Self-Assembly of Mesoscale Artificial Clathrin Mimics - ACS Nano

Sep 18, 2017 - Fluidic control and sampling in complex environments is an important ... of clathrin proteins allows cells to sample their external env...
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Self-Assembly of Mesoscale Artificial Clathrin Mimics Yifan Kong, Mina-Elraheb S. Hanna, Denys Zhuo, Katherine G. Chang, Tara Bozorg-Grayeli, and Nicholas A. Melosh* Department of Materials Science and Engineering, Stanford University, 476 Lomita Mall, Stanford, California 94305, United States S Supporting Information *

ABSTRACT: Fluidic control and sampling in complex environments is an important process in biotechnology, materials synthesis, and microfluidics. An elegant solution to this problem has evolved in nature through cellular endocytosis, where the dynamic recruitment, self-assembly, and spherical budding of clathrin proteins allows cells to sample their external environment. Yet despite the importance and utility of endocytosis, artificial systems which can replicate this dynamic behavior have not been developed. Guided by clathrin’s unusual structure, we created simplified metallic microparticles that capture the three-legged shape, particle curvature, and interfacial attachment characteristics of clathrin. These artificial clathrin mimics successfully recreate biomimetic analogues of clathrin’s recruitment, assembly, and budding, ultimately forming extended networks at fluid interfaces and invaginating immiscible phases into spheres under external fields. Particle curvature was discovered to be a critical structural motif, greatly limiting irreversible aggregation and inducing the legs’ selective tip-to-tip attraction. This architecture provides a template for a class of active self-assembly units to drive structural and dimensional transformations of liquid−liquid interfaces and microscale fluidic sampling. KEYWORDS: self-assembly, biomimetics, clathrin, three-dimensional assembly, colloids icroscale and nanoscale fluidic manipulation and sampling is a general problem in many different areas, from single cell analysis1,2 to materials 3,4 synthesis to microfluidics.5,6 Approaches such as microfluidics generally rely upon top-down designs but can be challenging in complex environments where surface reactivity or material compatibility are concerns. Biological cells provide an alternative design motif, using bottom-up clathrin protein assembly to dynamically bud spherical vesicles from the 2D membrane for environmental sampling. While artificial selfassembling systems which organize at 2D interfaces are welldescribed7−12 and recent research is making progress toward more complex behavior such as responsive DNA−nanoparticle lattices,13 kirigami membranes,14 and oscillating homeostatic processes,15 none have shown endocytosis-like behavior. Here we use clathrin proteins as an inspiration for engineering self-assembling metallic particles for biologically inspired fluid sampling. Clathrin monomers are curved, threelegged “triskelia” (Figure 1a)16,17 that are recruited to the interior 2D cell membrane surface through adapter proteins. Following recruitment, clathrin undergoes leg-to-leg assembly with nearby monomers into an extended 2D hexagonal network (Figure 1b).18 Once assembled, the network can be triggered by receptor binding to transform from a 2D sheet to a 3D vesicle and finally is driven to bud into a 3D vesicle while entrapping sampled cargo within itself.19−22

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Based on these observed traits, we designed ACMs as bimetallic Janus triskelia (Figure 1c, d), incorporating four key biomimetic elements: (1) three-leg shape, (2) leg-to-leg attraction, (3) particle curvature, and (4) 2D surface affinity. ACMs are nearly 1000 times larger than clathrin, yet endocytosis-like functionality could be reproduced by appropriate selection of shape, curvature, and surface energy in the presence of an external magnetic field. Particle curvature was discovered to be particularly important, dictating interparticle interaction strength as well as mitigating irreversible aggregation. Instead of irreversibly clumping, curved ACM particles associated into loose clusters through relatively weak and reversible point-to-point contacts (Figure 1c, d) which were still interfacially active. This study highlights the utility of particle curvature in controlling interaction and assembly characteristics and allows for particle designs leveraging these behaviors in interfacial self-assembly and fluidic manipulation. Inorganic systems with similar functionality would be a significant step toward designing nanosampling systems, such as “reverse-endocytosis”, where small vesicles are budded from the cell for chemical analysis. Received: May 28, 2017 Accepted: September 18, 2017 Published: September 18, 2017 9889

