In the present work, we developed a method for the direct determination of the hydrophobic character of NMs. The hydrophobicity is determined by the direct measurement of the binding affinity of the nanomaterial to the different surfaces or collectors. Each collector is characterised by a combination of surface energy components that according to the XDLVO theory will allow the determination of the surface energy components of the nanomaterials. The affinity is measured by calculating the surface density of nanoparticles immobilised on the specific collector after a given exposure time and after rinsing the sample thoroughly with water to remove loosely bound nanoparticles and possible salt residues.
The detection platform consists in a silicon surface modified with plasma polymer (pAA and PTFE) and layer-by-layer-deposited polyelectrolytes (PSS and PDDA) in order to generate areas with controlled properties. The combination of plasma deposition and polyelectrolytes self-assembly allows the tuning of the surface energy components in a relatively wide range, without dramatically affecting the surface morphology. The selectivity and specificity of the ENMs binding to the surfaces strongly depend on characteristic of the interaction forces such as force strength, range of interaction distance and attractiveness and repulsiveness. Other parameters for the tuning of the interaction forces were ionic strength and pH of the colloidal dispersion used.
Polystyrene particles (200 nm diameter) were used as model hydrophobic NM. The non-modified particles are stabilised by sulfonate groups and are thus negatively charged and hydrophobic. The same types of polystyrene particles modified with carboxyl groups were chosen as hydrophilic model. This surface modification confers to the nanoparticles a higher hydrophilicity.
The hydrodynamic diameter of the nanoparticles was measured by dynamic light scattering together with their zeta potential, which also plays an important role by modifying the electrostatic forces involved in the interactions. To evaluate the difference in hydrophobicity of the different types of NM, their contact angles with water were also measured. All the results are presented in Table 1 for measurements done at pH 7. The PS NMs have a slightly larger hydrodynamic diameter than the PS-COOH while their nominal range declared by the producer is similar (200 nm with a polydispersity index of 10%). The values for the Z-potential indicate that the PS particles, stabilised by sulfonate groups as declared by the producer, are more negatively charged than the PS-COOH particles. The large negative value for the zeta potential enables a colloidal stability for the hydrophobic PS particles even at high salt concentration.
A contact angle of 95° was measured for the PS particle monolayer and 23° for the PS-COOH. Those results confirmed that the modification of the polystyrene particles with carboxyl groups increases the hydrophilicity of the NM, corresponding to a lower contact angle. On the other hand, the contact angle (affinity) for the α-bromonaphtalene was very low for the PS NMs (12°) while increasing (22°) for the PS-COOH particles. Besides, water/octanol partition experiments have been performed and revealed that for both functionalised and non-functionalised materials, no nanoparticles were found in the non-polar octanol phase. This result indicates that this method does not enable to distinguish between hydrophobic and hydrophilic NMs.
To study the selective binding of NMs onto chemically modified surfaces, two sets of surfaces have been prepared. A first set of Si surface has been coated with a plasma-deposited layer of PTFE (hydrophobic) first, and then with several layers of polyelectrolytes (PE) (PSS/PDDA) to enhance the surface hydrophilic character. A second set of surfaces has been prepared with plasma-deposited PAA as starting layer (hydrophilic) and also further modified by the PSS/PDDA multilayers. A complete characterisation was performed on the different substrates to further determine their properties. The results are presented in Table 2.
The thickness and the refractive index of the plasma-deposited substrates and the PE layers were measured by ellipsometry after each step of PE deposition. The thickness of the PTFE and the PAA was respectively 123 ± 1 nm and 89 ± 1 nm, with a refractive index of 1.52 ± 0.02 and 1.38 ± 0.04, respectively. The thickness for each PE layer was then fitted using a refractive index of 1.38 (value declared by the producer). The values for the thickness of each PE during the LBL formation are reported in Table 2. The mechanism of formation of the LBL was different for the two polymer substrates: the LBL deposited PE formed on the PTFE is very homogenous (about 0.6 nm/layer) with an initial increase of the roughness (up to 0.85 nm for T3). After the formation of the 4th layer, the value of the roughness remains constant indicating the formation of a homogeneous polyelectrolyte layer. It is most likely due to the exposition of the hydrophobic domains of the first PE layer towards the substrate, leaving the positively charged groups directed towards the water solution. The successive PE mainly interacts through its negative charges to neutralise the positive ones, thus exposing the hydrophobic domains. This mechanism of formation of the PE explains why the T6 sample is not super-hydrophilic (CA water 45 ± 2°) since the external surface contains hydrophobic domains, which contribute to the reduction of the acid-base component of the surface free energy.
