is referred to refs 19 and 29 for further details. The consistent modeling of the NR electrokinetic properties as a function of c∞ and solution pH is reported in Figures 4 and 5. The analysis shows that the electrophoretic response of NR particle is equivalent to that of a core particle surrounded by a permeable shell composed by the succession of a cationic layer and an anionic layer (M = 2). This representation leads to the correct variation of μ and PZM over 7 pH units and 2 orders of magnitude in c∞. The modeling was performed with δ1/δ2 = 5.8, 1/λo ∼ δ2, and δ1 + δ2 = 4.1 nm, in good agreement with the NR surface layer thickness obtained independently from cryo-TEM (Figure 3). The additional model parameters pertaining to the innermost layer (j = 1) are pKa,1 = 4.2 ± 0.1, ρ1/F = 400 ± 10 mM, and α1/δ1 = 0.12 ± 0.05, and those relevant for the outermost layer (j = 2) are pKa,2 = 0.7 ± 0.1, ρ2/F = −575 ± 10 mM, and α2/δ2 = 0.33 ± 0.1. It is emphasized that the remarkable reconstruction of the NR electrokinetic features is here achieved with the minimum number of parameters required for explaining the dependence of PZM on c∞ and that of μ with pH and c∞. Obviously, the chemical composition of the NR shell is more complex than that suggested by eqs 9 and 10. An account of additional charging reactions would inevitably lead to an overdetermined parametric modeling. Consequently, the obtained pKa,j=1,2 should be viewed as effective dissociation constants pertaining to the components that predominantly govern the electrostatic properties of layers 1 and 2. The values of pKa,j=1,2 allow for deriving some information on the nature of the layer j = 1 and j = 2 within the NR shell. Namely, a literature survey on the composition of NR surface layer reveals that the main constitutive proteins of the shell are defined by isoelectric points in the range 4−4.6.7 These values are close to that we obtain for pKa,1. In addition, the reported pK value for the phosphate groups of the main phospholipidic constituents in the shell, α-lecithin (or phosphatidylcholine), is 0.8,39 which is in agreement with the magnitude of pKa,2 derived from electrokinetic analysis. These elements strongly suggest that the NR shell is composed of a protein-rich layer supported by the polyisoprene core of the NR particles and a second layer containing the hydrophilic heads of the lipids in contact with solution and that protrude from the innermost proteinaceous layer. A schematic representation of this proposed NR interfacial structure is given in Figure 7. In addition, we recall that the contrast value determined from cryo-TEM for layer 1 is intermediate between those expected

Figure 7. Schematic representation of the here-proposed NR interfacial structure. The corona of NR particles consists of a protein-rich layer with a typical thickness of δ1 = 3.5 nm and a second layer containing hydrophilic heads of the lipids in contact with solution (characteristic thickness δ2 = 0.6 nm) and that protrude from the innermost proteinaceous layer. For the sake of illustration, we report a typical distribution of the density of charges carried by NR shell at pH 2 for y(r) → 0.

for hevamine and α-lecithin (Table 2), having in mind that the thickness δ2 ≪ δ1 of layer 2 is of the order of the pixel size in the micrographs of Figure 3 and is thus not detectable by cryoTEM. This result indicates that layer 1 probably contains elements other than proteins, including phospholipidic residues possibly encountered at the core surface of the NR particles or adsorbed on the hydrophobic domains of the proteins. The thicknesses δ1 and δ2 obtained in this study qualitatively agree with the representation given in Figure 7. The value for δ1 is slightly lower than, e.g., the size reported for purified hevamine from X-ray crystallographic analysis (∼5 nm).40 This may indicate that the protein structure is somehow flattened when adsorbed at the NR core component. As already stated, we verified from DLS data that NR particles are stable over the range of pH and ionic strength conditions examined in this work (Supporting Information), including the sets of pH and ionic strength values that correspond to zero electrophoretic mobility condition. This observation supports the presence of a densely grafted corona at the NR surface, in line with an electrosteric stabilization of the particles for medium composition where the PZM condition is met. A refined representation of the molecular arrangements of proteinaceous and phospholipidic components at the NR/solution interface would surely require additional spatially resolved measurements. The current study has the merit to clearly evidence a spatial separation of charges of different types (presumably carried by protein-like materials or lipids) and thus to rule out the mixed lipid−protein NR surface structure representation often given in the literature.7−9 The results further suggest that the use of any proteolytic treatment of the NR surface will lead to a removal of not only the surface proteins but also that of the lipids present all across the shell, within the innermost protein-rich layer and in the outermost layer. This questions the applicability of the above enzymatic treatment for identifying unambiguously the respective contributions of lipids and proteins to NR surface properties. In addition, we note that the presence of negatively charged lipidic components in the peripheral region of NR shell is very well in line with repulsive interaction forces recently measured when approaching a (negatively charged) AFM tip toward a NR particle surface.6 The detection of tip jump-in and jump-off events further likely originates from hydrophobically driven adhesion processes favored by the presence of the lipidic 14663


