Mechanisms and Consequences of Macromolecular Phase Separation.

Over a century ago, colloidal phase separation of matter into non-membranous bodies was recognized as a fundamental organizing principal of cell "protoplasm." Recent insights into the molecular properties of such phase-separated bodies present challenges to our understanding of cellular protein interaction networks, as well as opportunities for interpreting and understanding of native and pathological genetic and molecular interactions. Here, we briefly review examples of and discuss physical principles of phase-separated cellular bodies and then reflect on how knowledge of these principles may direct future research on their functions.

Work hardening in colloidal crystals.

Colloidal crystals exhibit interesting properties1-4 that are in many ways analogous to their atomic counterparts. They have the same crystal structures2,5-7, undergo the same phase transitions8-10, and possess the same crystallographic defects11-14. In contrast to these structural properties, the mechanical properties of colloidal crystals are quite different from those of atomic systems. For example, unlike in atomic systems, the elasticity of hard-sphere colloidal crystals is purely entropic15; as a result, they are so soft that they can be melted just by stirring16,17. Moreover, crystalline materials deform plastically when subjected to increasing shear and become stronger because of the ubiquitous process of work hardening18; but this has so far never been observed in colloidal crystals, to our knowledge. Here we show that hard-sphere colloidal crystals exhibit work hardening. Moreover, despite their softness, the shear strength of colloidal crystals can increase and approach the theoretical limit for crystals, a value reached in very few other materials so far. We use confocal microscopy to show that the strength of colloidal crystals increases with dislocation density, and ultimately reaches the classic Taylor scaling behaviour for atomic materials19-21, although hard-sphere interactions lack the complexity of atomic interactions. We demonstrate that Taylor hardening arises through the formation of dislocation junctions22. The Taylor hardening regime, however, is established only after a transient phase, and it ceases when the colloidal crystals become so hard that the strain is localized within a thin boundary layer in which slip results from an unconventional motion of dislocations. The striking resemblance between colloidal and atomic crystals, despite the many orders of magnitude difference in particle size and shear modulus, demonstrates the universality of work hardening.

Digital colloid-enhanced Raman spectroscopy by single-molecule counting.

Quantitative detection of various molecules at very low concentrations in complex mixtures has been the main objective in many fields of science and engineering, from the detection of cancer-causing mutagens and early disease markers to environmental pollutants and bioterror agents1-5. Moreover, technologies that can detect these analytes without external labels or modifications are extremely valuable and often preferred6. In this regard, surface-enhanced Raman spectroscopy can detect molecular species in complex mixtures on the basis only of their intrinsic and unique vibrational signatures7. However, the development of surface-enhanced Raman spectroscopy for this purpose has been challenging so far because of uncontrollable signal heterogeneity and poor reproducibility at low analyte concentrations8. Here, as a proof of concept, we show that, using digital (nano)colloid-enhanced Raman spectroscopy, reproducible quantification of a broad range of target molecules at very low concentrations can be routinely achieved with single-molecule counting, limited only by the Poisson noise of the measurement process. As metallic colloidal nanoparticles that enhance these vibrational signatures, including hydroxylamine-reduced-silver colloids, can be fabricated at large scale under routine conditions, we anticipate that digital (nano)colloid-enhanced Raman spectroscopy will become the technology of choice for the reliable and ultrasensitive detection of various analytes, including those of great importance for human health.

Designer phospholipid capping ligands for soft metal halide nanocrystals.

