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Aqueous Suspensions of Steroid Nanotubules: Structural and Rheological Characterizations† Pierre Terech*,‡ and Yeshayahu Talmon§ UMR 5819 CEA-CNRS-Universite´ J. Fourier, De´ partement de Recherche Fondamentale sur la Matie` re Condense´ e, 17, rue des Martyrs, 38054 Grenoble Ce´ dex 09, France, and Department of Chemical Engineering, TechnionsIsrael Institute of Technology, Haifa 32000, Israel Received January 24, 2002. In Final Form: February 27, 2002 It has been known that lithocholic acid self-assembles in highly alkaline aqueous dispersions. By a combination of cryogenic-temperature transmission electron microscopy and small-angle neutron and X-ray scattering, we were able to characterize the long single-walled nanotubules that are formed in the system. We have found that the nanotubules appear quite monodisperse with cross sections of 52 nm outer diameter and an internal cylindrical cavity of 49 nm diameter. Such a steroid introduces a new class of tubule-forming systems with cavity dimensions in a not previously reported nanoscale range. The flowing properties of the lithocholate suspensions are analyzed, and it is concluded that the uniform nanotubules are interacting moderately in the suspensions. As these nanotubes are fairly insensitive to the presence of salts, they are potentially good candidates for metallization and mineralization templates.
Introduction The formation of organic tubular species from the aggregation of molecular species is an important field of research of the nanosciences since it may find applications in catalysis, selective separations, sensors, and conducting devices in nano-, opto-, or ionoelectronics. A variety of chemical structures can produce tubular aggregates in the micronic or atomic range.1 Candidates forming tubular rods in the nanoscopic range are much less numerous. Special lipids2-4 and complex mixtures of steroids5,6 are the best known examples. In addition to the advantages provided by the self-assembling route (reversibility, soft chemistry experimental conditions, easy processing), the fundamental context is also challenging. Recently, theoretical models,7 taking into account ingredients of the freeenergy like couplings between chirality and topology, have been able to describe the metastable intermediary species (helical ribbons) which precede the formation of tubules. This context might also be illustrated with completely different low-mass chiral systems such as aggregates formed with various glutamic acids studied almost two decades ago and for which the relations between tubular and helical morphologies were already guessed.8,9 * To whom correspondence should be addressed. † This article is part of the special issue of Langmuir devoted to the emerging field of self-assembled fibrillar networks. ‡ CEA-CNRS-Universite ´ J. Fourier. § TechnionsIsrael Institute of Technology. (1) Bong, D. T.; Clark, T. D.; Granja, J. R.; Ghadiri, M. R. Angew. Chem., Int. Ed. 2001, 40, 988-1011. (2) Schnur, J. M. Science 1993, 262, 1669-1676. (3) Schnur, J. M.; Shashidhar, R. Adv. Mater. 1994, 6, 971-974. (4) Spector, M. S.; Singh, A.; Messersmith, P. B.; Schnur, J. M. Nano Lett. 2001, 1, 375-378. (5) Chung, D. S.; Benedek, G. B.; Konikoff, F. M.; Donovan, J. M. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11341-11345. (6) Zastavker, Y. V.; Asherie, N.; Lomakin, A.; Pande, J.; Donovan, J. M.; Schnur, J. M.; Benedek, G. B. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7883-7887. (7) Selinger, J. V.; MacKintosh, F. C.; Schnur, J. M. Phys. Rev. E 1996, 53, 3804-3818. (8) Yamada, K.; Ihara, H.; Ide, T.; Fukumoto, T.; Hirayama, C. Chem. Lett. 1984, 1713-1716. (9) Nakashima, N.; Asakuma, S.; Kunitake, T. J. Am. Chem. Soc. 1984, 107, 509-510.