Langmuir

Article

Notes

components beyond the protein surface layer. In particular, measured adhesion forces upon retraction of the tip may reach values as large as 1−3 nN.6 This magnitude even exceeds that obtained for hydrophobic coatings of Streptococcus thermophilus bacteria, whose adhesion onto abiotic materials strongly depends on temperature following important modulations of the hydrophobic/hydrophilic balance of their polymeric surface layer.41 This comparison again supports the sole presence of charged lipidic components at the outer periphery of the NR shell structure.

The authors declare no competing financial interest.

■

5. CONCLUSIONS The structural organization of the soft layer located at the surface of natural rubber (NR) particles from Hevea brasiliensis is investigated by means of cryo-TEM and detailed interpretation of electrophoretic mobility measured over a broad range of pH and ionic strength conditions. Theoretical analysis of the cryo-TEM micrographs allows the evaluation of the soft surface layer thickness (2−4 nm). Electrokinetics further reveals a number of peculiar pH- and ionic-strengthdependent features that are all in line with the existence of a chemical stratification of the shell particle component. In detail, results suggest the presence of a first protein-rich layer in the vicinity of the polyisoprene particle core and the positive charge of that layer mainly stems from the presence of amino group residues. This layer further likely contains lipidic constituents anchored at the hydrophobic polyisoprene core or adsorbed at the hydrophobic domains of the proteins. The hydrophilic heads of these lipidic constituents further protrude from the proteinaceous layer, thus forming a negatively charged corona facing the outer electrolyte solution phase. This representation is formulated from the remarkable recovery of NR electrophoretic mobility collected over 7 pH units and 2 orders of magnitude in solution ionic strength, with the use of electrohydrodynamic theory for electrophoresis of ion-permeable and chemically stratified particles. The approach revisits previously reported shell structure organizations according to which the NR shell consists of either a mixed protein−lipid layer or a lipid monolayer surrounding the polyisoprene core beneath a peripheral protein film. In addition, the work provides the first successful and systematic confrontation between theory and experiments for analyzing NR electrokinetic features. As such, it underlines the shortcomings of previous work based either on the incorrect use of the ζ-potential concept or on that of approximate Ohshima’s expression strictly valid for homogeneous and poorly charged surface layers with thicknesses much larger than the Debye screening length and flow penetration length.

■

ASSOCIATED CONTENT

S Supporting Information *

Size distributions of NR particles for various sets of pH values, KNO3 electrolyte concentrations, and NR suspension equilibration times, as measured by dynamic light scattering (DLS), Figures S1−S3. Cryo-TEM micrograph of a 7 wt % NR colloidal particle suspension at pH 11.7 (no KNO3 salt added), Figure S4. This material is available free of charge via the Internet at http://pubs.acs.org.

