Self-assembly Step Research Articles

Dimerization and Dissociation: The Rate-Controlling steps of molecular Self-Assembly during solution nucleation determined by variation of gas media

To further probe into the link between solution chemistry and nucleation processes experimental and computational data are presented on unsaturated, saturated, and supersaturated solutions and in situ monitoring of nucleation chemistry of p-chlorobenzoic acid (PCBA), p-methoxybenzoic acid (PMBA) and p-nitrobenzoic acid (PNBA), three benzoic acid derivatives with different properties of para-substituents. By variation of the gas media, we were able to establish that hydrogen-bonded dimers (HD) are the basic building unite in solution for clusters and nucleuses despite of the gases and solvents, and there is a strong link between the supersaturated solution chemistry and the intrinsic supersaturation ratio, and the nucleation kinetics. Two different molecular self-assembly pathways and the rate-controlling steps were discussed, which paves new aspects for understanding the role of solution chemistry in directing nucleation processes.

Direct Bottom-Up In Situ Growth: A Paradigm Shift for Studies in Wet-Chemical Synthesis of Gold Nanoparticles.

Plasmonic gold nanoparticles have been used increasingly in solid-state systems because of their applicability in fabricating novel sensors, heterogeneous catalysts, metamaterials, and thermoplasmonic substrates. While bottom-up colloidal syntheses take advantage of the chemical environment to control size, shape, composition, surface chemistry, and crystallography of the nanostructures precisely, it can be challenging to assemble nanoparticles rationally from suspension onto solid supports or within devices. In this Review, we discuss a powerful recent synthetic methodology, bottom-up in situ substrate growth, which circumvents time-consuming batch presynthesis, ligand exchange, and self-assembly steps by applying wet-chemical synthesis to form morphologically controlled nanostructures on supporting materials. First, we briefly introduce the properties of plasmonic nanostructures. Then we comprehensively summarize recent work that adds to the synthetic understanding of in situ geometrical and spatial control (patterning). Next, we briefly discuss applications of plasmonic hybrid materials prepared by in situ growth. Overall, despite the vast potential advantages of in situ growth, the mechanistic understanding of these methodologies remains far from established, providing opportunities and challenges for future research.

Sequential Flash NanoPrecipitation for the scalable formulation of stable core-shell nanoparticles with core loadings up to 90%

Flash NanoPrecipitation (FNP) is a scalable, single-step process that uses rapid mixing to prepare nanoparticles with a hydrophobic core and amphiphilic stabilizing shell. Because the two steps of particle self-assembly – (1) core nucleation and growth and (2) adsorption of a stabilizing polymer onto the growing core surface – occur simultaneously during FNP, nanoparticles formulated at core loadings above approximately 70% typically exhibit poor stability or do not form at all. Additionally, a fundamental limit on the concentration of total solids that can be introduced into the FNP process has been reported previously. These limits are believed to share a common mechanism: entrainment of the stabilizing polymer into the growing particle core, leading to destabilization and aggregation. Here, we demonstrate a variation of FNP which separates the nucleation and stabilization steps of particle formation into separate sequential mixers. This scheme allows the hydrophobic core to nucleate and grow in the first mixing chamber unimpeded by adsorption of the stabilizing polymer, which is later introduced to the growing nuclei in the second mixer. Using this Sequential Flash NanoPrecipitation (SNaP) technique, we formulate stable nanoparticles with up to 90% core loading by mass and at 6-fold higher total input solids concentrations than typically reported.

Autonomous Non-Equilibrium Self-Assembly and Molecular Movements Powered by Electrical Energy.

The ability to exploit energy autonomously is one of the hallmarks of life. Mastering such processes in artificial nanosystems can open technological opportunities. In the last decades, light- and chemically driven autonomous systems have been developed in relation to conformational motion and self-assembly, mostly in relation to molecular motors. In contrast, despite electrical energy being an attractive energy source to power nanosystems, its autonomous harnessing has received little attention. Herein we consider an operation mode that allows the autonomous exploitation of electrical energy by a self-assembling system. Threading and dethreading motions of a pseudorotaxane take place autonomously in solution, powered by the current flowing between the electrodes of a scanning electrochemical microscope. The underlying autonomous energy ratchet mechanism drives the self-assembly steps away from equilibrium with a higher energy efficiency compared to other autonomous systems. The strategy is general and might be extended to other redox-driven systems.