We describe here the formation of tubules with monodisperse cross sections in the nanoscopic range formed by a very simple steroid derivative in alkaline aqueous solutions. We also take advantage of the complementarity between techniques to characterize the suspension of aggregates. Cryo-transmission electron microscopy observations are compared to small-angle scattering data obtained from brilliant sources of neutrons and X-rays. Rheological measurements quantify the level of interactions between the tubules. Materials and Methods Materials. Solutions of the sodium salt of lithocholic acid were prepared by direct dispersion of the lithocholic acid (Aldrich, 98% purity) in aqueous sodium hydroxide buffers at appropriate concentrations (in the range 0.10-0.25 M) resulting in alkaline (pH ∼ 12.3) solutions. The stabilized specimens (after at least 1 week) were submitted to the experiments. Cryo-Transmission Electron Microscopy (Cryo-TEM). Cryo-TEM has been described in detail by one of us.10 In brief, we placed a 3 µL drop of aqueous dispersion on a perforated carbon-film-coated TEM copper grid and blotted excess liquid with a filter paper to produce a thin (about 300 nm) liquid film supported on holes (several micrometers in diameter) in the carbon film. The grid was then plunged into liquid ethane at its freezing point and vitrified. We performed specimen preparation in the Controlled Environment Vitrification System (CEVS) at 25 °C and 100% relative humidity. The vitrified specimens were stored under liquid nitrogen and transferred to an Oxford CT3500 cooling-holder of a Philips CM120 transmission electron microscope, where they were examined at a temperature of about -180 °C. Low electron dose imaging (less than 20 electrons per Å2) was carried out using an acceleration voltage of 120 kV. Images were recorded with a Gatan MultiScan 791 CCD camera, using the Gatan DigitalMicrograph 3.1 software package. Small-Angle Scattering. Small-angle neutron scattering (SANS) experiments used the high flux neutron source of the Institut Laue Langevin (ILL, Grenoble). Three distances (20, 4, and 1.1 m) were used at λ ) 6 Å to cover the range of momentum transfer 0.002-0.13 Å-1 using the D11 camera. Small-angle X-ray scattering (SAXS) experiments were performed on the “High Brilliance” beamline ID2 of the European (10) Talmon, Y. In Modern Characterization Methods of Surfactant Systems; Binks, B. P., Ed.; Marcel Dekker: New York, 1999; pp 147178.
10.1021/la025574r CCC: $22.00 © 2002 American Chemical Society Published on Web 06/07/2002
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Figure 2. Low-magnification cryo-TEM image of a 0.1% SLC vitrified aqueous dispersion. Very long and uniform tubules form the suspension.
Figure 1. Cryo-TEM images of SLC vitrified aqueous dispersions. (A) 0.5% SLC; arrows point to tubule ends. (B) 0.1% SLC; arrows indicate a break in a tubule. The image in (B) was taken at a higher magnification and larger objective-lens defocus to enhance contrast. Synchrotron Facility (ESRF, Grenoble).11 The usual treatments were made before the standard radial averaging procedure. The lithocholate solution was enclosed in a 1 mm thick cell between Capton windows. Rheological Measurements. To analyze the flow properties of the suspensions, we used a Haake RS100 controlled stress rheometer, with cone-plate geometry (35 mm diameter, 2° angle). A glass cap limited water evaporation, and the temperature was kept at 20 ( 0.1 °C.