The formation of the first positively charged PE layer on the hydrophilic PAA results in an intermixing of positively charged groups and negatively charged carboxyl groups of the PAA. This is demonstrated by a dramatic change in zeta potential (from −78 ± 1 mV to −5 ± 1 mV) and by a slight reduction of the native roughness of the PAA. Furthermore, the thickness of the first layer measured by ellipsometry is lower than 0.3 nm. Subsequently, the surface becomes super-hydrophilic with a relatively large surface energy, and in particular with a relatively large acid-base component.
The AFM analysis shows that the morphology of the collectors is not strongly affected by the formation of the polyelectrolyte layers. The results show that the roughness increases from 0.29 to 0.76 nm for the PTFE substrate and from 0.23 to 1.87 nm for the PAA substrate. This increase in roughness is due to the formation of nano- and micro-clusters of polyelectrolytes during self-assembly. The surface chemical homogeneity at the nanoscale was investigated through AFM with force mapping, using different probes.
The adhesion force is mapped automatically by the instrument in the selected area with a resolution of 10 nm (the lateral resolution of the technique is limited by the radius of curvature of the used tip, which is nominally 10 nm). The force mapping revealed that the average adhesion of the PDDA-terminated layers was considerably higher than the average adhesion for the PSS-terminated layer, with an average adhesion value of 0.06 ± 0.01 nN for PSS vs. 0.78 ± 0.3 nN for PDDA. The observed increase of roughness was attributed to the formation of clusters due to the intermixing between the two polyelectrolytes during the multilayer formation. The most important information, which could be extracted from these experiments, was that the surfaces were homogeneous at the nanoscale in terms of adhesion forces, and hence they could be treated as flat homogenous plane in the modelling of the interaction forces with the nanoparticles. The results are shown in Fig. 1.
The chemical composition of the surface was studied by XPS and ToF-SIMS after the surface modifications by the PTFE film and layer-by-layer polyelectrolytes deposition. The spectra are presented in Fig. 2. The surface analysis through XPS measurements after plasma deposition is mainly characterised with a fluorine and a C1s peak having a characteristic PTFE shape (Balazs et al. 2005; Jaszewski et al. 1999) demonstrating the presence of a confluent PTFE layer that masks fully the silicon substrate. The ToF-SIMS static spectrum, which reveals chemical information about the outermost layer thanks to the high surface sensitivity of the technique, confirmed the XPS findings by the identification of carbon and carbon-fluorine clusters such as CF2
+ and CF3
+. The analysis after the six PE layer self-assembly showed the full coverage of the PTFE surface plasma. In fact, from the XPS survey spectra (Fig. 2a–c), it is possible to notice a drastic decrease of the signal given by the PTFE, such as fluorine, and the appearance of nitrogen and sulphur signal, belonging to the PDDA and PSS, respectively. Moreover, a comparison of the C1s core-level spectra collected on the plasma-polymerised PTFE film before and after the deposition of the polyectrolyte layers (Fig. 2d, e) reveals a strong decrease of the components related to the C-F moieties with a corresponding increase of the hydrocarbon, carboxyl, ester and amino compound components. The ToF-SIMS data support the XPS results as can be seen in Fig. 2i, j where positive spectra of the different substrates are illustrated. The appearance of the fragments such as+ (31 m/z) and CF3
+ (69 m/z) and the corresponding disappearance of the fragments related to the SiO
substrate after the PTFE deposition demonstrate that the film is uniform and pin-hole free. After the deposition of the PDDA and PSS, the PTFE fragments and the detection of C
+ mass peaks (e.g. C3H8N at 58 m/z) with the correspondent suppression of the peaks related to PTFE further vouching the successful functionalisation of the PTFE film with polyelectrolyte multilayers with a thickness greater than 2 nm. Those analyses demonstrated a good coverage of the substrate, indicating that each polyelectrolyte layer is covering the one underneath, and after siz layers (3 PDDA + 3 PSS alternatively) the PTFE substrate is not detectable.