The success of colloidal semiconductor nanocrystals (NCs) in science and optoelectronics is inextricable from their surfaces. The functionalization of lead halide perovskite NCs1-5 poses a formidable challenge because of their structural lability, unlike the well-established covalent ligand capping of conventional semiconductor NCs6,7. We posited that the vast and facile molecular engineering of phospholipids as zwitterionic surfactants can deliver highly customized surface chemistries for metal halide NCs. Molecular dynamics simulations implied that ligand-NC surface affinity is primarily governed by the structure of the zwitterionic head group, particularly by the geometric fitness of the anionic and cationic moieties into the surface lattice sites, as corroborated by the nuclear magnetic resonance and Fourier-transform infrared spectroscopy data. Lattice-matched primary-ammonium phospholipids enhance the structural and colloidal integrity of hybrid organic-inorganic lead halide perovskites (FAPbBr3 and MAPbBr3 (FA, formamidinium; MA, methylammonium)) and lead-free metal halide NCs. The molecular structure of the organic ligand tail governs the long-term colloidal stability and compatibility with solvents of diverse polarity, from hydrocarbons to acetone and alcohols. These NCs exhibit photoluminescence quantum yield of more than 96% in solution and solids and minimal photoluminescence intermittency at the single particle level with an average ON fraction as high as 94%, as well as bright and high-purity (about 95%) single-photon emission.

Shape memory in self-adapting colloidal crystals.

Reconfigurable, mechanically responsive crystalline materials are central components in many sensing, soft robotic, and energy conversion and storage devices1-4. Crystalline materials can readily deform under various stimuli and the extent of recoverable deformation is highly dependent upon bond type1,2,5-10. Indeed, for structures held together via simple electrostatic interactions, minimal deformations are tolerated. By contrast, structures held together by molecular bonds can, in principle, sustain much larger deformations and more easily recover their original configurations. Here we study the deformation properties of well-faceted colloidal crystals engineered with DNA. These crystals are large in size (greater than 100 µm) and have a body-centred cubic (bcc) structure with a high viscoelastic volume fraction (of more than 97%). Therefore, they can be compressed into irregular shapes with wrinkles and creases, and, notably, these deformed crystals, upon rehydration, assume their initial well-formed crystalline morphology and internal nanoscale order within seconds. For most crystals, such compression and deformation would lead to permanent, irreversible damage. The substantial structural changes to the colloidal crystals are accompanied by notable and reversible optical property changes. For example, whereas the original and structurally recovered crystals exhibit near-perfect (over 98%) broadband absorption in the ultraviolet-visible region, the deformed crystals exhibit significantly increased reflection (up to 50% of incident light at certain wavelengths), mainly because of increases in their refractive index and inhomogeneity.

Self-assembly of emulsion droplets through programmable folding.

In the realm of particle self-assembly, it is possible to reliably construct nearly arbitrary structures if all the pieces are distinct1-3, but systems with fewer flavours of building blocks have so far been limited to the assembly of exotic crystals4-6. Here we introduce a minimal model system of colloidal droplet chains7, with programmable DNA interactions that guide their downhill folding into specific geometries. Droplets are observed in real space and time, unravelling the rules of folding. Combining experiments, simulations and theory, we show that controlling the order in which interactions are switched on directs folding into unique structures, which we call colloidal foldamers8. The simplest alternating sequences (ABAB...) of up to 13 droplets yield 11 foldamers in two dimensions and one in three dimensions. Optimizing the droplet sequence and adding an extra flavour uniquely encodes more than half of the 619 possible two-dimensional geometries. Foldamers consisting of at least 13 droplets exhibit open structures with holes, offering porous design. Numerical simulations show that foldamers can further interact to make complex supracolloidal architectures, such as dimers, ribbons and mosaics. Our results are independent of the dynamics and therefore apply to polymeric materials with hierarchical interactions on all length scales, from organic molecules all the way to Rubik's Snakes. This toolbox enables the encoding of large-scale design into sequences of short polymers, placing folding at the forefront of materials self-assembly.

Open-channel metal particle superlattices.

Although tremendous advances have been made in preparing porous crystals from molecular precursors1,2, there are no general ways of designing and making topologically diversified porous colloidal crystals over the 10-1,000 nm length scale. Control over porosity in this size range would enable the tailoring of molecular absorption and storage, separation, chemical sensing, catalytic and optical properties of such materials. Here, a universal approach for synthesizing metallic open-channel superlattices with pores of 10 to 1,000 nm from DNA-modified hollow colloidal nanoparticles (NPs) is reported. By tuning hollow NP geometry and DNA design, one can adjust crystal pore geometry (pore size and shape) and channel topology (the way in which pores are interconnected). The assembly of hollow NPs is driven by edge-to-edge rather than face-to-face DNA-DNA interactions. Two new design rules describing this assembly regime emerge from these studies and are then used to synthesize 12 open-channel superlattices with control over crystal symmetry, channel geometry and topology. The open channels can be selectively occupied by guests of the appropriate size and that are modified with complementary DNA (for example, Au NPs).