Results and Discussion Cryo-TEM. Figure 1A,B shows two segments of cryoTEM images of vitrified specimens of C ) 0.1 and 0.5 wt % sodium lithocholate (SLC). The main features in both micrographs are long, stiff tubules with a mean diameter, D, of 52 nm. That those are tubules, not ribbons, is obvious from their contrast. Remember that the TEM image is a 2D magnified projection of the specimen on the film or detector. A projection of a ribbon should have a uniform optical density throughout, while a tube in projection has darker edges, because in that part electrons traverse up to about 2.5 times more material as compared to the center of the cylinder. Remember that in the electron microscope all the objects in the specimens are projected “in focus” on the film or detector. The apparent thickness of the edges, t, is about 1.5 nm, about the size of the lithocholic molecule, measured from its hydroxyl to its carboxyl group. The edges look darker in Figure 1B than in Figure 1A, as a result of more underfocus of the objective lens. Defocusing is used to enhance “phase contrast” in that system that has very little inherent contrast. The tubules appear to be noncrystalline. Crystallinity should give rise to “crystalline contrast” (darker and lighter areas, a function of the crystal orientation with respect to the electron beam) and to moire´ patterns where two or more tube areas are superposed. Also, we detected no crystallinity in the (11) Narayanan, T.; Diat, O.; Bo¨secke, P. Nucl. Instrum. Methods Phys. Res., Sect. A 2001, 467-8, 1005-1009.
Figure 3. Cryo-TEM images of a 0.1% SLC vitrified aqueous dispersion. Black arrows indicate twisted ribbons, while white arrows show where a tubule is disintegrating into (or formed from) a wide ribbon. Double arrows in (A) indicate inner structure in the tubule. White arrowheads in (B) indicate the strip (ribbon) that is the building element of the tube. The small dark objects (e.g., the one denoted by F in (A)) are small frost particles. The alignment of the tubules probably results from the blotting during specimen preparation.
diffraction mode of the TEM. Arrows in Figure 1A indicate tube ends. The tubes are stiff. They appear very straight, and occasionally, but infrequently, we observed breakage of tubules (arrows in Figure 1B), probably caused during specimen preparation. The average length of the tubules cannot be determined accurately, but it is certainly greater than many hundreds of nanometers. In some low magnification images, we observed tubules much longer than 1 µm. Figure 2 is a low-magnification image which illustrates the very large aspect ratio (.50) of the tubes and the remarkable uniformity of their diameters. Nanotubules are by far the dominant structure observed in the vitrified SLC solutions. However, narrow twisted ribbons are also seen, but in much smaller numbers. Examples are given in the micrographs of Figure 3 (black arrows). Areas of the twisted ribbons observed edge-on are denoted, for example, by black arrowheads in Figure 3B and appear relatively darker than areas imaged faceon, because in the former case electrons traverse a larger material thickness. In these two images and in those seen in Figure 1, the pitch of the twist is about 130 nm. While
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Figure 4. Two cryo-TEM images of 0.05% SLC with added NaCl at an equivalent ration of 1:2 (excess salt). (A) Aligned tubules including one open flexible tubule (darker). (B) Center of the former image shown magnified by a factor of 2. The structure of the twisted ribbon is clearly seen. (C) Another example of a twisted ribbon in the same excess NaCl. (D) The middle of the ribbon shown again at twice the magnification.
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center of this image is shown again magnified by a factor of 2 in Figure 4B. The structure of the helical ribbon is clearly seen in this object. Also note the flexibility of this tube as compared to the intact stiff ones. Another example of a helical ribbon in the same excess NaCl system is shown in Figure 4C. This particular ribbon is very open. Its middle is shown again at twice the magnification in Figure 4D. There is only little contrast between the ribbon and the background. This is not surprising, as the ribbon is one molecule thick. It should be emphasized that although we show here these two “open” examples, almost all nanotubes even in the salt systems, including those in 4:1 excess CuCl2 (not shown), are of the intact kind. Why only a very small number of the tubes we have seen in our specimens are open is not clear, but it does point to their very high stability. Small-Angle Scattering Measurements. The scattering curves at low angles for rather dilute specimens are known to constitute a valuable piece of information to elucidate the morphology of nanoaggregates. The SANS signal is produced by large-scale fluctuations of the neutron scattering length density F(r). Q is the scattering vector equal to (4π/λ) sin θ, θ being 1/2 the scattering angle, as usual for elastic scattering. Expression 1 describes the situation of a dilute suspension of particles with no correlation between their orientation and in concentration conditions such that the scattered waves by two particles do not interfere. These conditions are those of a “single particle scattering”.13,14 Expression 1 is for one particle and an incident flux of 1 neutron cm-2.