The surfaces were then characterised in terms of surface energy components using the two-liquid contact-angle technique and using the model of Owen, Wendt and Fowkes, also known as the OWRK theory. Briefly, we measured the advancing contact angle with water and α-bromonaphtalene, respectively. This combination of testing liquids allowed to take into account the polar component (AB) and the dispersive component (LW) of the surfaces. The surface energy components (Lishfitz-van der Waals and acid-base) were then calculated from the contact angles with the two liquids, solving a system of equations in two variables (Eq. 6). The surface energy components for the different collectors are shown in Table 2.
It can be observed that PTFE exhibits the lowest value for both LW and AB components (19.3 mJ/m2 and 0.9 mJ/m2, respectively). The presence of PE layers results in an increase of the LW and the AB components, with the AB component being larger on the PSS-terminated layers than on the PDDA-terminated layers. On PAA, the surface energy components are 41.8 mJ/m2 and 18.3 mJ/m2, respectively, and the addition of PSS and PDDA does not change these values dramatically.
To summarise this section, the experiments performed on the PTFE- and PAA-modified surfaces showed the same trend, even though the base layer of PTFE is hydrophobic and that of PAA more hydrophilic. We could indeed reach similar surface properties, with an increase in hydrophilicity on both substrates thanks to the formation of PE layers, a slight increase in roughness and a negative zeta potential for all conditions. The 6th layer enabled to obtain close surface properties with two substrates of different given properties, PTFE or PAA. In conclusion, the LBL-modified hydrophobic and hydrophilic polymers exhibit, after a given number of modification layers:
Relatively low surface roughness (RMS roughness <2 nm).
Surface chemical homogeneity, as indicated by the XPS and Tof-SIMS analysis and with homogeneous adhesion forces, as indicated by AFM.
A negative surface zeta potential (in order to avoid electrostatic attraction forces).
Two “wettable” surfaces exhibiting different combination of LW and AB components: T6 and P6 with a relatively low and very high value of the AB component, respectively.
To evaluate the binding capacity of the hydrophobic collector, a PTFE-coated surface, characterised by a contact angle of 105°, was tested against hydrophobic PS particles. The PS particles were incubated on the surface using 16 different conditions of pH and ionic strength, ranging from 0 to 100 mM NaCl and pH 2 to 10. The goal was to assess the influence of electrostatic forces on NP binding. The surfaces were then analysed via SEM, and the surface coverage was determined for the different conditions using ImageJ software. The results are presented as colour maps in Fig. 3.
The surface coverage of the hydrophobic particles on the hydrophobic collector was found to be low for all conditions, with a slight trend for a higher binding for low pH and high salt concentration. This result could be attributed to the poor wettability of the PTFE surface. Indeed, because of its high hydrophobicity, the immersion of the substrate in water may promote the formation of micro-bubbles (Attard 2003; Steitz et al. 2003), thus creating a physical barrier that impedes the contact between surface and particles in suspension and preventing the hydrophobic interactions to occur. Experiments using surface plasmon resonance was performed in order to confirm this hypothesis; this experiment and the results are presented in the Supporting information.
The modification of the PTFE surface by a multilayer of polyelectrolytes influences the properties of the substrate by drastically lowering the surface hydrophobicity (as shown by the decrease of contact angle down to 45° with water). Such a difference suppresses the formation of a physical barrier when in contact with water (SI.1). The same experiments were then performed with the same set of conditions of ionic strength and pH, with the PTFE surface covered by six layers of PE, which exhibit the lowest contact angle possible for this substrate. The SEM observation of the surfaces following the incubation of the particles showed an important NM binding in all conditions. The mapping of the surface coverage calculated with ImageJ under the different conditions is shown in Fig. 3b.
Compared with the results without PEs, the surface covered by the particles is dramatically increased.