Transmembrane transport in inorganic colloidal cell-mimics.

A key aspect of living cells is their ability to harvest energy from the environment and use it to pump specific atomic and molecular species in and out of their system-typically against an unfavourable concentration gradient1. Active transport allows cells to store metabolic energy, extract waste and supply organelles with basic building blocks at the submicrometre scale. Unlike living cells, abiotic systems do not have the delicate biochemical machinery that can be specifically activated to precisely control biological matter2-5. Here we report the creation of microcapsules that can be brought out of equilibrium by simple global variables (illumination and pH), to capture, concentrate, store and deliver generic microscopic payloads. Borrowing no materials from biology, our design uses hollow colloids serving as spherical cell-membrane mimics, with a well-defined single micropore. Precisely tunable monodisperse capsules are the result of a synthetic self-inflation mechanism and can be produced in bulk quantities. Inside the hollow unit, a photoswitchable catalyst6 produces a chemical gradient that propagates to the exterior through the membrane's micropore and pumps target objects into the cell, acting as a phoretic tractor beam7. An entropic energy barrier8,9 brought about by the micropore's geometry retains the cargo even when the catalyst is switched off. Delivery is accomplished on demand by reversing the sign of the phoretic interaction. Our findings provide a blueprint for developing the next generation of smart materials, autonomous micromachinery and artificial cell-mimics.

Hopping and crawling DNA-coated colloids.

Understanding the motion of particles with multivalent ligand-receptors is important for biomedical applications and material design. Yet, even among a single design, the prototypical DNA-coated colloids, seemingly similar micrometric particles hop or roll, depending on the study. We shed light on this problem by observing DNA-coated colloids diffusing near surfaces coated with complementary strands for a wide array of coating designs. We find colloids rapidly switch between 2 modes: They hop-with long and fast steps-and crawl-with short and slow steps. Both modes occur at all temperatures around the melting point and over various designs. The particles become increasingly subdiffusive as temperature decreases, in line with subsequent velocity steps becoming increasingly anticorrelated, corresponding to switchbacks in the trajectories. Overall, crawling (or hopping) phases are more predominant at low (or high) temperatures; crawling is also more efficient at low temperatures than hopping to cover large distances. We rationalize this behavior within a simple model: At lower temperatures, the number of bound strands increases, and detachment of all bonds is unlikely, hence, hopping is prevented and crawling favored. We thus reveal the mechanism behind a common design rule relying on increased strand density for long-range self-assembly: Dense strands on surfaces are required to enable crawling, possibly facilitating particle rearrangements.

Surface-mediated spontaneous emulsification of the acylated peptide, semaglutide.

Acylated peptides composed of glucagon-like peptide-1 receptor agonists modified with a fatty acid side chain are an important class of therapeutics for type 2 diabetes and obesity but are susceptible to an unusual physical instability in the presence of hydrophobic surfaces, i.e., spontaneous emulsification, also known as ouzo formation in practice. In this work, light scattering, small-angle X-ray scattering, and circular dichroism measurements are used to characterize the physical properties of the semaglutide colloidal phase, including size distribution, shape, secondary structure, internal structure, and internal composition, as a function of solution physico-chemical conditions. The existence and size of the colloids formed are successfully predicted by a classical Rayleigh model, which identifies the parameters controlling their size and formation. Colloid formation is found to be catalyzed by hydrophobic surfaces, and formation rates are modeled as an autocatalytic reaction, enabling the formation of a master curve for various surfaces that elucidates the mechanism. Surfaces differ due to differences in surface wettability, which can be correlated with Hansen solubility parameters. This work provides insights into this unusual colloidal phenomenon and guides the peptide synthesis process and drug product formulation in the pharmaceutical industry.

Colloidal Approaches to Zinc Oxide Nanocrystals.