DOI: 10.1021/acsnano.7b03739 ACS Nano 2017, 11, 9889−9897

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Figure 1. Artificial clathrin mimics are based on clathrin’s structure. (a) Illustration of single clathrin subunit, reproduced using Chimera.16,17 (b) Electron micrograph of clathrin network (scale bar 33 nm), reprinted with permission from ref 18. Copyright 1987 The Rockefeller University Press. (c) Illustration of ACM liftoff and aggregation at silicon wafer surface. (d) Optical image of ACMs after release in water, showing high native curvature. Scale bar 20 μm.

ACMS The ACMs were fabricated using standard photolithography techniques, as shown in Figure 2a−c. By using a simple bimetallic physical vapor deposition to construct the ACMs, they could both be orthogonally functionalized and create curvature from the different intrinsic stresses between layers (see the Methods). ACM release was facilitated by the use of germanium as a sacrificial underlayer, which allows for broad CMOS compatible processing and organic solvent use. The ACM release was triggered by immersion into a 0.3% hydrogen peroxide solution. As the oxidizing aqueous solution attacked the germanium layer, the ACMs individually released from the wafer. The flat to curved transformations of the ACMs were immediately apparent as they scattered light away from the microscope. As individual ACMs detached from the interface, their appearance transformed from primarily bright to dark (Figure 2f−h). The full video can be seen in Supplementary Video S2.

Figure 2. Fabrication and release of ACMs. (a) Fabrication of Au/ Ni ACMs on a Ge lift-off layer. (b) The upper Au layer is selectively functionalized with alkanethiols to make a Janus ACM. (c) The Ge is dissolved in water, releasing ACMs from the silicon surface. (d) Many alternate geometries, such as hydrophobic bands, are facile with additional processing steps. (e) SEM image of ACMs fabricated on a silicon wafer, scale bar 10 μm. (f) Optical image of ACMs on chip. (g) ACM liftoff begins as germanium is dissolved. Originally bright, flat ACMs become dark as they are released and adopt their native curvature. (h) The germanium is dissolved, and ACMs begin to loosely aggregate as they leave the surface. Scale bar f−h: 20 μm.

scatter reflected light off-axis and block background illumination. When an ACM is recruited to the interface it becomes bright, as there is sufficient interfacial energy to both remove the ACM from the aggregate and also flatten it, increasing the reflectivity. This process provides a facile way to monitor the progression of surface attachment and density. As shown in Figure 3b,c, the particles adsorb sequentially rather than en masse in an aggregate, highlighting that the particles within the aggregates are loosely bound and easily separate from their neighbors. Nonspecific aggregation is limited by the open, curved structure that ACMs share with clathrin, preventing large surface-to-surface contacts. The Janus hydrophobicity further prevents top-to-bottom “dish-type” stacking due to the opposite hydrophobicity of the upper and lower faces. The association of ACM surfaces of similar hydrophobicity is therefore hindered by the small, curved contact area, with hydrophobic interaction distances on the order of 100 nm or less.26 The open nature of ACMs further limits aggregation as ACMs often rotate such that the legs lie perpendicular to one another, forming point contact leg-leg cross junctions. These results are in contrast to planar self-assembling units, where face-to-face stacking often drives three-dimensional assembly,27−29 creating aggregates that can be difficult to

RESULTS AND DISCUSSION Recruitment. In endocytosis attachment, proteins provide the lipophilic anchor necessary for the recruitment of clathrin proteins to lipid membranes and mediate leg-to-leg clathrin assembly.19,21 Instead of using complex attachment proteins, ACMs are attached to a fluid−fluid interface via functionalization with an alkanethiol to create a hydrophobic upper gold layer and a hydrophilic nickel base (Figure 2a−c). This Janus particle behavior has been extensively explored in selfassembly.23−25 As ACMs move from solution to the interface, the total surface energy of the system is reduced by matching hydrophobic and hydrophilic faces and fluid phases. Note that once at the interface the ACMs will have translational and rotational freedom in the 2D plane but will not desorb from the surface as the energy penalty of moving back into water is roughly 3.5 × 10−12 J per ACM, much higher than thermal fluctuations. The recruitment of ACMs to an air−water interface from their aggregated state is shown in Figure 3b,c. Dark, loose aggregates of ACMs are visible in the background, as they 9890

