12738
Langmuir 2008, 24, 12738-12741
Imaging New Transient Nanostructures Using a Microfluidic Chip Integrated with a Controlled Environment Vitrification System for Cryogenic Transmission Electron Microscopy Jinkee Lee,† Ashish K. Jha,‡ Arijit Bose,‡ and Anubhav Tripathi*,† DiVision of Engineering, Brown UniVersity, ProVidence, Rhode Island 02912, and Department of Chemical Engineering, UniVersity of Rhode Island, Kingston, Rhode Island 02881 ReceiVed August 21, 2008. ReVised Manuscript ReceiVed October 1, 2008 Nanostructures (vesicles, micelles, bilayers) are important in nanomedicine and biochemical processes. They are agents for encapsulation and eventual release of drugs, flavors, and fragrances. The structural transition from micelles to vesicles through disk-like intermediate states has been demonstrated previously. Here, we disclose a new route for the micelle-vesicle transition, where micelles aggregate to first form long tubules that become unstable, and break up into vesicles. A simple theory, based on energy principles, is presented to explain the tubule-vesicle transition. Observation of this new tubular intermediate state has been facilitated by the development of an integrated microfluidic chip/cryogenic transmission electron microscopy (cryo-TEM) unit. Although this transition has been observed in a specific amphiphilic system where micellar solutions of cetyltrimethylammonium bromide (CTAB) and dodecylbenzene sulfonic acid (HDBS) are mixed to form vesicles, this new tool can be applied broadly to study transient structures in nanoscale systems under the very controlled conditions provided by microfluidics.
Aqueous mixtures of cationic and anionic surfactants form a rich variety of composition-dependent nanostructures in solution, including spherical and wormlike micelles, bilayer membranes, vesicles, and a variety of lamellar phases.1,2 A detailed understanding of the process of self-assembly is necessary to exploit the use of these structures as carriers and to control their size distribution.3-7 One well-known mechanism for the transformation of micelles to vesicles is illustrated in Figure 1a,b,c. The micelles of each surfactant aggregate to form disk-like structures. The exposed edges are energetically unfavorable, and, at a critical disk size, the energy penalty corresponding to folding these membranes into closed spheres of curvature away from the spontaneous one is compensated by the lowering of energy due to the removal of the exposed edges.4,5,7-10 Here, we show a new route where mixed micellar aggregates form long cylindrical tubules, which then become unstable11 and transform into vesicles (Figure 1,a,d,e,f). This observation is enabled by a new tool, shown in Figure 2a, that combines a microfluidic chip with a controlled environment vitrification system, permitting direct visualization of intermediate structures by cryogenic transmission electron microscopy (cryo-TEM). * Corresponding author. E-mail:
[email protected] (A.T.);
[email protected] (A.B.). † Brown University. ‡ University of Rhode Island. (1) Gelbart, W. M.; Ben-Shaul, A.; Roux, D. Micelles, Membranes, Microemulsions, and Monolayers; Springer-Verlag: New York, 1994. (2) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. N. Science 1989, 245(4924), 1371–1374. (3) O’connor, A. J.; Hatton, T. A.; Bose, A. Langmuir 1997, 13(26), 6931– 6940. (4) Leng, J.; Egelhaaf, S. U.; Cates, M. E. Biophys. J. 2003, 85(3), 1624–1646. (5) Shioi, A.; Hatton, T. A. Langmuir 2002, 18(20), 7341–7348. (6) Weiss, T. M.; Narayanan, T.; Wolf, C.; Gradzielski, M.; Panine, P.; Finet, S.; Helsby, W. I. Phys. ReV. Lett. 2005, 94(3), 038303. (7) Noguchi, H.; Gompper, G. J. Chem. Phys. 2006, 125(16), 164908. (8) Schmolzer, S.; Grabner, D.; Gradzielski, M.; Narayanan, T. Phys. ReV. Lett. 2002, 88(25), 258301. (9) Xia, Y.; Goldmints, I.; Johnson, P. W.; Hatton, T. A.; Bose, A. Langmuir 2002, 18(10), 3822–3828. (10) Zhu, K.; Brubaker, G.; SmCith, J. D. Biochemistry 2007, 46(21), 6299– 6307. (11) Granek, R. Langmuir 1996, 12(21), 5022–5027.