Zinc oxide is an extensively studied semiconductor with a wide band gap in the near-UV. Its many interesting properties have found use in optics, electronics, catalysis, sensing, as well as biomedicine and microbiology. In the nanoscale regime the functional properties of ZnO can be precisely tuned by manipulating its size, shape, chemical composition (doping), and surface states. In this review, we focus on the colloidal synthesis of ZnO nanocrystals (NCs) and provide a critical analysis of the synthetic methods currently available for preparing ZnO colloids. First, we outline key thermodynamic considerations for the nucleation and growth of colloidal nanoparticles, including an analysis of different reaction methodologies and of the role of dopant ions on nanoparticle formation. We then comprehensively review and discuss the literature on ZnO NC systems, including reactions in polar solvents that traditionally occur at low temperatures upon addition of a base, and high temperature reactions in organic, nonpolar solvents. A specific section is dedicated to doped NCs, highlighting both synthetic aspects and structure-property relationships. The versatility of these methods to achieve morphological and compositional control in ZnO is explicated. We then showcase some of the key applications of ZnO NCs, both as suspended colloids and as deposited coatings on supporting substrates. Finally, a critical analysis of the current state of the art for ZnO colloidal NCs is presented along with existing challenges and future directions for the field.

Geometrically programmed self-limited assembly of tubules using DNA origami colloids.

Self-assembly is one of the most promising strategies for making functional materials at the nanoscale, yet new design principles for making self-limiting architectures, rather than spatially unlimited periodic lattice structures, are needed. To address this challenge, we explore the tradeoffs between addressable assembly and self-closing assembly of a specific class of self-limiting structures: cylindrical tubules. We make triangular subunits using DNA origami that have specific, valence-limited interactions and designed binding angles, and we study their assembly into tubules that have a self-limited width that is much larger than the size of an individual subunit. In the simplest case, the tubules are assembled from a single component by geometrically programming the dihedral angles between neighboring subunits. We show that the tubules can reach many micrometers in length and that their average width can be prescribed through the dihedral angles. We find that there is a distribution in the width and the chirality of the tubules, which we rationalize by developing a model that considers the finite bending rigidity of the assembled structure as well as the mechanism of self-closure. Finally, we demonstrate that the distributions of tubules can be further sculpted by increasing the number of subunit species, thereby increasing the assembly complexity, and demonstrate that using two subunit species successfully reduces the number of available end states by half. These results help to shed light on the roles of assembly complexity and geometry in self-limited assembly and could be extended to other self-limiting architectures, such as shells, toroids, or triply periodic frameworks.

The Wide World of Coacervates: From the Sea to Neurodegeneration.

The formation of immiscible liquid phases or coacervates is a phenomenon widely observed in biology. Marine organisms, for instance, use liquid-liquid phase separation (LLPS) as the precursor phase to form various fibrillar or crustaceous materials that are essential for surface adhesion. More recently, the importance of LLPS has been realized in the compartmentalization of living cells and in obtaining ordered but dynamic partitions that can be reversed according to necessity. Here, we compare the properties, features, and peculiarities of intracellular and extracellular coacervates, drawing parallels and learning from the differences. A more general view of the phenomenon may in the future inform new studies to allow a better comprehension of its laws.

A colloidal model for the equilibrium assembly and liquid-liquid phase separation of the reflectin A1 protein.