DOI: 10.1021/acsnano.7b03739 ACS Nano 2017, 11, 9889−9897

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Figure 3. Recruitment, assembly, and budding of ACMs. (a) ACM recruitment to an interface from solution. (b) Reflected light microscopy image of ACM recruitment to the water−air interface. Dark subsurface aggregates are visible in background along with a bright (flat) attached ACM. (c) A previously aggregated ACM (circled) detaches, binds and flattens to the interface, becoming bright. (d) ACM assembly at the planar interface. (e) ACM from b and c are driven together by capillary forces. Scale bar b, c, e: 20 μm. (f) Large ACM networks assemble, and residual aggregates are washed away. Scale bar 40 μm. (g) ACM budding from an interface driven by magnetic fields. (h−k) Progression of ACM budding on a butanol droplet in water as a magnet approaches (bottom right, dark region). (i, j) As the magnet draws closer, the ACM network begins to collapse in discrete stages into its final budded state. (k) Butanol droplet after ACM-coated bud has detached from the surface. Scale bar h−k: 400 μm. (l) A budded droplet on the magnet surface, loaded with fluorophore. Scale bar 200 μm.

used surface tension driven attractions,25,31 including selective hydrophobic functionalization of particle edges and faces.7−9,23,24 Unlike these strategies, ACMs appear to create localized liquid curvature at the tips of their legs (see Modeling). These localized high energy interfacial deformations drive strong tip-to-tip interactions, which lead to longrange network self-assembly through the 3-fold symmetry of the ACMs (Figure 3d−f). An example of two ACMs snapping into contact through curvature induced tip-to-tip attractions is shown in Figure 3d,e.

redisperse once they are formed. Prefunctionalization with polymers can reduce aggregation30 but limits further functionalization of the particle surface. The out-of-plane curvature and Janus functionality of ACMs removes the head-to-tail symmetry for stacking, providing a useful design template for avoiding nonspecific aggregation and precipitation even for strongly interacting particles. 2D Assembly. After recruitment, ACMs rapidly selfassemble into a 2D honeycomb lattice (Figure 3f) through selective tip-to-tip attachment. Previous studies have extensively 9891

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Figure 4. Budding and collapse of the ACM network. (a) As the magnet approaches the bud, the ACM network deforms and the base of the bud grows smaller. Discrete events where the ACM network collapses under increasing magnetic fields are indicated, which result in a denser, more compact network at each stage (Figure 3h−j and Supplementary Video S1). (b) A magnified image of Figure 3l, where a postbudded droplet filled with fluorophore is shown. The network is highly disrupted and defective in this postbudded state. Scale bar is 50 μm.

perpendicular to a butanol−water interface. As the strength of the magnetic field was systematically increased, the budding was driven in progressive stages. Initial ACM-mediated bending of the interface (Figure 3h,i) is followed by the formation of a highly curved protovesicle (Figure 3i) ultimately leading to the pull-off of a discrete droplet coated with ACMs (Figure 3j,k). Budding occurs in discrete stages as the magnetic force is increased, resulting in an initial bud diameter with a base of 470 μm (see Supplementary Video S1), then suddenly collapsing to 280 μm, then 200 μm, and finally pinching off (Figure 3i,j,k, respectively). This transition can be seen in Figure 5a. Despite its disordered arrangement, the network continues to provide structural support to the droplet (Figure 3l and the magnified Figure 4b), and the deformed water−butanol interface visibly distorts so as to maintain contact with the ACMs. The relative size of the droplet is larger than that of clathrin; a butanol bud has a leg-to-bud diameter ratio of 13.6 here, while clathrin’s leg to bud ratio can vary from about 1.6 to 3.4.19,32,33 The larger effective size for a butanol bud is consistent with the amount of energy to create a curved fluid/fluid interface and is larger than that required to form a vesicle (see the Supporting Information). Figure 3l shows one ACM-coated bud sampled from water loaded with fluorescently conjugated DNA. The ACM network at the interface has lost its ordered packing and has collapsed into a compact state. Network reorganization was expected as it is not topologically possible to curve a hexagonal network into a sphere without defects; in clathrin, this requires breaking its symmetry to form odd numbered rings34 (Figure 1b). Here, it appears the particles do not leave the interface but lose tip-totip attachment and honeycomb organization. As seen in Figure 4b, the ACM network does not retain order after budding, demonstrating that the specific hexagonal organization does not play the same role in the budding process as it does in clathrin where ordered cages are retained. However, the budding behavior in Figure 4a provides evidence that budding is not entirely independent of ACM design, and differs from what would be expected in the budding of the simplest networks of close packed spheres.