Moreover, one can observe an important increase of the NM binding with the decrease in pH and the increase of salt concentration (red areas on the map 3b) since these experimental conditions favour the screening of the electrostatic repulsive forces. At the opposite, at low ionic strength and high pH, the electrostatic repulsive forces are preponderant impeding the NM to approach the surface leading to a very low binding rate. By screening the repulsive forces, hydrophobic particles can approach and bind on the PTFE covered by six PE layers, demonstrating that the hydrophobic forces generated by the hydrophobic properties of the substrate act at a long range overcrossing the polyelectrolyte layers (Meyer et al. 2006).
Further experiments were performed using the hydrophilic substrate (PAA), with and without PE modification to compare the binding rate obtained with the hydrophobic substrate and to verify the validity of the theory of the long-range interactions in our experimental conditions. The degree of hydrophobicity of the PAA-modified surfaces was tuned from around 48° of contact angle without polyelectrolyte to 18° with six layers of polyelectrolytes. The different surfaces were analysed by SEM after incubation of hydrophobic polystyrene particles in the conditions previously described. The results are presented in the maps in Fig. 3 in terms of surface coverage for PAA alone (Fig. 3c) and PAA with six layers of polyelectrolytes (Fig. 3d). The SEM images are available in Supporting information S.2.
As it can be observed for both PAA alone and PAA + PE surfaces, the surface coverage is extremely low for most conditions, with salt concentration between 0 and 10 mM and for all pH on PAA alone and pH 4 to 10 on PAA + PE. The trend already observed before, with an increase in the binding rate with the increase of ionic strength and decrease of pH is even more evident than before since particle binding only increases in those extreme conditions.
Comparing the results on the PAA and PTFE substrates without polyelectrolytes (Fig. 3a, c), it can be assumed that, with the highest ionic strength, the binding is more important on PAA than on PTFE because of the physical barrier existing due to the highly hydrophobic properties of PTFE. The main binding difference occurs between PTFE + PE and PAA + PE (Fig. 3b, d). Indeed, the addition of PE layers induced a large increase in the binding onto the hydrophobic substrate, whereas the binding change with polyelectrolyte on PAA is observed only for pH 2 with salt. This shows that the hydrophilic superficial layer on hydrophobic substrate allows long-range hydrophobic interactions to take place, resulting in the binding of the hydrophobic particles only onto the hydrophobic substrate.
To summarise, when the hydrophobic PTFE substrate was in direct contact with the aqueous medium containing the nanoparticles, an air interface was generated, limiting the contacts with water, and thus preventing the physical interaction with particles. The most favourable conditions for the binding of hydrophobic particles onto the hydrophobic substrate is obtained by using a superficial hydrophilic layer on top of the hydrophobic substrate and by minimising the electrostatic repulsion thanks to low pH and high salt concentration. The hydrophilic layer has to be thin enough (here around 5 nm) to allow long-range hydrophobic interactions to take place. The use of a polyelectrolyte layer was in this case a good method to obtain a high hydrophilicity with a thin coverage of the hydrophobic substrate. The superficial hydrophilicity degree, representing the wettability of the substrate, was therefore not driving the binding but enabling the hydrophobic interactions, present only with the underlying hydrophobic substrate PTFE to take place.
To assess the selectivity of the collector towards hydrophobic particles, the same experiments were performed with the particles presenting a higher hydrophilicity. For this purpose, polystyrene particles modified with carboxyl groups were used, providing them hydrophilic properties as shown by a contact angle value in water of 23° and a zeta potential of −53.7 mV. The PS-COOH particles dispersed in 16 conditions of pH and ionic strength were incubated on the different surfaces. The analysis by SEM enabled to calculate the surface coverage for the different conditions. The results are presented as a colour map in Fig. 4.
As it can be seen in Fig. 4, the hydrophilic particles have an extremely low binding rate (<1% of surface coverage in most of the cases) for all conditions on the hydrophobic substrate with or without modification by the polyelectrolyte layers. A significant binding is observed only for high salt concentration and low pH, similar on the modified and non-modified surface. These results confirmed the absence of hydrophobic interactions explaining why no particle is bound to the different surfaces.
From the systematic study of NM binding onto the different collectors, it is possible to determine the surface energy components for the nanoparticles using the XLDVO theory.