Hybrid Hydrogels of FKF-Peptide Assemblies and Gelatin for Sustained Antimicrobial Activity.

The growing resistance of pathogenic bacteria to conventional antibiotics promotes the development of new antimicrobial agents, including peptides. Hydrogels composed of antimicrobial peptides (AMPs) may be applied as topical treatments for skin infection and wound regeneration. The unique antimicrobial and ultrashort-peptide FKF (Phe-Lys-Phe) was recently demonstrated to form bactericidal hydrogels. Here, we sought to improve the cyto-biocompatibility of FKF by combining FKF hydrogels with gelatin. Homogeneous hybrid hydrogels of FKF:gelatin were developed based on a series of self-assembly steps that involved mixing solutions of the two components with no covalent cross-linkers. The hydrogels were characterized for their structural features, dissolution, cyto-biocompatibility, and antibacterial properties. These hybrid hydrogels first release the antibacterial FKF assemblies, leaving the gelatinous fraction of the hydrogel to serve as a scaffold for tissue regeneration. Sponges of these hybrid hydrogels, obtained by lyophilization and rehydrated prior to application, exhibited enhanced antimicrobial activity compared to the hydrogels' formulations.

Scalable and Uniform Length-Tunable Biodegradable Block Copolymer Nanofibers with a Polycarbonate Core via Living Polymerization-Induced Crystallization-Driven Self-assembly.

Uniform 1D block copolymer (BCP) nanofibers prepared by the seeded-growth approach termed living crystallization-driven self-assembly (CDSA) offer promising potential for various applications due to their anisotropy, length tunability, and variable core and coronal chemistries. However, this procedure consists of a multi-step process involving independent BCP synthesis and self-assembly steps, where the latter is performed at low solution concentrations (<1 wt %), hindering scale-up. Here, we demonstrate the use of a one-pot BCP synthesis and self-assembly process, polymerization-induced CDSA (PI-CDSA), to access length-disperse nanofibers with a biodegradable crystalline poly(fluorenetrimethylenecarbonate) (PFTMC) core and a hydrophilic poly(ethylene glycol) (PEG) corona derived from PEG-b-PFTMC at concentrations up to 20 wt %, 400 times higher than those previously reported. Furthermore, living PI-CDSA could be used to access scalable, low dispersity, and length-tunable 1D PEG-b-PFTMC nanofibers at concentrations of up to 10 wt %. This provides the first example of living PI-CDSA involving an all-organic and biodegradable BCP that utilizes a conveniently implemented BCP synthesis protocol and does not involve living anionic polymerization. Significantly, samples of low-dispersity nanofibers of controlled lengths from 100 to 660 nm (Lw/Ln = 1.08-1.20) were prepared, allowing for upscaled access to well-defined biodegradable nanofibers at useful length-scales for applications in nanomedicine. Interestingly, detailed studies revealed a key role for PFTMC homopolymer impurities in the BCP prepared in situ in the formation of nanofibers under the reaction conditions used.

Multicolor Patterning of 2D Semiconductor Nanoplatelets.

Nanocrystal micro/nanoarrays with multiplexed functionalities are of broad interest in the field of nanophotonics, cellular dynamics, and biosensing due to their tunable electrical and optical properties. This work focuses on the multicolor patterning of two-dimensional nanoplatelets (NPLs) via two sequential self-assembly and direct electron-beam lithography steps. By using scanning electron microscopy, atomic force microscopy, and fluorescence microscopy, we demonstrate the successful fabrication of fluorescent nanoarrays with a thickness of only two or three monolayers (7-11 nm) and a feature line width of ∼40 nm, which is three to four NPLs wide. To this end, first, large-area thin films of red-emitting CdSe/ZnyCd1-yS and green-emitting CdSe1-xSx/ZnyCd1-yS core/shell NPLs are fabricated based on Langmuir-type self-assembly at the liquid/air interface. By varying the concentration of ligands in the subphase, we investigate the effect of interaction potential on the film's final characteristics to prepare thin superlattices suitable for the patterning step. Equipped with the ability to fabricate a uniform superlattice with a controlled thickness, we next perform nanopatterning on a thin film of NPLs utilizing a direct electron-beam lithography (EBL) technique. The effect of acceleration voltage, aperture size, and e-beam dosage on the nanopattern's resolution and fidelity is investigated for both of the presented NPLs. After successfully optimizing EBL parameters to fabricate single-color nanopatterns, we finally focus on fabricating multicolor patterns. The obtained micro/nanoarrays provide us with an innovative experimental platform to investigate biological interactions as well as Förster resonance energy transfer.