Since the force is not a whole-body interaction but acts on the tips of each ACM leg, the ACMs are often observed to rotate into a linear leg-to-leg orientation during approach. Note that tip-to-body contacts are relatively rare. Liquid curvature is also evident from the dark spots near the end of each leg, visible in Figure 3g, and will be discussed below. 2D assembly proceeds by sequential addition of new ACMs to form extended networks with p6mm symmetry (Figure 3f), which is the same space group as honeycomb lattices and clathrin. After the interface assembly is complete, the excess unattached ACMs are washed away by passing through another water−air interface (see the Methods). The ACM network itself is sufficiently robust to survive the transfer as well as remain ordered during this process, and the packing density between many ACM islands as seen in Supplementary Figure S3a is approximately 6700 ACMs/mm2, which compares favorably to a maximum packing density of 7700 ACMs/mm2. While ACM networks are relatively well-ordered, some overlap and tip to body defects exist. Overlaps occur when one additional particle is attached to the underside of a network. Due to the ACM curvature limiting aggregation, these defects are relatively rare at a rate of approximately 421 ACMs/mm2, approximately 6.3% of the assembled ACMs. The second type of defect is leg-body assembly between ACMs. These defects do not generally occur during individual particle addition but rather when two islands merge together to satisfy a larger number of tip-to-tip assemblies. Both types of defects can be seen in Figure 3f, and an extended view of ACM networks can be found in Supplementary Figure S3a. Budding. During the budding process, the interface (a lipid bilayer in the case of clathrin or a fluid/fluid interface in the case of ACMs) is deformed by the assembled network into a necked shape encapsulating one phase, ultimately pinching off due to surface tension and forming a budded droplet. In clathrin, this process is actively driven by regulatory proteins such as epsins and dynamin.20−22 ACM networks are driven to bud in a similar process by the application of an external force, such as magnetism. We fabricated the ACM’s base metal layer from nickel and applied a magnetic field to create a force on the ACMs 9892

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Figure 5. Modeling and analysis of ACM assembly. (a) Interaction between two ACMs. The highest liquid surface energy is at the tip of each ACM, and the overall energy is reduced when two tips come into contact. Dotted and dashed lines represent the cross sections from b and c, respectively. (b) Cross-section line plot for the particle profile (red) and liquid interface (blue) of an unbound ACM tip. Inset is 1:1 scaling of axis. (c) Line plot of the particle and interface profile (red and blue, respectively) between two ACMs spaced by 1 μm. Black line shows the experimental intensity profile across a leg-leg junction denoted by the yellow dotted line in e. Note that the X-axis scaling is doubled compared to b. (d) Six ACMs in proximity create maximum distortion at the center. (e) These distortions are experimentally visible as dark regions due to refraction of background light. Sites of one to six ACM interactions are circled. Dotted yellow line indicates intensity profile from (c). Scale bar 20 μm.

