Self-Sorting in Diastereomeric Mixtures of Functionalized Dipeptides.

Self-sorting in functionalized dipeptide systems can be driven by the chirality of a single amino acid, both at a high pH in the micellar state and at a low pH in the gel state. The structures formed are affected to some degree by the relative concentrations of each component showing the complexity of such an approach. The structures underpinning the gel network are predefined by the micellar structures at a high pH. Here, we describe the systems prepared from two dipeptide-based gelators that differ only by the chirality of one of the amino acids. We provide firm evidence for self-sorting in the micellar and gel phases using small-angle neutron scattering and cryo-transmission electron microscopy (cryo-TEM), showing that complete self-sorting occurs across a range of relative concentrations.

Lipid doping of the sponge (L3) mesophase.

The polymorphism of lipid aggregates has long attracted detailed study due to the myriad factors that determine the final mesophase observed. This study is driven by the need to understand mesophase behaviour for a number of applications, such as drug delivery and membrane protein crystallography. In the case of the latter, the role of the so-called 'sponge' (L3) mesophase has been often noted, but not extensively studied by itself. The L3 mesophase can be formed in monoolein/water systems on the addition of butanediol to water, which partitions the headgroup region of the membrane, and decreases its elastic moduli. Like cubic mesophases, it is bicontinuous, but unlike them, has no long-range translational symmetry. In our present study, we show that the formation of the L3 phase can delicately depend on the addition of dopant lipids to the mesophase. While electrostatically neutral molecules similar in shape to monoolein (DOPE, cholesterol) have little effect on the general mesophase behaviour, others (DOPC, DDM) significantly reduce the composition at which it can form. Additionally, we show that by combining cholesterol with the anionic lipid DOPG, it is possible to form the largest stable L3 mesophases observed to date, with characteristic lengths over 220 Å.

Polymorphic protein phase transitions driven by surface anisotropy.

Phase transitions of proteins are strongly influenced by surface chemical modifications or mutations. Human γD-crystallin (HGD) single-mutants have been extensively studied because they are associated with the onset of juvenile cataract. However, they have also provided a rich library of molecules to examine how specific inter-protein interactions direct protein assembly, providing new insights and valuable experimental data for coarse-grained patchy-particle models. Here, we demonstrate that the addition of new inter-protein interactions by mutagenesis is additive and increases the number and variety of condensed phases formed by proteins. When double mutations incorporating two specific single point mutations are made, the properties of both single mutations are retained in addition to the formation of a new condensed phase. We find that the HGD double-mutant P23VC110M self-assembles into spherical particles with retrograde solubility, orthorhombic crystals, and needle/plate shape crystals, while retaining the ability to undergo liquid-liquid phase separation. This rich polymorphism is only partially predicted by the experimental data on the constituent single mutants. We also report a previously un-characterized amorphous protein particle, with unique properties that differ from those of protein spherulites, protein particulates previously described. The particles we observe are amorphous, reversible with temperature, tens of microns in size, and perfectly spherical. When they are grown on pristine surfaces, they appear to form by homogeneous nucleation, making them unique, and we believe a new form of protein condensate. This work highlights the challenges in predicting protein behavior, which has frustrated rational assembly and crystallization but also provides rich data to develop new coarse-grained models to explain the observed polymorphism.

A de novo peroxidase is also a promiscuous yet stereoselective carbene transferase.

By constructing an in vivo-assembled, catalytically proficient peroxidase, C45, we have recently demonstrated the catalytic potential of simple, de novo-designed heme proteins. Here, we show that C45's enzymatic activity extends to the efficient and stereoselective intermolecular transfer of carbenes to olefins, heterocycles, aldehydes, and amines. Not only is this a report of carbene transferase activity in a completely de novo protein, but also of enzyme-catalyzed ring expansion of aromatic heterocycles via carbene transfer by any enzyme.

A Self-Assembling Flavin for Visible Photooxidation.