many ribbons do show pitches of similar length, values as large as 500 nm are also seen occasionally. Practically all the nanotubes we have imaged were extremely uniform in appearance and featureless. The very few exceptions we have observed in the many hundreds of micrographs taken may provide some clues to the mechanism of formation of those remarkably uniform nanotubules. In the center of Figure 3A, a white arrow indicates a swollen domain in a nanotube. There and below it, one can see hints of inner structure in the tube, that is, vague fine lines at about 45° to the tube long axis (white double arrows). A more striking example is seen in Figure 3B, where the white arrow indicates a segment of the tube either in the process of assembly or disintegrating. Above and below that segment is the open ribbon (denoted by the white arrowheads), which could be the building element of the tube, much like a cardboard tube rolled up from a cardboard strip. This is reminiscent of the tubes we had seen as intermediates in crystallization of gallstones in bile.12 The angle of the ribbon edge with respect to the tube long axis is here, too, 45°. The full significance of these last findings is to be established by the continuation of the present work. Those uniform, self-aggregating nanotubes are natural candidates for metallization or mineralization for hosts of applications, and thus we probed their stability in relatively high concentrations of salts that could be involved in such processes. As demonstrated below, the aggregates were found to be remarkably stable in the presence of 2:1 (equivalent ratio) of NaCl and 4:1 CuCl2. Figure 4A shows a cryo-TEM image of 0.05% SLC with added NaCl at an equivalent ratio of 1:2 (excess salt). The same nanotubules described above are seen here, too. In this particular image, however, we see another example of a tube in the process of assembly or disintegration. The
The integration is over the volume V of the particle, and Fs is the mean scattering length density of the solvent. Expression 1 recalls that the scattered intensity varies according to the contrast of the system (particle/solvent). For a given contrast situation and resolution conditions of the diffraction experiment (2π/Qmax), the intensity may also depend on the spatial inhomogeneities of the particle. Presently, the low-angle scattering signal for a suspension at rest is analyzed with reference to the above TEM data. The most frequent morphology observed with TEM micrographs being that of long tubular fibers, the theoretical modeling will use the corresponding theoretical form factor of expression 2. The internal structure and the refined contrast profile (or heterogeneity) of the wall cannot be reached by the present scattering data since the resolution is beyond its thickness (t ∼ 15 Å). As a consequence, the prefactor of the scattered intensity (to get the absolute intensities per unit volume in cm-1) involves a constant contrast term F(r) - Fs. The contrast is changed from neutron scattering to X-ray scattering experiments since it is proportional to the isotopic composition of the particle (through the neutron scattering length of the constitutive atoms) or to the amount of electrons, respectively. As long as the domain of intermediate and large Q values is considered, the scattering intensities between SANS and SAXS should be only vertically shifted. The tubular particles are considered as isolated, homogeneous particles with an internal medium identical to the surrounding medium (alkaline buffer). The rodlike particles are long enough (L . D) so that nonzero contributions to the scattered intensity IF(Q) can be seen only for Q normal to the fiber axis which leads to
(12) Kaplun, A.; Konikoff, F. M.; Eitan, A.; Rubin, M.; Vilan, A.; Lichtenberg, D.; Gilat, T.; Talmon, Y. Microsc. Res. Tech. 1997, 39, 85-96.
(13) Glatter, O.; Kratky, O. Small-Angle X-ray Scattering; Academic Press: London, 1982. (14) Jacrot, B. Rep. Prog. Phys. 1976, 39, 911-953.
