Lipid membranes supported by polydimethylsiloxane substrates with designed geometry.

The membrane curvature of cells and intracellular compartments continuously adapts to enable cells to perform vital functions, from cell division to signal trafficking. Understanding how membrane geometry affects these processes in vivo is challenging because of the biochemical and geometrical complexity as well as the short time and small length scales involved in cellular processes. By contrast, in vitro model membranes with engineered curvature would provide a versatile platform for this investigation and applications to biosensing and biocomputing. Here, we present a strategy that allows fabrication of lipid membranes with designed shape by combining 3D micro-printing and replica-molding lithography with polydimethylsiloxane to create curved micrometer-sized scaffolds with virtually any geometry. The resulting supported lipid membranes are homogeneous and fluid. We demonstrate the versatility of the system by fabricating structures of interesting combinations of mean and Gaussian curvature. We study the lateral phase separation and how local curvature influences the effective diffusion coefficient. Overall, we offer a bio-compatible platform for understanding curvature-dependent cellular processes and developing programmable bio-interfaces for living cells and nanostructures.

Micrometer-sized TPM emulsion droplets with surface-mobile binding groups.

Colloids coated with lipid membranes have been widely employed for fundamental studies of lipid membrane processes, biotechnological applications such as drug delivery and biosensing, and more recently, for self-assembly. The latter has been made possible by inserting DNA oligomers with covalently linked hydrophobic anchors into the membrane. The lateral mobility of the DNA linkers on micrometer-sized droplets and solid particles has opened the door to creating structures with unprecedented structural flexibility. Here, we investigate micro-emulsions of TPM (3-(trimethoxysilyl)propyl methacrylate) as a platform for lipid monolayers and further functionalization with proteins and DNA oligonucleotides. TPM droplets can be produced with a narrow size distribution and are polymerizable, thus providing supports for model lipid membranes with controlled size and curvature. With fluorescence recovery after photobleaching, we observed that droplet-attached lipids, NeutrAvidin proteins, as well as DNA oligonucleotides all show mobility on the surface. We explored the assembly of micron-sized particles on TPM-droplets by exploiting either avidin-biotin interactions or double-stranded DNA with complementary single-stranded end groups. While the single molecules are mobile, the particles that are attached to them are not. We propose that this is caused by the heterogeneous nature of emulsified TPM, which forms an oligomer network that limits the collective motion of linkers, but allows the surface mobility of individual molecules.

Microparticle Assembly Pathways on Lipid Membranes.

Understanding interactions between microparticles and lipid membranes is of increasing importance, especially for unraveling the influence of microplastics on our health and environment. Here, we study how a short-ranged adhesive force between microparticles and model lipid membranes causes membrane-mediated particle assembly. Using confocal microscopy, we observe the initial particle attachment to the membrane, then particle wrapping, and in rare cases spontaneous membrane tubulation. In the attached state, we measure that the particle mobility decreases by 26%. If multiple particles adhere to the same vesicle, their initial single-particle state determines their interactions and subsequent assembly pathways: 1) attached particles only aggregate when small adhesive vesicles are present in solution, 2) wrapped particles reversibly attract one another by membrane deformation, and 3) a combination of wrapped and attached particles form membrane-mediated dimers, which further assemble into a variety of complex structures. The experimental observation of distinct assembly pathways, induced only by a short-ranged membrane-particle adhesion, shows that a cytoskeleton or other active components are not required for microparticle aggregation. We suggest that this membrane-mediated microparticle aggregation is a reason behind reported long retention times of polymer microparticles in organisms.

Surfactant-free Colloidal Particles with Specific Binding Affinity.

Colloidal particles with specific binding affinity are essential for in vivo and in vitro biosensing, targeted drug delivery, and micrometer-scale self-assembly. Key to these techniques are surface functionalizations that provide high affinities to specific target molecules. For stabilization in physiological environments, current particle coating methods rely on adsorbed surfactants. However, spontaneous desorption of these surfactants typically has an undesirable influence on lipid membranes. To address this issue and create particles for targeting molecules in lipid membranes, we present here a surfactant-free coating method that combines high binding affinity with stability at physiological conditions. After activating charge-stabilized polystyrene microparticles with EDC/Sulfo-NHS, we first coat the particles with a specific protein and subsequently covalently attach a dense layer of poly(ethyelene) glycol. This polymer layer provides colloidal stability at physiological conditions as well as antiadhesive properties, while the protein coating provides the specific affinity to the targeted molecule. We show that NeutrAvidin-functionalized particles bind specifically to biotinylated membranes and that Concanavalin A-functionalized particles bind specifically to the glycocortex of Dictyostelium discoideum cells. The affinity of the particles changes with protein density, which can be tuned during the coating procedure. The generic and surfactant-free coating method reported here transfers the high affinity and specificity of a protein onto colloidal polystyrene microparticles.

Preparation of Colloidal Organosilica Spheres through Spontaneous Emulsification.