Constitutional Dynamic Selection at Low Reynolds Number in a Triple Dynamic System: Covalent Dynamic Adaptation Driven by Double Supramolecular Self-Assembly.

A triple dynamic complex system has been designed, implementing a dynamic covalent process coupled to two supramolecular self-assembly steps. To this end, two dynamic covalent libraries (DCLs), DCL-1 and DCL-2, have been established on the basis of dynamic covalent C═C/C═N organo-metathesis between two Knoevenagel derivatives and two imines. Each DCL contains a barbituric acid-based Knoevenagel constituent that may undergo a sequential double self-organization process involving first the formation of hydrogen-bonded hexameric supramolecular macrocycles that subsequently undergo stacking to generate a supramolecular polymer SP yielding a viscous gel state. Both DCLs display selective self-organization-driven amplification of the constituent that leads to the SP. Dissociation of the SP on heating causes reversible randomization of the constituent distributions of the DCLs as a function of temperature. Furthermore, diverse distribution patterns of DCL-2 were induced by modulation of temperature and solvent composition. The present dynamic systems display remarkable self-organization-driven constitutional adaption and tunable composition by coupling between dynamic covalent component selection and two-stage supramolecular organization. In more general terms, they reveal dynamic adaptation by component selection in low Reynolds number conditions of living systems where frictional effects dominate inertial behavior.

Zwitterionic dendrimer – Polymer hybrid copolymers for self-assembling antifouling coatings

In this work, we show two different routes to synthesize polymer-dendrimer hybrids by the coupling of poly(l-lysine) and zwitterionic dendrimers (ZIDs). Poly(l-lysine) (PLL) is used because of its advantageous self-assembly properties onto silicon oxide by charged-based interactions between the lysine groups and the negatively charged surface, whilst the coupled ZIDs provide antifouling properties. The first route yields network-like structures in which PLL and ZIDs are crosslinked by multiple amide bonds. By using different ratios of PLL and ZID, we vary the size of the formed networks. A more defined, linear PLL-ZID macromolecule is formed via coupling of multiple ZIDs to PLL in a controlled way by a copper-catalyzed azide/alkyne cycloaddition (CuAAC) “click” reaction. Following synthesis and characterization of the two different types of PLL-ZID macromolecules, they are self-assembled on silicon oxide surfaces from aqueous solutions in a single step, to form thin, hydrophilic coatings. Their potential use as antifouling coatings is tested by fluorescence microscopy and quartz crystal microbalance (QCM) with foulants such a single proteins and diluted human serum. Finally, by performing an on-surface biofunctionalization step by biotin we demonstrate it is possible to use these polymer-dendrimer hybrids for selective detection of target analytes (here: streptavidin), while the underlying coating maintains its antifouling properties.This method presents a new, straightforward approach for the manufacturing of PLL-ZID based coatings that can be pre-synthesized partly or fully and applied as coating in a single self-assembly step. Both steps can take place in aqueous solution and under ambient conditions, and result in stable coatings that not only display antifouling properties but also maintain the possibility of further functionalization.

Hydrogen-Bond-Driven Chemical Separations: Elucidating the Interfacial Steps of Self-Assembly in Solvent Extraction

Chemical separations, particularly liquid extractions, are pervasive in academic and industrial laboratories, yet a mechanistic understanding of the events governing their function are obscured by interfacial phenomena that are notoriously difficult to measure. In this work, we investigate the fundamental steps of ligand self-assembly as driven by changes in the interfacial H-bonding network using vibrational sum frequency generation. Our results show how the bulk pH modulates the interfacial structure of extractants at the buried oil/aqueous interface via the formation of unique H-bonding networks that order and bridge ligands to produce self-assembled aggregates. These extended H-bonded structures are key to the subsequent extraction of Co2+ from the aqueous phase in promoting micelle formation and subsequent ejection of said micelle into the oil phase. The combination of static and time resolved measurements reveals the mechanisms underlying complexities of liquid extractions at high [Co2+]:[ligand] ratios by showing an evolution of interfacially assembled structures that are readily tuned on a chemical basis by altering the compositions of the aqueous phase. The results of this work point to new mechanistic principles to design separations through the manipulation of surface charge, electrostatic screening, and the associated H-bonding networks that arise at the interface to facilitate organization and subsequent extraction.