∫
I(Q) ) 〈| (F(r) - Fs) exp(iQ‚r) d3r|2〉
(1)
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a separation of the axial contribution (proportional to Q-1) from the cross-sectional factor. For such a hollow cylinder (external diameter Do) with an inner core (diameter Di), the cross-sectional form-factor intensity IFC(Q) can be modeled by expression 2 in which J1(QD/2) are Bessel functions of the first kind.
IFC(Q) ∝ [(J1(QD0/2)/(QD0/2)) - (Di/D0)2(J1(QDi/2)/ (QDi/2))]2 (2) Oscillations are generated in the intermediate and large Q domain by the arithmetic subtraction of Bessel functions. Figure 5A shows the neutron scattering curve in which 4-5 of such oscillations can be easily observed. These latter are found to be typical of concentric cylindrical morphologies since a simulation according to the simplified theoretical expression 2 reproduces correctly the experimental data with Do ) 52 nm and Di ) 49 nm. The same calculation can be made with SAXS data obtained at the ESRF high-flux synchrotron source (Figure 5B). The experimental data exhibit better resolved oscillations. On the basis of the Q positions of the apexes of the oscillations, a satisfactory agreement is observed with a vertical shift of the theoretical SANS adjustment, as expected (vide supra). The present data are not appropriate for a determination of the cross-sectional radius of gyration Rc of the tubular cylinders (see expression 3) since their large cross-sectional dimensions would require ultra-small-angle conditions of measurements. Indeed, to satisfy the so-called Guinier condition Qrc < 1, so as to expand expression 2 to the low-Q limit and get the Gaussian decay from which Rc could be extracted, would require experiments at Q < 0.004 Å-1 (ultra-small-angle scattering).
Rc2 ) (Ro2 + Ri2)/2
(3)
The small-angle scattering data are in agreement with cryo-TEM observations of the tubular species. The wall thickness, t, estimated from TEM or SAXS, ca. 1.5 nm, corresponds to a monomolecular length of the steroid for the present specimens. The experimental oscillations (Figure 5B) are less sharp than the theoretical ones which suggests that fluctuations of the thin wall have to be taken into account in a refined calculation. Figure 5C shows a comparison between the theoretical scattering curves of tubular cylinders with either a monomolecular or a bimolecular wall. A shift to larger Q values of the apexes is expected from the mono- to the bimolecular situation, which is more pronounced for higher order oscillations. The difference, while moderate, can be appreciated with the synchrotron data. Complementary studies are under way to better characterize this aspect of the molecular packing using infrared spectroscopy, circular dichroism measurements, and SAXS during the kinetics of preparation and evolution of the suspensions. Rheological Measurements. The objective of the rheological characterization is to show that the SLC system behaves more like a suspension of weakly interacting species than as a solidlike interconnected network as in a gel-like system. The shear viscosity η as a function of the shear rate γ˘ (Figure 6A) shows not a Newtonian plateau but a maximum followed by a decay with a slope of -1 in a double logarithmic plot. This profile resembles the socalled shear thinning behavior observed with polymeric
Figure 5. (A) Small-angle neutron scattering curve log I versus log Q of a SLC solution at C ) 1.26 wt % in D2O. Full circles are data points, and the full line is the theoretical (expression 2) scattering curve for homogeneous and cross-sectionally monodisperse tubules with Do ) 52 nm and Di ) 49 nm. Vertical bars indicate the Q positions of the apexes of the theoretical form-factor oscillations. (B) Small-angle X-ray scattering curve log I versus log Q of a SLC solution at C ) 3 wt % in H2O. Full circles are data points, and the full line is the theoretical scattering curve for homogeneous and cross-sectionally monodisperse tubules with Do ) 52 nm and Di ) 49 nm. Vertical arrows indicate the Q positions of the apexes of the theoretical form-factor oscillations (expression 2). The dotted curve corresponds to Do ) 52 nm and Di ) 46 nm, i.e., for a bimolecular wall thickness. (C) Enlargement of the SAXS data of (B).
solutions. The Carreau expression15 cannot be used to describe the shear rate dependence of the viscosity of this system. These features characterize a system with a very limited linear viscoelastic domain and a weak yield stress value separating elasto-plastic and viscous behavior. Such nonlinear behavior is linked to microstructural modifications, and certain theories of the thixotropy (15) Carreau, P. J.; DeKee, D.; Daroux, M. Can. J. Chem. Eng. 1979, 57, 135-140.