ACM budding is a balance between the magnetic force on the ACMs and the surface tension of the fluid which opposes deformation. The effect of the ACM network on the bud surface can be viewed as a solid surfactant, similar to a Pickering emulsion. Yet unlike a densely packed surfactant layer, the ACMs and the formed network are open structures, and it is the tip−tip capillary interactions which initially hold the network together. As the magnetic field increases, the magnetic force pulling the ACMs away from the interface increases beyond the strength of interparticle capillary and steric mechanical forces. As a result, the network collapses and densifies, resulting in a number of changes. First, the more densely packed network covers a smaller area, allowing the underlying fluid experiencing budding to take on a more minimal shape. Second, the network collapse also decreases the local surface energy by increasing the density of ACMs acting as solid surfactant. Third, the higher density of ACMs increases the magnetic force per unit area on the underlying fluid, favoring stronger budding states. In opposition to these forces which provide impetus for the budding, the denser network is more mechanically rigid; the ability of the network to resist deformation counterbalances “runaway” collapse and pull-off.

Together, these result in the progressively smaller bud sizes and higher curvatures seen in Figure 3h−k and Supplementary Video S1. Each stage of the ACM collapse represents a mechanically stable ACM network overcoming its rigidity and collapsing into a denser, more defect laden state. The first “collapse” in Figure 4a at lower magnetic fields and long magnet ranges (880 μm) represents the condensation of larger islands on the bud of the droplet. The subsequent sequential collapse states at (230 μm) and (140 μm) are indicative of inter-ACM linkages breaking and reforming into more compact states, seen most clearly in Supplementary Video S1. Between collapse events, the ACM bud modestly compresses in size in response to an increased magnetic field. Due to the strong retention of ACMs on postbudded droplets and the lack of individual desorption events from the interface, it seems likely that during network collapse the ACMs primarily slip and overlap with one another. This type of defect and network failure can be seen in compressed 2D networks of ACMs, shown in Supplementary Figure S3. While the specific symmetry of the ACM network may not be critical to budding, open networks modulate the interparticle bond strength and govern how the mechanical 9893

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apparent at the fully coordinated 6-fold network binding sites where lensing is strongest (Figure 5e), yet dimples are visible even for single legs. The dimples are present throughout the network (e.g., Figure 5f) and are not optical illusions. The black trace in Figure 5c shows the intensity profile from a leg−leg junction in Figure 5e (yellow dashed line). The curvature of the interface is clearly visible and largely matches the predicted profile (note that they should not match exactly, as the reflected light intensity is a nonlinear function of height). There are bright spots in the center of the dimple and on the middle of the ACM bodies, corresponding to regions where the surface is flat, limiting refraction or increasing reflection, respectively. To estimate the interparticle attraction strength, an interfacial energy map of two ACMs interacting with each other is shown in Figure 6a where an ACM is rotated around a fixed particle at