Reflectin is an intrinsically disordered protein known for its ability to modulate the biophotonic camouflage of cephalopods based on its assembly-induced osmotic properties. Its reversible self-assembly into discrete, size-controlled clusters and condensed droplets are known to depend sensitively on the net protein charge, making reflectin stimuli-responsive to pH, phosphorylation, and electric fields. Despite considerable efforts to characterize this behavior, the detailed physical mechanisms of reflectin's assembly are not yet fully understood. Here, we pursue a coarse-grained molecular understanding of reflectin assembly using a combination of experiments and simulations. We hypothesize that reflectin assembly and phase behavior can be explained from a remarkably simple colloidal model whereby individual protein monomers effectively interact via a short-range attractive and long-range repulsive (SA-LR) pair potential. We parameterize a coarse-grained SA-LR interaction potential for reflectin A1 from small-angle x-ray scattering measurements, and then extend it to a range of pH values using Gouy-Chapman theory to model monomer-monomer electrostatic interactions. The pH-dependent SA-LR interaction is then used in molecular dynamics simulations of reflectin assembly, which successfully capture a number of qualitative features of reflectin, including pH-dependent formation of discrete-sized nanoclusters and liquid-liquid phase separation at high pH, resulting in a putative phase diagram for reflectin. Importantly, we find that at low pH size-controlled reflectin clusters are equilibrium assemblies, which dynamically exchange protein monomers to maintain an equilibrium size distribution. These findings provide a mechanistic understanding of the equilibrium assembly of reflectin, and suggest that colloidal-scale models capture key driving forces and interactions to explain thermodynamic aspects of native reflectin behavior. Furthermore, the success of SA-LR interactions presented in this study demonstrates the potential of a colloidal interpretation of interactions and phenomena in a range of intrinsically disordered proteins.

Biomarker-Driven DNA-Functionalized Colloidal Programmed Simultaneous Assembly and Disassembly in Cells.

Complex structures and devices, both natural and artificial, can often undergo assembly and disassembly. Assembly and disassembly allow multiple stimuli to initiate, for example, the assembly and disassembly of primary cilia under the control of E3 ubiquitin ligases and deubiquitinases. Although biology relies on such schemes, they are rarely available in materials science. Here, we demonstrate a DNA-functionalized colloidal Au response to endogenous biomarkers to trigger simultaneous assembly and disassembly techniques. Colloidal Au is initially inert because the starting DNA strands are paired and prehybridized. TK1 mRNA competes to bind one of the paired strands and release its complement. The released complement binds to the next colloidal Au to initiate assembly, and APE1 can shear the colloidal Au assembly binding site to initiate disassembly. Our strategy provides temporal and spatial logic control during colloidal Au assembly and disassembly, and this simultaneous assembly and disassembly process can be used for sequential detection and cellular imaging of two biomarkers, effectively reducing signal false-positive results and shortening detection time. This work highlights biomarker-controlled colloidal Au simultaneous assembly and disassembly in ways that are simple and versatile, with the potential to enrich the application scope of DNA nanotechnology and provide an idea for the application of precision medicine testing.

DNA-Based Replication of Programmable Colloidal Assemblies.

Nature uses replication to amplify the information necessary for the intricate structures vital for life. Despite some successes with pure nucleotide structures, constructing synthetic microscale systems capable of replication remains largely out of reach. Here, a functioning strategy is shown for the replication of microscale particle assemblies using DNA-coated colloids. By positioning DNA-functionalized colloids using capillary forces and embedding them into a polymer layer, programmable sequences of patchy particles are created that act as a primer and offer precise binding of complementary particles from suspension. The strings of complementary colloids are cross-linked, released from the primer, and purified via flow cytometric sorting to achieve a purity of up to 81% of the replicated sequences. The replication of strings of up to five colloids and non-linear shapes is demonstrated with particles of different sizes and materials. Furthermore, a pathway for exponential self-replication is outlined, including preliminary data that shows the transfer of patches and binding of a second-generation of assemblies from suspension.

Tracking colloidal silica particles to evaluate their dispersion and interactions in concentrated suspensions under shear force applications.

This study aimed to characterize interactions within colloidal silica particles in their concentrated suspensions, using rheo-confocal measurements and imaging, followed by image analysis. We studied the effect of shear rate (0-500 s-1) and solution pH (6, 10) on the dispersion degree of colloidal silica particles via the determination and comparison of interparticle distances and their modeling. Images corresponding to different shear rates were analyzed to identify the coordinates of the particles. These coordinates were further analyzed to calculate the distance among the particles and then their surface-to-surface distance normalized by the particle diameter (H/D). It was found that the population of the particles per unit area of the image and H/D varied with increasing shear rate. The comparison between experimentally measured and theoretically calculated H/D identified that for some particles, the former was shorter than the latter, indicating the unexpected attractions among them against the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Then, the modification of previously reported equations for H/D was suggested and confirmed its validity. Assuming pair potential interaction and hydrodynamic interaction were the main non-DLVO interactions, their magnitudes were calculated and confirmed the significance of pH and shear application strength on particle dispersion/coagulation.