A new flavin-based gelator is reported which forms micellar structures at high pH and gels at low pH. This flavin can be used for the photooxidation of thiols under visible light, with the catalytic efficiency being linked to the self-assembled structures present.

Synthesis and characterisation of diketopyrrolopyrrole-based hydrogels.

Diketopyrrolopyrrole (DPP) based materials can be easily tuned by functionalising with groups that extend the conjugation and thus alter the electronic properties. When attaching thiophenes to give dithiophene-diketopyrrolopyrroles (DTDPPs), a donor-acceptor-donor system is created that is suitable for charge-transfer applications. This core also promotes π-stacking and hydrophobic interactions. Here, we describe a number of DTDPPs functionalised with amino acids that undergo pH-trigerred gelation. We show that the optical properties of our DTDPPs are affected by whether the amino acids have aromatic or aliphatic side chains. We also describe the effect of solvent polarity. We have successfully produced hydrogels via a pH trigger with examples containing phenylalanine (F), valine (V), leucine (L) and alanine (A) amino acids. Viscosity and small angle X-ray scattering measurements show the presence of micellar structures in solution in water at pH 10.5, with gelation starting at a pH less than 7 due to the formation of a fibrous network.

Using small angle scattering to understand low molecular weight gels.

The material properties of a gel are determined by the underpinning network that immobilises the solvent. When gels are formed by the self-assembly of small molecules into a so-called low molecular weight gel, the network is the result of the molecules forming one-dimensional objects such as fibres or nanotubes which entangle or otherwise cross-link to form a three-dimensional network. Characterising the one-dimensional objects and the network is difficult. Many conventional techniques rely on drying to probe the network, which often leads to artefacts. An effective tool to probe the gel in the solvated state is small angle scattering. Both small angle X-ray scattering (SAXS) and small angle neutron scattering (SANS) can be used. Here, we discuss these approaches and provide a tutorial review to describe how these approaches work, what opportunities there are and how the data treatment should be approached. We aim to show the power of this approach and provide enabling information to make them accessible to the non-specialist.

Scale-invariance in miniature coarse-grained red blood cells by fluctuation analysis.

To accurately represent the morphological and elastic properties of a human red blood cell, Fu et al. [Fu et al., Lennard-Jones type pair-potential method for coarse-grained lipid bilayer membrane simulations in LAMMPS, 2017, 210, 193-203] recently developed a coarse-grained molecular dynamics model with particular detail in the membrane. However, such a model accrues an extremely high computational cost for whole-cell simulation when assuming an appropriate length scaling - that of the bilayer thickness. To date, the model has only simulated "miniature" cells in order to circumvent this, with the a priori assumption that these miniaturised cells correctly represent their full-sized counterparts. The present work assesses the validity of this approach, by testing the scale invariance of the model through simulating cells of various diameters; first qualitatively in their shape evolution, then quantitatively by measuring their bending rigidity through fluctuation analysis. Cells of diameter of at least 0.5 µm were able to form the characteristic biconcave shape of human red blood cells, though smaller cells instead equilibrated to bowl-shaped stomatocytes. Thermal fluctuation analysis showed the bending rigidity to be constant over all cell sizes tested, and consistent between measurements on the whole-cell and on a planar section of bilayer. This is as expected from the theory on both counts. Therefore, we confirm that the evaluated model is a good representation of a full-size RBC when the model diameter is ≥0.5 µm, in terms of the morphological and mechanical properties investigated.

Understanding gel-to-crystal transitions in supramolecular gels.