rigidity of the network changes as it collapses, ultimately determining the kinetic nature of budding. Comparatively, if the ACMs began in a densely packed state (such as found in a layer of traditional close-packed hydrophobic spheres) the network would have much more difficulty exhibiting discrete collapse states, due to the high initial density and mechanical stability of the network. Modeling ACM Interactions. To understand ACM assembly we modeled the ACM monomer behavior on the water surface, pairwise interaction strength, and effect of curvature. While capillary attraction between particles based purely on hydrophobicity has been previously explored,7,8,35,36 capillary attraction based on particle curvature has only received limited treatment in the context of capillary arrows.37 Fundamentally, a curved particle experiences a competition between the deformation energy of water which must maintain contact with the curved particle, and the particle’s mechanical strain due to the water tending to pull it flat. This balance leads to a dimpling of the water surface and thus capillary attraction. When a free ACM comes into contact with the interface, the lower interfacial energy of the Janus surface drives it into contact. Once in contact, the Janus nature of the boundary allows the interface to assume any contact angle between that of the hydrophobic and hydrophilic sides.38,39 However, adopting the particle’s curvature increases the liquid surface area, along with a surface energy of ΔE = γΔA (Figure 5a). The competition between this energy and the particle’s spring energy, E = ∼1/2κΔh2, leads to a flattened particle shape. The large increased interfacial area near the ACM center is balanced by the localized interfacial curvature around the ACM tips (Figure 5a,b) creating strong tip-to-tip attraction. The equilibrium particle shape, liquid surface energy, and pairwise interaction potentials were modeled using COMSOL.40 Figure 5a−c shows the calculated shape and curvature energy of a water−air surface with two ACM particles after energy minimization. The particle is significantly flattened, with small regions of high curvature and negative displacement in the liquid near the tips of each leg, Figure 5b. The inset at 1:1 scale shows the dimple in the liquid near the tips is about ∼5% of the leg length. As two ACM’s approach one another the curvature around their legs is reduced, (Figure 5a,c,d) in turn reducing the total interfacial energy. Curved particles on liquid interfaces have been described previously.25,38 showing periodic liquid deformations distributed about circular or cylindrical particles resulting in quadrapolar interactions. Certain insects also utilize this mechanism for “capillary climbing”.41 Here, the three-legged shape and Janus hydrophobicity promotes strong liquid displacements at the leg tips. As the system attempts to lower its total free energy, the liquid bulge energy under the particle body competes with the penalty of creating a depression near each tip. Since the bulge has much larger area, the equilibrium configuration is an intermediate bulge height with localized dimples of high curvature at the tips. These dimples provide strong directional interactions between particles. The triskelia shape also dictates a p6mm 2D network assembly, unlike rodlike particles which often pack in onedimensional chains but without long-range order. Remarkably, the curved interface near the particle tips can be clearly observed experimentally. Near the tips of each ACM or junction between ACM legs dark spots appear (Figure 5e) due to refraction of background illumination through the curved liquid surface, acting as a lens and refracting the light out of the microscope objective’s collection cone. These spots are most

Figure 6. Modeling of energies and forces felt between ACMs. (a) Net interfacial energy and force directions (arrows) as one ACM (not shown) approaches another at various angles. Repulsive forces due to mismatched regions of high and low curvature occur near the center of each ACMs (red), while attractive forces guide ACMs to assemble tip-to-tip (blue). (b) Net energy and attractive force as a function of separation distance between two ACMs as they approach tip to tip. (c) Attractive force between ACMs at 1 μm separation at an air−water interface as their native curvature is varied. As the ACM curvature height is increased, the interfacial energy rapidly drives stronger ACM binding forces. Dotted line represents a parabolic fit for the attractive force with a quadratic constant of 1 kN/m2. 9894

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lithography or self-assembled fabrication may also be able to recruit and assemble on a cell, driving “reverse endocytosis” the sampling of the membrane and cytoplasmic contents by a protein-inspired nanomachine. Ultimately, the form and function of ACM type self-assembling strategies offer a general scheme for bioinspired interfacial self-assembly.

different separations. There is a repulsive interaction between ACM body and tips due to the opposite sign of the menisci curvature,36 seen as the red-shaded areas indicating high energy. The tip-to-tip attraction drives ACMs together and orients them such that they approach from the lowest energy direction. This is visible from the convergence of the force directionality (arrows) to the ACM tips for different angles of approach. This is corroborated by the experimental observation that the particles rotate to align their legs, seen in the sequential motion of the two particles in Figure 3c and e. As ACMs approach one another the attractive force between them increases dramatically (Figure 6b) and the absolute value is much larger than colloidal particles of similar size. The attractive forces between ACMs 1 μm apart is on the order of 1 nN, while 4−10 μm spherical colloids with rough contact lines tend to experience attractive capillary forces on the order of 1 pN.42,43 The large calculated force between ACMs is instead in the range of particles which curve the interface strongly44 and much larger particles which deform interfaces via gravity35 even though gravity is negligible at the ACM’s size scale.42,43 Localized curvature thus provides a mechanism for very strong, yet orientationally selective interactions at the micron scale. The interaction energy between particles was calculated to be a strong function of the initial particle curvature. The mechanical energy of an ACM was approximated as a simple cantilever via κh2, where κ is the spring constant and h is the tip displacement. The calculated force between ACM separated by 1 μm as a function of curvature is shown in Figure 6c. The attractive force increases as the square of ACM height h and therefore proportionally with the total stress energy. This relationship provides a mechanism for tuning the particle interaction strength through composition, deposition conditions, or changing the width/leg ratio of the ACM and is orthogonal to modifying the surface energy.