Cryo-TEM is a powerful tool for direct visualization of amphiphilic nanoaggregates present in solution in their native states. It has been used to image steady-state nanostructures or transient states in very slowly evolving materials, with evolution time constants larger than a few minutes.9,12-18 In some systems, the time scales of the underlying structural reorganization may be on the order of several seconds, and intermediate nanostructures remain inaccessible using current preparation techniques. The identification of intermediate nanostructures is important for completing our understanding of how amphiphiles assemble into complex nanostructures dictated by their molecular architecture and well-defined solvent conditions.19 Microfluidics offers exquisite control of flow rates and residence times in the second to subsecond time scales, critical for understanding temporal evolution in many nanoscale systems. In this work, we have integrated a microfluidic chip with a controlled environment vitrification system (µf-CEVS), allowing us to take advantage of the key features of each technique. Figure 2a shows a schematic of the µf-CEVS unit. The microfluidic chip is fabricated using standard polydimethylsiloxane (PDMS) soft lithography.20 The microfluidics channel was coated with dodecylchlorosilane (obtained from Gelest, Inc., Morrisville, PA) to suppress the adsorption of surfactant. The microchannel walls (12) Shih, W. M.; Quispe, J. D.; Joyce, G. F. Nature 2004, 427(6975), 618– 621. (13) Jung, H. T.; Coldren, B.; Zasadzinski, J. A.; Iampietro, D. J.; Kaler, E. W. Proc. Natl. Acad. Sci. U.S.A. 2001, 98(4), 1353–1357. (14) Jung, H. T.; Lee, S. Y.; Kaler, E. W.; Coldren, B.; Zasadzinski, J. A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99(24), 15318–15322. (15) Liu, S. Y.; Gonzalez, Y. I.; Kaler, E. W. Langmuir 2003, 19(26), 10732– 10738. (16) Yaacob, I. I.; Bose, A. J. Colloid Interface Sci. 1996, 178(2), 638–647. (17) Pitard, B.; Aguerre, O.; Airiau, M.; Lachages, A. M.; Boukhnikachvili, T.; Byk, G.; Dubertret, C.; Herviou, C.; Scherman, D.; Mayaux, J. F.; Crouzet, J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94(26), 14412–14417. (18) Zheng, Y.; Lin, Z.; Zakin, J. L.; Talmon, Y.; Davis, H. T.; Scriven, L. E. J. Phys. Chem. B 2000, 104(22), 5263–5271. (19) Haberkorn, H.; Franke, D.; Frechen, T.; Goesele, W.; Rieger, J. J. Colloid Interface Sci. 2003, 259(1), 112–126. (20) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70(23), 4974–4984.
10.1021/la8027379 CCC: $40.75 2008 American Chemical Society Published on Web 10/24/2008
Letters
Langmuir, Vol. 24, No. 22, 2008 12739
Figure 1. Two different surfactant self-assembly routes are illustrated in amphiphilic systems. (1) The evolution of micelles (a) into disk-like structures (b) that close to form into vesicles (c). (2) Evolution of micelles (a) to cylindrical tubules (d) that become unstable (e) and break up into spherical vesicles (f).
Figure 2. (a) The microfluidics integrated controlled environment vitrification system. Surfactant solutions are supplied though the microfluidics channel (iv) and mixed in the capillary (iii). The T-shaped channel consists of 20 mm of supply channels, a 5 mm length mixing channel, and is 100 µm deep. The mixing channel is connected to the glass capillary which has a length of 100 mm and an inner diameter of 500 µm. By controlling the capillary length and the linear velocity of the fluid, the mixing/incubation time is controlled. The solution ejects on the holey carbon grid (ii) held by a tweezer (i). The grid is subsequently plunged into liquid ethane (v) to complete the vitrification, placed on a cold stage (vi), and imaged in a TEM with the sample at -170 C. (b) Solution of the convective diffusion equation in the microfluidic channel and capillary indicate the CTAB and HDBS concentrations at the exit of capillary when Q ) 100 µL/min. To increase the evolution time, the flow was stopped for a period called the “wait time”. Red denotes 100% CTAB, and blue denotes 100% HDBS. Green denotes a weight ratio of 1:1.