■

REFERENCES

(1) Mooibroek, H.; Cornish, K. Alternative sources of natural rubber. Appl. Microbiol. Biotechnol. 2000, 53, 355−365. (2) Amari, T.; Themelis, N. J.; Wernick, I. K. Resource recovery from used tires. Resour. Policy 1999, 25, 179−188. (3) Allmers, H.; Brehler, R.; Chen, Z.; Raulf-Heismoth, M.; Fels, H.; Baur, X. Reduction of latex aeroallergens and latex-specific IgE antibodies in sensitized workers after removal of powdered natural rubber latex gloves in a hospital. J. Allergy Clin. Immunol. 1998, 102, 841−846. (4) Singh, A. P.; Wi, S. G.; Chung, G. C.; Kim, Y. S.; Kang, H. The micromorphology and protein characterization of rubber particles in Ficus Carica, Ficus Benghalensis and Hevea Brasiliensis. J. Exp. Bot. 2003, 54, 985−992. (5) Cornish, K. Biochemistry of natural rubber, a vital raw material, emphasizing biosynthetic rate, molecular weight and compartmentalization, in evolutionary divergent plant species. Nat. Prod. Rep. 2001, 18, 182−189. (6) Gaboriaud, F.; de Gaudemaris, B.; Rousseau, T.; Derclaye, S.; Dufrene, Y. F. Unravelling the nanometer-scale stimuli responsive properties of natural rubber latex particles using atomic force microscopy. Soft Matter 2012, 8, 2724−2729. (7) Sansatsadeekul, J.; Sakdapipanich, J.; Rojruthai, P. Characterization of associated proteins and phospholipids in natural rubber latex. J. Biosci. Bioeng. 2011, 6, 628−634. (8) Cornish, K.; Wood, D. F.; Windle, J. J. Rubber particles from four different species, examined by transmission electron microscopy and electron-paramagnetic-resonance spin labeling, are found to consist of a homogeneous rubber core enclosed by a contiguous monolayer membrane. Planta 1999, 210, 85−96. (9) Nawamawat, K.; Sakdapipanich, J. T.; Ho, C. C.; Ma, Y.; Song, J.; Vancso, J. G. Surface nanostructure of Hevea Brasiliensis natural rubber latex particles. Colloids Surf., A 2011, 390, 157−166. (10) Tanaka, Y.; Kawahara, S.; Tangpakdee, J. Structural characterization of natural rubber. KGK, Kautsch. Gummi Kunstst. 1997, 50, 6− 11. (11) Francius, G.; Polyakov, P.; Merlin, J.; Abe, Y.; Ghigo, J.-M.; Merlin, C.; Beloin, C.; Duval, J. F. L. Bacterial surface appendages strongly impact nanomechanical and electrokinetic properties of Escherichia coli cells subjected to osmotic stress. PLoS One 2011, 6 (5), e20066. (12) Gaboriaud, F.; Gee, M. L.; Strugnell, R.; Duval, J. F. L. Coupled electrostatic, hydrodynamic and mechanical properties of bacterial interfaces in aqueous media. Langmuir 2008, 24, 10988−10995. (13) Ohshima, H. Electrophoresis of soft particles. Adv. Colloid Interface Sci. 1995, 62, 189−235. (14) Duval, J. F. L.; Ohshima, H. Electrophoresis of diffuse soft particles. Langmuir 2006, 22, 3533−3546. (15) Hill, R. J.; Saville, D. A.; Russel, W. B. Electrophoresis of spherical polymer-coated particles. J. Colloid Interface Sci. 2003, 258, 56−74. (16) Lopez-Garcia, J. J.; Grosse, C.; Horno, J. Numerical study of colloidal suspensions of soft spherical particles using the network method: 1. DC electrophoretic mobility. J. Colloid Interface Sci. 2003, 265, 327−340. (17) Duval, J. F. L.; Gaboriaud, F. Progress in electrohydrodynamics of soft microbial particle interphases. Curr. Opin. Colloid Interface Sci. 2010, 15, 184−195. (18) Ho, C. C.; Kondo, T.; Muramatsu, N.; Ohshima, H. Surface structure of natural rubber particles from electrophoretic mobility data. J. Colloid Interface Sci. 1996, 178, 442−445. (19) Langlet, J.; Gaboriaud, F.; Gantzer, C.; Duval, J. F. L. Impact of chemical and structural anisotropy on the electrophoretic mobility of spherical soft multilayer particles: the case of bacteriophage MS2. Biophys. J. 2008, 94, 3293−3312.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (J.F.L.D.). 14664


Langmuir

Article

Streptococcus thermophilus Δrgg0182 strain. Langmuir 2013, 29, 4847−4856.