Most supramolecular gels are stable or assumed to be stable over time, and aging effects are often not studied. However, some gels do show clear changes on aging, and a small number of systems exhibit gel-to-crystal transitions. In these cases, crystals form over time, typically at the expense of the network underpinning the gel; this leads to the gel falling apart. These systems are rare, and little is known about how these gel-to-crystal transitions occur. Here, we use a range of techniques to understand in detail a gel-to-crystal transition for a specific functionalised dipeptide based gelator. We show that the gel-to-crystal transition depends on the final pH of the medium which we control by varying the amount of glucon-δ-lactone (GdL) added. In the gel phase, at low concentrations of GdL, and at early time points with high concentrations of GdL, we are able to show the nanometre scale dimensions of the self-assembled fibre using SAXS; however there is no evidence of molecular ordering of the gel fibres in the WAXS. At low concentrations of GdL, these self-assembled fibres stiffen with time but do not crystallise over the timescale of the SAXS experiment. At high concentrations of GdL, the fibres are already stiffened, and then, as the pH drops further, give way to the presence of crystals which appear to grow preferentially along the direction of the fibre axis. We definitively show therefore that the gel and crystal phase are not the same. Our work shows that many assumptions in the literature are incorrect. Finally, we also show that the sample holder geometry is an important parameter for these experiments, with the rate of crystallisation depending on the holder in which the experiment is carried out.

Decorated networks of native proteins: nanomaterials with tunable mesoscopic domain size.

Natural and artificial proteins with designer properties and functionalities offer unparalleled opportunity for functional nanoarchitectures formed through self-assembly. However, to exploit this potential we need to design the system such that assembly results in desired architecture forms while avoiding denaturation and therefore retaining protein functionality. Here we address this challenge with a model system of fluorescent proteins. By manipulating self-assembly using techniques inspired by soft matter where interactions between the components are controlled to yield the desired structure, we have developed a methodology to assemble networks of proteins of one species which we can decorate with another, whose coverage we can tune. Consequently, the interfaces between domains of each component can also be tuned, with potential applications for example in energy - or electron - transfer. Our model system of eGFP and mCherry with tuneable interactions reveals control over domain sizes in the resulting networks.

Protein-polymer mixtures in the colloid limit: Aggregation, sedimentation, and crystallization.

While proteins have been treated as particles with a spherically symmetric interaction, of course in reality, the situation is rather more complex. A simple step toward higher complexity is to treat the proteins as non-spherical particles and that is the approach we pursue here. We investigate the phase behavior of the enhanced green fluorescent protein (eGFP) under the addition of a non-adsorbing polymer, polyethylene glycol. From small angle x-ray scattering, we infer that the eGFP undergoes dimerization and we treat the dimers as spherocylinders with aspect ratio L/D - 1 = 1.05. Despite the complex nature of the proteins, we find that the phase behavior is similar to that of hard spherocylinders with an ideal polymer depletant, exhibiting aggregation and, in a small region of the phase diagram, crystallization. By comparing our measurements of the onset of aggregation with predictions for hard colloids and ideal polymers [S. V. Savenko and M. Dijkstra, J. Chem. Phys. 124, 234902 (2006) and Lo Verso et al., Phys. Rev. E 73, 061407 (2006)], we find good agreement, which suggests that the behavior of the eGFP is consistent with that of hard spherocylinders and ideal polymers.

Isotopic Control over Self-Assembly in Supramolecular Gels.

It is common to switch between H2O and D2O when examining peptide-based systems, with the assumption being that there are no effects from this change. Here, we describe the effect of changing from H2O to D2O in a number of low-molecular-weight dipeptide-based gels. Gels are formed by decreasing the pH. In most cases, there is little difference in the structures formed at high pH, but this is not universally true. On lowering the pH, the kinetics of gelation are affected and, in some cases, the structures underpinning the gel network are different. Where there are differences in the self-assembled structures, the resulting gel properties are different. We, therefore, show that isotopic control over gel properties is possible.

Bénard-Marangoni Dendrites upon Evaporation of a Reactive ZnO Nanofluid Droplet: Effect of Substrate Chemistry.