Colloidal particles of controlled size are promising building blocks for the self-assembly of functional materials. Here, we systematically study a method to synthesize monodisperse, micrometer-sized spheres from 3-(trimethoxysilyl)propyl methacrylate (TPM) in a benchtop experiment. Their ease of preparation, smoothness, and physical properties provide distinct advantages over other widely employed materials such as silica, polystyrene, and poly(methyl methacrylate). We describe that the spontaneous emulsification of TPM droplets in water is caused by base-catalyzed hydrolysis, self-condensation, and the deprotonation of TPM. By studying the time-dependent size evolution, we find that the droplet size increases without any detectable secondary nucleation. Resulting TPM droplets are polymerized to form solid particles. The particle diameter can be controlled in the range of 0.4 to 2.8 µm by adjusting the volume fraction of added monomer and the pH of the solution. Droplets can be grown to diameters of up to 4 µm by adding TPM monomer after the initial emulsification. Additionally, we characterize various physical parameters of the TPM particles, and we describe methods to incorporate several fluorescent dyes.

Colloidal joints with designed motion range and tunable joint flexibility.

The miniaturization of machines towards the micron and nanoscale requires the development of joint-like elements that enable and constrain motion. We present a facile method to create colloidal joints, that is, anisotropic colloidal particles functionalized with surface mobile DNA linkers that control the motion range of bonded particles. We demonstrate quantitatively that we can control the flexibility of these colloidal joints by tuning the DNA linker concentration in the bond area. We show that the shape of the colloidal joint controls the range of motion of bonded particles through a maximisation of the bond area. Using spheres, cubes, and dumbbells, we experimentally realize spherical joints, planar sliders, and hinges, respectively. Finally we demonstrate the potential of the colloidal joints for programmable bottom-up self-assembly by creating flexible colloidal molecules and colloidal polymers. The reconfigurability and motion constraint offered by our colloidal joints make them promising building blocks for the development of switchable materials and nanorobots.

Automated tracking of colloidal clusters with sub-pixel accuracy and precision.

Quantitative tracking of features from video images is a basic technique employed in many areas of science. Here, we present a method for the tracking of features that partially overlap, in order to be able to track so-called colloidal molecules. Our approach implements two improvements into existing particle tracking algorithms. Firstly, we use the history of previously identified feature locations to successfully find their positions in consecutive frames. Secondly, we present a framework for non-linear least-squares fitting to summed radial model functions and analyze the accuracy (bias) and precision (random error) of the method on artificial data. We find that our tracking algorithm correctly identifies overlapping features with an accuracy below 0.2% of the feature radius and a precision of 0.1 to 0.01 pixels for a typical image of a colloidal cluster. Finally, we use our method to extract the three-dimensional diffusion tensor from the Brownian motion of colloidal dimers.

Lipid membrane-mediated attraction between curvature inducing objects.

The interplay of membrane proteins is vital for many biological processes, such as cellular transport, cell division, and signal transduction between nerve cells. Theoretical considerations have led to the idea that the membrane itself mediates protein self-organization in these processes through minimization of membrane curvature energy. Here, we present a combined experimental and numerical study in which we quantify these interactions directly for the first time. In our experimental model system we control the deformation of a lipid membrane by adhering colloidal particles. Using confocal microscopy, we establish that these membrane deformations cause an attractive interaction force leading to reversible binding. The attraction extends over 2.5 times the particle diameter and has a strength of three times the thermal energy (-3.3 kBT). Coarse-grained Monte-Carlo simulations of the system are in excellent agreement with the experimental results and prove that the measured interaction is independent of length scale. Our combined experimental and numerical results reveal membrane curvature as a common physical origin for interactions between any membrane-deforming objects, from nanometre-sized proteins to micrometre-sized particles.

Colloidal Recycling: Reconfiguration of Random Aggregates into Patchy Particles.

The key ingredients to the successful bottom-up construction of complex materials are believed to be colloids with anisotropic shapes and directional, or patchy, interactions. We present an approach for creating such anisotropic patchy particles based on reconfiguring randomly shaped aggregates of colloidal spheres. While colloidal aggregates are often undesirable in colloidal dispersions due to their random shapes, we exploit them as a starting point to synthesize patchy particles. By a deliberate destabilization of the colloidal particles, diffusion-limited aggregation is induced which partitions the particles into randomly shaped aggregates with controlled size distribution. We achieve a reconfiguration of the aggregates into uniform structures by swelling the polymer spheres with an apolar solvent. The swelling lowers the attractive van der Waals forces, lubricates the contact area between the spheres, and drives the reorganization through minimization of the interfacial energy of the swollen polymer network. This reorganization process yields patchy particles whose patch arrangement is uniform for up to five patches. For particles with more patches, we find that the patch orientation depends on the degree of phase separation between the spheres and the monomer. This enables the synthesis of patchy particles with unprecedented patch arrangements. We demonstrate the broad applicability of this recycling strategy for making patchy particles as well as clusters of spheres by varying the swelling ratio, swelling solvent, surfactant concentration, and swelling time.