Intra- and intermolecular self-assembly of a 20-nm-wide supramolecular hexagonal grid.

For the past three decades, the coordination-driven self-assembly of three-dimensional structures has undergone rapid progress; however, parallel efforts to create large discrete two-dimensional architectures-as opposed to polymers-have met with limited success. The synthesis of metallo-supramolecular systems with well-defined shapes and sizes in the range of 10-100 nm remains challenging. Here we report the construction of a series of giant supramolecular hexagonal grids, with diameters on the order of 20 nm and molecular weights greater than 65 kDa, through a combination of intra- and intermolecular metal-mediated self-assembly steps. The hexagonal intermediates and the resulting self-assembled grid architectures were imaged at submolecular resolution by scanning tunnelling microscopy. Characterization (including by scanning tunnelling spectroscopy) enabled the unambiguous atomic-scale determination of fourteen hexagonal grid isomers.

Symmetry-breaking transitions in the early steps of protein self-assembly.

Protein misfolding and subsequent self-association are complex, intertwined processes, resulting in development of a heterogeneous population of aggregates closely related to many chronic pathological conditions including Type 2 Diabetes Mellitus and Alzheimer's disease. To address this issue, here, we develop a theoretical model in the general framework of linear stability analysis. According to this model, self-assemblies of peptides with pronounced conformational flexibility may become, under particular conditions, unstable and spontaneously evolve toward an alternating array of partially ordered and disordered monomers. The predictions of the theory were verified by atomistic molecular dynamics (MD) simulations of islet amyloid polypeptide (IAPP) used as a paradigm of aggregation-prone polypeptides (proteins). Simulations of dimeric, tetrameric, and hexameric human-IAPP self-assemblies at physiological electrolyte concentration reveal an alternating distribution of the smallest domains (of the order of the peptide mean length) formed by partially ordered (mainly β-strands) and disordered (turns and coil) arrays. Periodicity disappears upon weakening of the inter-peptide binding, a result in line with the predictions of the theory. To further probe the general validity of our hypothesis, we extended the simulations to other peptides, the Aβ(1-40) amyloid peptide, and the ovine prion peptide as well as to other proteins (SOD1 dimer) that do not belong to the broad class of intrinsically disordered proteins. In all cases, the oligomeric aggregates show an alternate distribution of partially ordered and disordered monomers. We also carried out Surface Enhanced Raman Scattering (SERS) measurements of hIAPP as an experimental validation of both the theory and in silico simulations.

Control of Molecular Bonding Strength on Metal Catalysts with Organic Monolayers for CO2 Reduction.

The development of separate levers for controlling the bonding strength of different reactive species on catalyst surfaces is challenging but essential for the design of highly active and selective catalysts. For example, during CO2 reduction, production of CO often requires balancing a trade-off between the adsorption strength of the reactant and product states: weak binding of CO is desirable from a selectivity perspective, but weak binding of CO2 leads to low activity. Here, we demonstrate a new method of controlling both CO2 adsorption and CO desorption over supported metal catalysts by employing a single self-assembly step where organic monolayer films were deposited on the catalyst support. Binding of phosphonic acid monolayers on supported Pt and Pd catalysts weakened CO binding via a through-support effect. The weakened CO adsorption was generally accompanied by decreased adsorption and reactivity of CO2. However, by the incorporation of basic amine functions at controlled positions in the modifying film, strong CO2 adsorption and hydrogenation reactivity could be restored. Thus, both through-surface and through-space interactions could be manipulated by design of the organic modifiers. After surface modification, the catalysts exhibited significantly improved selectivity (up to ∼99% at conversions near 50%) and activity toward CO production. Moreover, the rate of deactivation was notably reduced due to prevention of CO poisoning.

Heterocomponent ternary supramolecular complexes of porphyrins: A review

Porphyrins are prominent host molecules which are widely used due to their structural characteristics and directional interaction sites. This review summarizes non-covalently bound ternary complexes of porphyrins, constructed from at least three non-identical species. Progress in supramolecular chemistry allows the creation of complex molecular machinery tools, such as rotors, motors and switches from relatively simple structures in a single self-assembly step. In the current review, we highlight the collection of sophisticated molecular ensembles including sandwich-type complexes, cages, capsules, tweezers, rotaxanes, and supramolecular architectures mediating oxygen-binding and oxidation reactions. These diverse structures have high potential to be applied in sensing, production of new smart materials as well as in medical science.