Combining Scattering Experiments and Colloid Theory to Characterize Charge Effects in Concentrated Antibody Solutions.

Charges and their contribution to protein-protein interactions are essential for the key structural and dynamic properties of monoclonal antibody (mAb) solutions. In fact, they influence the apparent molecular weight, the static structure factor, the collective diffusion coefficient, or the relative viscosity, and their concentration dependence. Further, charges play an important role in the colloidal stability of mAbs. There exist standard experimental tools to characterize mAb net charges, such as the measurement of the electrophoretic mobility, the second virial coefficient, or the diffusion interaction parameter. However, the resulting values are difficult to directly relate to the actual overall net charge of the antibody and to theoretical predictions based on its known molecular structure. Here, we report the results of a systematic investigation of the solution properties of a charged IgG1 mAb as a function of concentration and ionic strength using a combination of electrophoretic measurements, static and dynamic light scattering, small-angle X-ray scattering, and tracer particle-based microrheology. We analyze and interpret the experimental results using established colloid theory and coarse-grained computer simulations. We discuss the potential and limits of colloidal models for the description of the interaction effects of charged mAbs, in particular pointing out the importance of incorporating shape and charge anisotropy when attempting to predict structural and dynamic solution properties at high concentrations.

Deciphering the Lipid-Random Copolymer Interactions and Encoding Their Properties to Design a Hybrid System.

Lipid/copolymer colloidal systems are deemed hybrid materials with unique properties and functionalities. Their hybrid nature leads to complex interfacial phenomena, which have not been fully encoded yet, navigating their properties. Moving toward in-depth knowledge of such systems, a comprehensive investigation of them is imperative. In the present study, hybrid lipid/copolymer structures were fabricated and examined by a gamut of techniques, including dynamic light scattering, fluorescence spectroscopy, cryogenic transmission electron microscopy, microcalorimetry, and high-resolution ultrasound spectroscopy. The biomaterials that were mixed for this purpose at different ratios were 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine and four different linear, statistical (random) amphiphilic copolymers, consisting of oligo(ethylene glycol) methyl ether methacrylate as the hydrophilic comonomer and lauryl methacrylate as the hydrophobic one. The colloidal dispersions were studied for lipid/copolymer interactions regarding their physicochemical, morphological, and biophysical behavior. Their membrane properties and interactions with serum proteins were also studied. The aforementioned techniques confirmed the hybrid nature of the systems and the location of the copolymer in the structure. More importantly, the random architecture of the copolymers, the hydrophobic-to-hydrophilic balance of the nanoplatforms, and the lipid-to-polymer ratio are highlighted as the main design-influencing factors. Elucidating the lipid/copolymer interactions would contribute to the translation of hybrid nanoparticle performance and, thus, their rational design for multiple applications, including drug delivery.

Nanoporous Polystyrene Inverse Opal Materials with Optical Interference Properties for Label-Free Biosensing.

Colloidal crystal nanomaterials have been proven to be valuable substrates for optical-based biosensing due to their ordered macroporous nanostructure and brilliant optical properties. In this work, silica colloidal crystal (SCC) thin films, as well as polystyrene-SCC composite films and inverse opal (IO) polystyrene films fabricated using SCC as templates, are investigated for their application as substrate materials in optical interferometric biosensors. The SCC films formed by the self-assembly of silica colloidal crystals have the most densely packed nano-3D structure, also known as the opal structure. IO films are fabricated by filling the opal pores of SCC with polystyrene and then removing the template, resulting in an interconnected nano-3D ordered macroporous structure, as indicated by the name inverse opal. The performance of the three materials was compared and discussed based on an ordered porous layer interferometry optical platform, focusing on refractive index response, protein adsorption response, and biomolecular interaction response. These results could potentially offer innovative material support for the advancement of label-free optical biosensors, which can be used for more biological/biochemical/biomolecular reaction monitoring studies.