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Figure 6. Rheology of two tubule suspensions: C1 ) 0.296 wt % (b, curve 1) and C2 ) 2.4 wt % (O, curve 2). Comparison with two other reference systems: (4) organometallic “equilibrium polymer” (curve 3), C ) 0.7 wt % in tert-butyl cyclohexane; (2) fatty acid organogel (curve 4), C ) 0.7 wt % in nitrobenzene. (A) Shear viscosity η (Pa s) vs shear rate dγ/dt (s-1). (B) Stress σ (Pa) vs shear rate.
describing the shear thinning and structural recovery phenomena consider a time-dependent yield stress concept which might be appropriate here.16 The interactions between tubules are responsible for the level of viscosity at zero shear and reveal a balance of structural breakdown and recovery of rheologically active units of significant but moderate strength. At this stage, the values of the maximum of viscosity can be compared (η1 ) 1058 Pa s and η2 ) 93 600 Pa s, respectively) and indicate that (16) Toorman, E. A. Rheol. Acta 1997, 36, 56-65.
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interactions between nanotubules at rest are weak enough to be easily broken under shear, accounting for the absence of a Newtonian regime in the flow curve. The rheological behavior is quantitatively compared to that of other fibrillar systems with different mechanisms of interactions between the 1D particles. Thus, a “true” gel made of a hydroxy-12 stearic acid in nitrobenzene17 is taken as a reference state for a soft solid with high yield stress (curve 4 in Figure 6). The σ versus d(γ)/dt profile (Figure 6B) illustrates the progressive rupture of the network cohesion which is known to be accounted for by crystalline microdomains. On the other hand, a system made of organometallic monomolecular wire undergoing fast breaking/ recombination processes is shown as a reference for a pseudo-Maxwellian liquid of interacting and breakable 1D species (curve 3 in Figure 6).18 The Newtonian plateau is clearly seen with the η versus d(γ)/dt profile (Figure 6A), while the overall signature σ versus d(γ)/dt (Figure 6B) is reminiscent of the so-called shear banding phenomenology. It is interesting to observe in Figure 6A that 10 decades of viscosity and 8 decades in shear rates are covered which illustrate well the specific intermediate situation of the SLC nanotubule suspensions. To conclude, a simple preparation of nanotubular aggregates in water is described using a common bile salt steroid. The characterization of the tubular morphology and associated structural features is carried out by cryoTEM experiments and confirmed by SAXS and SANS measurements. It appears that in such conditions of preparation, the most frequent morphology involves an outer diameter of 52 nm and an inner diameter of 49 nm. The rheological behavior qualifies the lithocholate/brine solutions as suspensions of tubules which are significantly interacting but not enough to form permanent networks at room temperature (and C < 3 wt %). Acknowledgment. The cryo-TEM work was performed at the Hannah and George Krumholz Advanced Microscopy Laboratory, part of the Technion Project of Complex Fluids, Microstructure and Macromolecules. We thank Dr. Ellina Kesselman for the images of Figure 4. ILL and ESRF facilities (Grenoble, France) are acknowledged for providing access to the neutron and synchrotron beams. We deeply thank Dr. O. Diat for interesting discussions and for the scattering curve of Figure 5B obtained on the ID2 diffractometer at ESRF. LA025574R (17) Terech, P.; Pasquier, D.; Bordas, V.; Rossat, C. Langmuir 2000, 16, 4485-4494. (18) Terech, P.; Coutin, P. J. Phys. Chem. B 2001, 105, 5670-5676.