Modulating the Mechanical Properties of Supercrystalline Nanocomposite Materials via Solvent-Ligand Interactions.

Supercrystalline nanocomposite materials with micromechanical properties approaching those of nacre or similar structural biomaterials can be produced by self-assembly of organically modified nanoparticles and further strengthened by cross-linking. The strengthening of these nanocomposites is controlled via thermal treatment, which promotes the formation of covalent bonds between interdigitated ligands on the nanoparticle surface. In this work, it is shown how the extent of the mechanical properties enhancement can be controlled by the solvent used during the self-assembly step. We find that the resulting mechanical properties correlate with the Hansen solubility parameters of the solvents and ligands used for the supercrystal assembly: the hardness and elastic modulus decrease as the Hansen solubility parameter of the solvent approaches the Hansen solubility parameter of the ligands that stabilize the nanoparticles. Moreover, it is shown that self-assembled supercrystals that are subsequently uniaxially pressed can deform up to 6 %. The extent of this deformation is also closely related to the solvent used during the self-assembly step. These results indicate that the conformation and arrangement of the organic ligands on the nanoparticle surface not only control the self-assembly itself but also influence the mechanical properties of the resulting supercrystalline material. The Hansen solubility parameters may therefore serve as a tool to predict what solvents and ligands should be used to obtain supercrystalline materials with good mechanical properties.

α-Helical Motif as Inhibitors of Toxic Amyloid-β Oligomer Generation via Highly Specific Recognition of Amyloid Surface.

SummaryAmyloid fibril surfaces can convert soluble proteins into toxic oligomers and are attractive targets for intervention of protein aggregation diseases. Thus far, molecules identified with inhibitory activity are either large proteins or flat cyclic compounds lacking in specificity. The main design difficulty is flatness of amyloid surfaces and the lack of knowledge on binding interfaces. Here, we demonstrate, for the first time, a rational design of alpha-helical peptide inhibitors targeting the amyloid-beta 40 (Aβ40) fibril surfaces, based on our in silico finding that a helical fragment of Aβ40 interacts in a unique way with side-chain arrays on the fibril surface. We strengthen the fragment's binding capability through mutations and helicity enhancement with our Terminal Aspartic acid strategy. The resulting inhibitor shows micromolar affinity for the fibril surface, effectively impedes the surface-mediated oligomerization of Aβ40, and mitigates its cytotoxicity. This work opens up an avenue to designing aggregation modulators for amyloid diseases.

Deciphering Z-scheme Charge Transfer Dynamics in Heterostructure NiFe-LDH/N-rGO/g-C3N4 Nanocomposite for Photocatalytic Pollutant Removal and Water Splitting Reactions

A series of heterostructure NiFe LDH/N-rGO/g-C3N4 nanocomposite were fabricated by combining calcinations-electrostatic self-assembly and hydrothermal steps. In this method, negatively charged N-rGO was electrostaticaly bonded to the self-assembled interface of n-n type g-C3N4/NiFe LDH hybrid. XRD and AFM results revealed successful formation of heterostructure nanocomposite due to the coupling effect of exfoliated NiFe LDH nanosheets with N-rGO and g-C3N4. Among the as synthesized heterostructure, CNNG3LDH performed superior photocatalytic activities towards 95 and 72% mineralization of RhB and phenol. Furthermore, CNNG3LDH could achieve the highest photocatalytic H2 evolution rate of 2508 μmolg−12h−1 and O2 evolution rate of 1280 μmolg−12h−1 under visible light irradiation. The CNNG3LDH possess lowest PL intensity, reduced arc of the Nyquist plot (43.8 Ώ) and highest photocurrent density (−0.97 mA cm−2) which revealed effective charge separation for superior photocatalytic activities. TRPL spectral results reveal the synergistic effect of layered component in CNNG3LDH for achievable higher life time of excitons of ~16.52 ns. In addition, N-rGO mediator based Z-scheme charge transfer mechanisms in CNNG3LDH were verified by the ESR and TA-PL studies. Enriched oxygen vacancy type defects in NiFe LDH and N-rGO mediated Z-scheme charge transfer mechanistic path strongly manifest the superior photocatalytic activities of the heterostructure materials.