(20) Crassous, J. J.; Rochette, C. N.; Wittemann, A.; Schrinner, M.; Ballauff, M.; Drechsler, M. Quantitative analysis of polymer colloids by cryo-transmission electron microscopy. Langmuir 2009, 25, 7862− 7871. (21) Langmore, J. P.; Smith, M. F. Quantitative energy-filtered electron microscopy of biological molecules in ice. Ultramicroscopy 1992, 46, 349−373. (22) Smith, M. F.; Langmore, J. P. Quantitation of molecular densities by cryo-electron microscopy: determination of the radial density distribution of tobacco mosaic virus. J. Mol. Biol. 1992, 226, 763−774. (23) Reimer, L. Transmission electron microscopy: Physics of image formation and microanalysis, 4th ed.; Springer Series in Optical Sciences 36; Springer: Berlin, 1997. (24) Wall, J.; Isaacson, M.; Langmore, J. P. The Collection of Scattered Electrons in Dark Field Electron Microscopy. Optik 1974, 39, 359−374. (25) Sceats, M. G.; Rice, S. A. Water; Plenum Press: New York, 1982; Vol. 7. (26) Sciortino, F.; Poole, P. H.; Essmann, U.; Stanley, H. E. Line of compressibility maxima in the phase diagram of supercooled water. Phys. Rev. E 1997, 55, 727−737. (27) Mayol, R.; Salvat, F. Total and transport cross sections for elastic scattering of electrons by atoms. At. Data Nucl. Data Tables 1997, 65, 55−154. (28) Duval, J. F. L.; Merlin, J.; Anantha, P. Electrostatic interactions between soft diffuse multi-layered (bio)particles: beyond DebyeHückel approximation and Deryagin formulation. Phys. Chem. Chem. Phys. 2011, 13, 1037−1053. (29) Duval, J. F. L.; Küttner, D.; Werner, C.; Zimmermann, R. Electrohydrodynamics of soft polyelectrolyte multilayers: point of zero-streaming current. Langmuir 2011, 27, 10739−10752. (30) Ascher, U.; Christiansen, J.; Russell, R. D. Collocation software for boundary value ODE’s. ACM Trans. Math. Software 1981, 7, 209− 222. (31) Jekel, P. A.; Hartmann, J. B. H.; Beintema, J. J. The primary structure of hevamine, an enzyme with lysozyme/chinitase activity from Hevea brasiliensis latex. Eur. J. Biochem. 1991, 200, 123−130. (32) Dennis, M. S.; Light, D. R. Rubber elongation factor from Hevea Brasiliensis: identification, characterization and role in rubber biosynthesis. J. Biol. Chem. 1989, 264, 18608−18617. (33) Van Parijs, J.; Broeckaert, W. F.; Goldstein, I. J.; Peumans, W. J. Hevein: an antifungal protein from rubber-tree (Hevea Brasiliensis) latex. Planta 1991, 264, 258−264. (34) Dennis, M. S.; Henzel, W. J.; Kohr, W.; Light, D. R. Amino acid sequence of rubber elongation factor protein associated with rubber particles in hevea latex. J. Biol. Chem. 1989, 264, 18618−18628. (35) Yeang, H. Y.; Ward, M. A.; Zamri, A. S. M.; Dennis, M. S.; Light, D. R. Amino acid sequence similarity of Hev b 3 to two previously reported 27- and 23-kDa latex proteins allergenic to spina bifida patients. Allergy 1998, 53, 513−519. (36) Lyklema, J. Electric double layers. In Fundamentals of Interface and Colloid Science. Vol. II: Solid-Liquid Interfaces; Academic Press: London, 1995; Chapter 3. (37) Shinagawa, T.; Ohshima, H.; Kondo, T. Isoelectric point of an ion-penetrable membrane. Biophys. Chem. 1992, 43, 149−56. (38) Nagahama, T.; Muramatsu, N.; Ohshima, H.; Kondo, T. Surface electric characteristics of guinea-pig polymorphonuclear leucocytes. Colloids Surf. 1992, 67, 61−65. (39) Moncelli, M. R.; Becucci, L.; Guidelli, R. The intrinsic pKa values for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in monolayers deposited on mercury electrodes. Biophys. J. 1994, 66, 1969−1980. (40) Rozeboom, H. J.; Budiani, A.; Beintema, J. J.; Dijkstra, B. W. Crystallization of hevamine, an enzyme with lysozyme/Chitinase activity from Hevea brasiliensis latex. J. Mol. Biol. 1990, 212, 441−443. (41) Francius, G.; Henry, R.; Duval, J. F. L.; Bruneau, E.; Merlin, J.; Fahs, A.; Leblond-Bourget, N. A. Thermo-regulated adhesion of the 14665


Shell Structure of Natural Rubber Particles - American Chemical Society

Recommend Documents