Evaluation of the interaction potential using the XLDVO theory
The experimental observation enabled us to detect the particles exhibiting a good affinity for the surface. We measured the surface density of the particles firmly adsorbed onto the different surfaces after a fixed incubation time. This value is a function of the affinity of the different particles for the different collectors, i.e. for the corresponding interaction potential, which depends on the surrounding solution conditions (pH and salt concentration). The XDLVO equation of the interaction potential between particles and surfaces enables to predict, in the different conditions, if the formation of the particle-surface interface is energetically favourable and if potential barriers are present at different distances from the surface. The potential barriers are able to physically repulse the particles from the surface, influencing their observed affinity for the collectors’ surfaces.
Moreover, only PSS-terminated collectors (negatively charged) were used for the determination of the NM surface properties in order to avoid misinterpretations of the adhesion due to electrostatic absorption between the amines of the PDDA-terminated collectors, with the PS NM having a negative zeta potential. The model presented here consists in the interaction of a hard (not deformable) sphere, with the nominal radius of the NMs, approaching by diffusion a flat and homogenous surface (characterised by the surface energy components measured by the contact angle technique). No interactions between spheres are taken into account.
The XDLVO potentials as a function of the distance for a sphere-surface physical model have been calculated, and the results are shown in Fig. 5. The contribution of the different forces to the resulting potential is also shown as separated curves. The electrostatic potential between two surfaces of the same charge is always repulsive and corresponds to positive values of energy (kT). The potential barrier is already active at 10 nm and increases close to the surface. On the other hand, the VdW potential is acting at very short distances and is always attractive, with a strength of attraction regulated by the Hamaker constant, which depends on the particle-surface-medium system, and is directly related to the dispersive (LW) components of the interacting entities. With only these two potentials, the hydrophilic/phobic character of the surfaces or in other words their polar contribution to the surface energy is not taken into account. This polar interaction potential is represented by the AB potential, which depends on the polar component (AB) of the surface energy of the particle, of the surface and the medium. The AB potential is exponentially decreasing from the surface with a typical decay length (λ) of 0.6 nm, as reported in literature (van Oss 1994).
The AB potential in Fig. 5b is calculated for a hydrophobic spherical surface interacting with a relatively hydrophilic surface in water. Under these conditions, the AB potential is mainly contributing with an attractive force at about 1 nm distance from the surface. This potential is able to decrease the potential barrier created by the electrostatic potential, increasing the affinity of the particles for the surface. The action of the AB potential is able to explain the observed differences in affinity between the different particles with the used collectors. If we compare the interaction potential at pH = 7 and 10 mM [NaCl] (Fig. 5), we observe that for z < 1 nm (so very close to the surface), the potential barrier for the hydrophilic NMs (COOH) with both collectors is positive (around 350 kT). The same is occurring for the interaction potential between the hydrophobic particles (PS) and the pAA-6PE surface (highly hydrophilic). The only potential exhibiting a negative value (−500 kT) at that distance range is the one calculated between the PS particles and the PTFE-6PE. This potential is dominated in this range by the AB component as already explained.
By plotting in a colour map the value of the interaction potential between the particles and the surfaces as a function of the pH and salt concentration, we obtain a direct mapping of the affinity of the particles with the different surfaces (Fig. 6). The areas in red represent the conditions (of salt concentration and pH) at which, at a distance of 0.5 nm from the surface, the potential is negative and consequently the stable adsorption of NMs is favourable. On the other hand, the areas in blue represent a barrier of potential hindering the adsorption of NMs. At pH 7 and [NaCl] = 10 mM, the potential is negative only for the PS (hydrophobic) particles in contact with the PTFE-6PE surface.
The model is therefore in a good agreement with the experimental results, enabling to explain the different affinities of the hydrophobic PS particles compared with the hydrophilic ones for the surfaces characterised by relatively low values of the surface free energy (as the PTFE plus six PE layers). This method can then be used to rapidly and qualitatively characterise the NM hydrophobicity and to empirically evaluate the effect of the surface functionalisation of NMs on their surface properties, which have a tremendous effect on the NM interaction with biological media, the formation of the corona and the subsequent bio-response and toxicity.