Visualizing the Initial Step of Self-Assembly and the Phase Transition by Stereogenic Amphiphiles with Aggregation-Induced Emission.

Many highly ordered structures with smart functions are generated by self-assembly with stimuli responsiveness. Despite that electron microscopes enable us to directly observe the end products, it is hard to visualize the initial step and the kinetic stimuli-responsive behavior of self-assembly. Here, we report the design and synthesis of stereogenic amphiphiles, namely, ( Z)-TPE-OEG and ( E)-TPE-OEG, with aggregation-induced emission (AIE) characteristics from the hydrophobic tetraphenylethene core and thermoresponsive behavior from the hydrophilic oligoethylene glycol monomethyl ether chain. The two isomers can be easily isolated by high-performance liquid chromatography and characterized by 2D NMR spectroscopy. While ( Z)-TPE-OEG self-assembles into vesicles, its ( E)-cousin forms micelles in water. The initial step of their self-assembly processes can be visualized based on AIE characteristics, with a sensitivity much higher than the method based on transmittance measurement. The entrapment and release capabilities of the ( Z)-stereogenic amphiphile are demonstrated by employing pyrene as a guest. The thermoresponsive behavior of the ( Z)-amphiphile results in its continuous phase transition from microscopic self-assembly to macroscopic aggregation, which is successfully visualized in situ by confocal laser scanning microscopy accompanied by the AIE technique. Such a kinetic process shows different stages according to the microscopic visualization, and these stages have never been monitored through roughly observing the appearance of precipitates. It is anticipated that this study can deepen the understanding of the self-assembly processes for better monitoring and controlling them in different systems.

Multivalent, multiflavored droplets by design

Nature self-assembles functional materials by programming flexible linear arrangements of molecules and then folding them to make 2D and 3D objects. To understand and emulate this process, we have made emulsion droplets with specific recognition and controlled valence. Uniquely monovalent droplets form dimers: divalent lead to polymer-like chains, trivalent allow for branching, and programmed mixtures of different valences enable a variety of designed architectures and the ability to subsequently close and open structures. Our functional building blocks are a hybrid of micrometer-scale emulsion droplets and nanoscale DNA origami technologies. Functional DNA origami rafts are first added to droplets and then herded into a patch using specifically designated "shepherding" rafts. Additional patches with the same or different specificities can be formed on the same droplet, programming multiflavored, multivalence droplets. The mobile patch can bind to a patch on another droplet containing complementary functional rafts, leading to primary structure formation. Further binding of nonneighbor droplets can produce secondary structures, a third step in hierarchical self-assembly. The use of mobile patches rather than uniform DNA coverage has the advantage of valence control at the expense of slow kinetics. Droplets with controlled flavors and valences enable a host of different material and device architectures.

Hydrogelation Kinetics Measured in a Microfluidic Device with in Situ X-ray and Fluorescence Detection.

Efficient hydrogelators will gel water fast and at low concentrations. Small molecule gelling agents that assemble into fibers and fiber networks are particularly effective hydrogelators. Whereas it is straightforward to determine their critical concentration for hydrogelation, the kinetics of hydrogelation is more difficult to study because it is often very fast, occurring on the subsecond time scale. We used a 3D focusing microfluidic device combined with fluorescence microscopy and in situ small-angle X-ray scattering (SAXS) to study the fast pH-induced gelation of a model small molecule gelling agent at the millisecond time scale. The gelator is a 1,3,5-benzene tricarboxamide which upon acidification assembles into nanofibrils and fibril networks that show a characteristic photoluminescence. By adjusting the flow rates, the regime of early nanofibril formation and gelation could be followed along the microfluidic reaction channel. The measured fluorescence intensity profiles were analyzed in terms of a diffusion-advection-reaction model to determine the association rate constant, which is in a typical range for the small molecule self-assembly. Using in situ SAXS, we could determine the dimensions of the fibers that were formed during the early self-assembly process. The detailed structure of the fibers was subsequently determined by cryotransmission electron microscopy. The study demonstrates that 3D focusing microfluidic devices are a powerful means to study the self-assembly on the millisecond time scale, which is applied to reveal early state of hydrogelation kinetics. In combination with in situ fluorescence and X-ray scattering, these experiments provide detailed insights into the first self-assembly steps and their reaction rates.