were then saturated with surfactants prior to any run.21 The chip design and operating conditions can be manipulated to probe the effects of controlled diffusive transport rates and residence time on nanostructure formation and evolution. The combination of a microfluidic setup with cryo-TEM allows the direct visualization of aggregates that are formed within the first few seconds (this time can be controlled by varying the residence time) after mixing. Mixing and structural evolution are concurrent processes. The newly formed structures appear first at the middle of the capillary tube, whereas regions of the capillary furthest from the mixed zone still contains micelles of each surfactant. Figure 2b is a visualization of concentration profiles at two different points in the capillary when the two surfactant solutions are mixed in the microfluidic setup. Here, the convective diffusion equation describing the transport of the surfactants was solved numerically using the computational software COMSOL (Multiphysics, COMSOL, Inc., Sweden). The molecular diffusivities of cetyltrimethylammonium bromide (CTAB) and dodecylbenzene sulfonic acid (HDBS) were set at 450 µm2/s.21 For the shortest residence time, approximately 75% of the cross-section of the tube has an exit composition of CTAB and HDBS that is between 40 and 60% (the starting compositions are 100% of each (21) Lee, J.; Bose, A.; Tripathi, A. Langmuir 2006, 22(26), 11412–11419.
surfactant). Diffusion effectively mixes the two surfactant solutions, and the structures observed by cryo-TEM accurately reflect the predominant transient morphologies existing at that time point. Note that exact exit compositions may be obtained by using the correct dependence of molecular diffusivities on the concentration and extent of mixing of CTAB and HDBS. Figure 3a is a cryo-TEM image of a sample in which the two surfactant solutions were mixed by very gentle agitation for 3 h in a scintillation vial and manually prepared in the CEVS. Spherical vesicles with a mean size of 65.2 ( 33.2 nm are observed (sample size of 80). In another experiment, a premixed vesicle solution was loaded into the microfluidic chip reservoir. The sample was then delivered (flow rate 100 µL/min, linear velocity ∼8.0 mm/s, shear rate ∼2.4/sec), to the cryo-TEM grid for imaging. Figure 3b is an image of this sample, showing spherical vesicles of size 64.6 ( 35.2 nm (sample size of 81). Since these structures are very similar to those obtained using the conventional preparation of samples (Figure 3a), the shear flow in the microchannel has no measurable effects on these nanostructures. CTAB and HDBS solutions (0.8 wt %) were loaded in the chip reservoirs. A total flow rate of 100 µL/min (total travel time ∼11.7 s) was established in the capillary. Except for the first experiment in this set, the flow was switched off after 6 s, and
12740 Langmuir, Vol. 24, No. 22, 2008
Figure 3. The CTAB/HDBS vesicles formed using (a) conventional sample preparation and manual loading of premixed samples and (b) premixed samples loaded through the microfluidic chip. Images show the formation of spherical vesicles for both samples. The scale bar is 100 nm.
Letters
Figure 5. Cryo-TEM image of CTAB/HDBS vesicles with manual preparation on the grid. The image clearly shows cylindrical tubules. The scale bar is 100 nm.
vesicle transition takes about 5 min. The transformation to tubules is rapid, but the transition of these unstable tubules to vesicles is slow. The dynamics of this latter transition is controlled by several factors, including the bending stiffness of the bilayer and the viscosity of the solvent. In order to eliminate any artifacts arising from possible surface interactions or shear of surfactants during microchannel flow, an additional set of experiments were performed. Aqueous 0.8 wt % CTAB and 0.8 wt % HDBS (2.5 µL each) were manually but sequentially loaded on the grid held on tweezers inside the CEVS chamber (T ) 25 °C). To expedite mixing, the pipet tip was moved very gently within the deposited drop. After waiting for ∼5 s to further allow diffusion-driven mixing and letting any shear induced structures relax, the sample was vitrified and imaged. Figure 5 clearly shows cylindrical tubules in these samples, confirming that the formation of cylindrical tubules is a material property of the surfactant system. A simple energy argument can be used to understand the transition from tubules to vesicles. If a cylindrical tubule of radius Rt and length Lt breaks up into N spherical vesicles of radius Req, then by mass conservation, we obtain
RtLt ) 2Req2N
(1)
The total curvature elastic energy of the cylindrical tubules is Figure 4. Self-assembly dynamics of CTAB/HDBS vesicles. Images clearly show (a) the cylindrical tubules at times