Evaporation of a particle laden sessile drop can lead to complex surface patterns with structural hierarchy. Most commonly, the dispersed particles are inert. We have recently reported that when the sessile drop contains reactive ZnO nanoparticles, solidified Bénard-Marangoni (BM) cells with dendritic micromorphology were formed in the residual surface pattern from in situ-generated nanoclusters. Here, we report the effect of substrate chemistry on the residual pattern from the evaporation of nanofluids containing ZnO particles dispersed in a mixture of cyclohexane and isobutylamine, by comparing three different substrates: glass, silicon, and hydrophilized silicon. In particular, we performed a quantitative analysis of the BM cell size, distribution, and the cell morphological characteristics via the fractal dimension analysis. We find that the size dimension λBM of the dendritic Bénard-Marangoni cells varied on the different substrates, attributed to their different hydrophilicity and affinity for water molecules, evident from the different polar components γP in their surface free energy from the Owens-Wendt analysis. The average BM cell size was the smallest for the glass substrate (λBM = 289 µm) and comparable for the unmodified and UV/ozone-treated silicon wafers (with λBM = 466 and 423 µm, respectively). The fractal dimension analysis provided a quantitative description of the BM cells with complex structural hierarchy, highlighting the differences in the geometric features of the surface patterns resulting from different substrate chemistry. We also found that the fractal dimensions depended on the BM cell size, attributing it to two different regimes: the growing fractals and the maturing fractals.

Hierarchical Surface Patterns upon Evaporation of a ZnO Nanofluid Droplet: Effect of Particle Morphology.

Surface structures with tailored morphologies can be readily delivered by the evaporation-induced self-assembly process. It has been recently demonstrated that ZnO nanorods could undergo rapid chemical and morphological transformation into 3D complex structures of Zn(OH)2 nanofibers as a droplet of ZnO nanofluid dries on the substrate via a mechanism very different from that observed in the coffee ring effect. Here, we have investigated how the crystallinity and morphology of ZnO nanoparticles would affect the ultimate pattern formation. Three ZnO particles differing in size and shape were used, and their crystal structures were characterized by powder X-ray diffraction (XRD) and transmission electron microscopy (TEM). Their dispersions were prepared by sonication in a mixture of isobutylamine and cyclohexane. Residual surface patterns were created by drop casting a droplet of the nanofluid on a silicon substrate. The residual surface patterns were analyzed by scanning electron microscopy (SEM) and microfocus grazing incidence X-ray diffraction (µGIXRD). Nanofluid droplets of the in-house synthesized ZnO nanoparticles resulted in residual surface patterns consisting of Zn(OH)2 nanofibers. However, when commercially acquired ZnO powders composed of crystals with various shapes and sizes were used as the starting material, Zn(OH)2 fibers were found covered by ZnO crystal residues that did not fully undergo the dissolution and recrystallization process during evaporation. The difference in the solubility of ZnO nanoparticles was linked to the difference in their crystallinity, as assessed using the Scherrer equation analysis of their XRD Bragg peaks. Our results show that the morphology of the ultimate residual pattern from evaporation of ZnO nanofluids can be controlled by varying the crystallinity of the starting ZnO nanoparticles which affects the nanoparticle dissolution process during evaporation.

Opposed flow focusing: evidence of a second order jetting transition.

We propose a novel microfluidic "opposed-flow" geometry in which the continuous fluid phase is fed into a junction in a direction opposite to the dispersed phase. This pulls out the dispersed phase into a micron-sized jet, which decays into micron-sized droplets. As the driving pressure is tuned to a critical value, the jet radius vanishes as a power law down to sizes below 1 µm. By contrast, the conventional "coflowing" junction leads to a first order jetting transition, in which the jet disappears at a finite radius of several µm, to give way to a "dripping" state, resulting in much larger droplets. We demonstrate the effectiveness of our method by producing the first microfluidic silicone oil emulsions with a sub micron particle radius, and utilize these droplets to produce colloidal clusters.

P-Type Low-Molecular-Weight Hydrogelators.

As the use of low-molecular-weight gelators (LMWGs) as components in single and multicomponent systems for optoelectronic and solar cell applications increases, so does the need for more functional gelators. There are relatively few examples of p-type gelators that can be used in such systems. Here, the synthesis and characterization of three amino-acid-functionalized p-type gelators based on terthiophene, tetrathiafulvalene, and oligo(phenylenevinylene) are described. The cores of these molecules are already used as electron donors in optoelectronic applications. These newly designed molecules can gel water to form highly organized structures, which can be dried into thin films that show p-type behavior.