MATERIALS AND METHODS Artificial Clathrin Mimic Design and Fabrication. The structure of an ACM is a curved three-legged microparticle composed of a 25 nm gold layer on top of an 80 nm nickel layer. Each leg is 10 μm long and 1 μm wide with a 120° angle between legs (Figure 1c,d and Figure 2). This structure possess similar symmetry to clathrin, which has legs of 44 nm length and 4 nm width20,32 in a curved triskelion with 3-fold rotational symmetry. ACMs are fabricated by optical photolithography on a silicon wafer with a thin (20 nm) layer of germanium as a lift-off layer, deposited by electron beam evaporation. Photoresist is then patterned on top, and 80 nm of nickel and 25 nm of gold are subsequently deposited by evaporation. The photoresist is lifted-off to leave the ACM, and the wafer was descummed by oxygen plasma. The gold layer is then rendered hydrophobic by immersion for 2 h in a 10 mM solution of 1Octadecanethiol in ethanol and finally sonicated and rinsed in 2propanol to remove excess thiols (Figure 2b). This process is similar to that of lithoparticles48 and is highly flexible; the particle shape as well as the thickness, sequence, number, and type of the layers can be readily varied. See the Supporting Information for more details. ACM Liftoff, Assembly, and Imaging. ACM assembly in solution is initiated by dissolving the underlying germanium by either immersion in a droplet of 0.3% hydrogen peroxide or pure water with for 2 or 20 h, respectively. Once released, a curvature of approximately 17 μm in radius is observed for each ACM leg due to the bimetallic nickel/gold structure. This phenomenon is due to the difference in intrinsic stresses stemming from electron-beam evaporation. The high intrinsic stress of the evaporated nickel layer compared to the gold49 results in curvature toward the nickel side once the ACMs are released, and this technique has been widely exploited in the construction of curved and 3D microstructures.50,51 The released ACMs spontaneously associate into loose, nonspecific aggregates (Figure 2f−h) and are subsequently driven by gravity or a magnetic field to an air−water, air−oil, or liquid−liquid interface. For ACMs at an air−water interface, they are washed by passing the droplet through an air−water interface for imaging. For budding experiments, a syringe filled with butanol was used to generate a droplet inside of a water dish with free ACMs. Once ACMs were assembled on the droplet surface, a magnet was brought into proximity to stimulate budding. For fluorescent budding experiments, an ACM network was assembled at a planar butanol water interface and pulled off vertically with a magnet before imaging. ACMs at an air−water interface were imaged by upright reflected light microscopy, while ACMs at butanol−water interfaces were imaged by transmission microscopy on an inverted microscope. See the Supporting Information for more details. ACM Modeling. Simulation of the ACM mechanical properties was done in the COMSOL40 Multiphysics structural mechanics package. The bimetallic stress between ACM layers and total ACM strain energy was found by fitting the modeled ACM curvatures to observed values. Interfacial energy of the ACM was calculated numerically by solving the Young−Laplace equation around the ACM (see the Supporting Information) in COMSOL. The structural energy and surface distortion energy values were iterated against one another until they were equal; this was taken to be the equilibrium ACM curvature. The equilibrium ACM curvature was then used to numerically solve for interaction energies between ACM by varying distance and orientation.

CONCLUSIONS Biomimetic artificial clathrin mimics were successfully fabricated, and they replicated several clathrin-like activities. ACMs supported the transition of a two-dimensional interface into a closed three-dimensional droplet, and the concept of bottom-up endocytosis has been generalized to inorganic microscale particles for fluidic control. Two key insights were made that are applicable to general self-assembly. First, the curved shape of ACMs largely prevented irreversible aggregation in solution. Second, particle curvature provided a surface-energy based interparticle attraction for selective assembly of three-legged micro particles into extended networks, with 87% packing density and 6.3% defect rate. The ACM leg curvature offers a versatile route to control the interaction between multiple ACMs both sterically and energetically. The general strategy of curvature controlled self-assembly to create biomimetic behavior is not limited to clathrin. For example, the engineering of both attractive and repulsive interactions in surface tension mediated selfassembly,36 which would allow assembly of multiparticle networks with different responsive elements. Nanoscale ACMs could also provide a route to cell-interfacing systems such as artificial pores in lipid membranes.45,46 Due to the versatile fabrication method, both size and functionality of ACMs are highly flexible, and structures such as “stealth band”47 ACMs in Figure 2d could be made to potentially promote insertion into the hydrophobic core of lipid bilayers. Nanoscale ACMs made by high resolution photo9895