Oil-in-water microfluidics on the colloidal scale: new routes to self-assembly and glassy packings.

We have developed norland optical adhesive (NOA) flow focusing devices, making use of the excellent solvent compatibility and surface properties of NOA to generate micron scale oil-in-water emulsions with polydispersities as low as 5%. While current work on microfluidic oil-in-water emulsification largely concerns the production of droplets with sizes on the order of 10s of micrometres, large enough that Brownian motion is negligible, our NOA devices can produce droplets with radii ranging from 2 µm to 12 µm. To demonstrate the utility of these emulsions as colloidal model systems we produce fluorescently labelled polydimethylsiloxane droplets suitable for particle resolved studies with confocal microscopy. We analyse the structure of the resulting emulsion in 3D using coordinate tracking and the topological cluster classification and reveal a new mono-disperse thermal system.

Opening a Can of Worm(-like Micelle)s: The Effect of Temperature of Solutions of Functionalized Dipeptides.

A simple heat/cool cycle can be used to significantly affect the properties of a solution of a low-molecular-weight gelator at high pH. The viscosity and extensional viscosity are increased markedly, leading to materials with very different properties than when the native solution is used.

Self-Assembly of a Functional Oligo(Aniline)-Based Amphiphile into Helical Conductive Nanowires.

A tetra(aniline)-based cationic amphiphile, TANI-NHC(O)C5H10N(CH3)3(+)Br(-) (TANI-PTAB) was synthesized, and its emeraldine base (EB) state was found to self-assemble into nanowires in aqueous solution. The observed self-assembly is described by an isodesmic model, as shown by temperature-dependent UV-vis investigations. Linear dichroism (LD) studies, combined with computational modeling using time-dependent density functional theory (TD-DFT), suggests that TANI-PTAB molecules are ordered in an antiparallel arrangement within nanowires, with the long axis of TANI-PTAB arranged perpendicular to the nanowire long axis. Addition of either S- or R- camphorsulfonic acid (CSA) to TANI-PTAB converted TANI to the emeraldine salt (ES), which retained the ability to form nanowires. Acid doping of TANI-PTAB had a profound effect on the nanowire morphology, as the CSA counterions' chirality translated into helical twisting of the nanowires, as observed by circular dichroism (CD). Finally, the electrical conductivity of CSA-doped helical nanowire thin films processed from aqueous solution was 2.7 mS cm(-1). The conductivity, control over self-assembled 1D structure and water-solubility demonstrate these materials' promise as processable and addressable functional materials for molecular electronics, redox-controlled materials and sensing.

Experimental confirmation of transformation pathways between inverse double diamond and gyroid cubic phases.

A macroscopically oriented double diamond inverse bicontinuous cubic phase (QII(D)) of the lipid glycerol monooleate is reversibly converted into a gyroid phase (QII(G)). The initial QII(D) phase is prepared in the form of a film coating the inside of a capillary, deposited under flow, which produces a sample uniaxially oriented with a ⟨110⟩ axis parallel to the symmetry axis of the sample. A transformation is induced by replacing the water within the capillary tube with a solution of poly(ethylene glycol), which draws water out of the QII(D) sample by osmotic stress. This converts the QII(D) phase into a QII(G) phase with two coexisting orientations, with the ⟨100⟩ and ⟨111⟩ axes parallel to the symmetry axis, as demonstrated by small-angle X-ray scattering. The process can then be reversed, to recover the initial orientation of QII(D) phase. The epitaxial relation between the two oriented mesophases is consistent with topology-preserving geometric pathways that have previously been hypothesized for the transformation. Furthermore, this has implications for the production of macroscopically oriented QII(G) phases, in particular with applications as nanomaterial templates.