DOI: 10.1021/acsnano.7b03739 ACS Nano 2017, 11, 9889−9897

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b03739. Video of ACM liftoff (AVI) Video of ACM budding (AVI) Additional detailed methods and materials information; Supplementary Figure S3 (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Yifan Kong: 0000-0002-8785-8370 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, under contract DE-AC02-76SF00515. Work was performed in part in the nano@Stanford labs, which are supported by the National Science Foundation as part of the National Nanotechnology Coordinated Infrastructure under award ECCS-1542152. Part of this work was performed at the Stanford NeuroFab. Molecular graphics and analyses were performed with the UCSF Chimera package. Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco. REFERENCES (1) Guillaume-Gentil, O.; Grindberg, R. V.; Kooger, R.; DorwlingCarter, L.; Martinez, V.; Ossola, D.; Pilhofer, M.; Zambelli, T.; Vorholt, J. A. Tunable Single-Cell Extraction for Molecular Analyses. Cell 2016, 166, 506−516. (2) Actis, P.; Maalouf, M. M.; Kim, H. J.; Lohith, A.; Vilozny, B.; Seger, R. A.; Pourmand, N. Compartmental Genomics in Living Cells Revealed by Single-Cell Nanobiopsy. ACS Nano 2014, 8, 546−553. (3) Elvira, K. S.; i Solvas, X. C.; Wootton, R. C. R.; deMello, A. J. The Past, Present and Potential for Microfluidic Reactor Technology in Chemical Synthesis. Nat. Chem. 2013, 5, 905−915. (4) Hu, X.; Zrazhevskiy, P.; Gao, X. Encapsulation of Single Quantum Dots with Mesoporous Silica. Ann. Biomed. Eng. 2009, 37, 1960−1966. (5) Hokkanen, A.; Stuns, I.; Schmid, P.; Kokkonen, A.; Gao, F.; Steinecker, A.; Budczies, J.; Heimala, P.; Hakalahti, L. Microfluidic Sampling System for Tissue Analytics. Biomicrofluidics 2015, 9, 54109. (6) Bhargava, K. C.; Thompson, B.; Malmstadt, N. Discrete Elements for 3D Microfluidics. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 15013− 15018. (7) Duan, H.; Wang, D.; Kurth, D. G.; Möhwald, H. Directing SelfAssembly of Nanoparticles at Water/oil Interfaces. Angew. Chem., Int. Ed. 2004, 43, 5639−5642. (8) Bowden, N.; Terfort, A.; Carbeck, J.; Whitesides, G. SelfAssembly of Mesoscale Objects into Ordered Two-Dimensional Arrays. Science (Washington, DC, U. S.) 1997, 276, 233−235. (9) Böker, A.; He, J.; Emrick, T.; Russell, T. P. Self-Assembly of Nanoparticles at Interfaces. Soft Matter 2007, 3, 1231. (10) Niu, Z.; He, J.; Russell, T. P.; Wang, Q. Synthesis of Nano/ microstructures at Fluid Interfaces. Angew. Chem., Int. Ed. 2010, 49, 10052−10066. (11) Kocabey, S.; Kempter, S.; List, J.; Xing, Y.; Bae, W.; Schiffels, D.; Shih, W. M.; Simmel, F. C.; Liedl, T. Membrane-Assisted Growth of DNA Origami Nanostructure Arrays. ACS Nano 2015, 9, 3530−3539. 9896

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DOI: 10.1021/acsnano.7b03739 ACS Nano 2017, 11, 9889−9897