In Situ Photophysical Characterization of π-Conjugated Oligopeptides Assembled via Continuous Flow Processing.

Bioinspired materials have been developed with the aim of harnessing natural self-assembly for precisely engineered functionality. Microfluidics is poised to play a key role in the directed assembly of advanced materials with ordered nano and mesoscale features. More importantly, there is a strong need for understanding the kinetics of continuous assembly processes. In this work, we describe a continuous microfluidic system for the assembly and alignment of synthetic oligopeptides with π-conjugated cores using a three-dimensional (3D) flow focusing of inlet reactant streams. This system facilitates in situ confocal fluorescence microscopy and in situ fluorescence lifetime imaging microscopy (FLIM), which can be used in unprecedented capacity to characterize the integrity of peptides during the assembly process. To achieve continuous assembly, we integrate chevron patterns in the ceiling and floor of the microdevice to generate a 3D-focused sheath flow of the reactant peptide. Consequently, the peptide stream is directed toward an acidic triggering stream in a cross-slot geometry which mediates assembly into higher-order fiber-like structures. Using this approach, the focused peptide stream is assembled using a planar extensional flow, which ensures high degrees of microstructural alignment within the assembled material. We demonstrate the efficacy of this approach using three different synthetic oligopeptides, and in all cases, we observe the efficient and continuous assembly of oligopeptides. In addition, finite element simulations are used to guide device design and to validate 3D focusing. Overall, this approach presents an efficient and effective method for the continuous assembly and alignment of ordered materials using microfluidics.

Evidence for Prenucleated Fibrilogenesis of Acid-Mediated Self-Assembling Oligopeptides via Molecular Simulation and Fluorescence Correlation Spectroscopy.

An important step in controlling biomimetic amyloid systems is understanding the self-assembly reaction kinetics. We are interested in a family of such materials characterized by symmetric sequences of amino acids flanking a π-conjugated functional core. Many of these materials rapidly self-assemble into long fibers upon protonation in an acidic environment. Despite extensive investigation of these materials' properties, little is yet understood regarding their reaction kinetics. Based on previous studies, we have chosen DFAG-4T-GAFD as a representative system and conducted molecular dynamics simulations to show that although large-scale assembly is induced by lowering pH, some degree of assembly is thermodynamically favorable in high-pH nonprotonating environments. These results are consistent with findings for other systems such as DFAG-OPV-GAFD. The nonprotonated aggregation also appears to be concentration dependent, occurring at concentrations of 100 nM and above. Single molecule measurements using fluorescence correlation spectroscopy provide experimental support for these computational predictions. We find evidence of spontaneous aggregation in aqueous solutions of peptides with concentrations as low as 100 nM; however, 10 nM solutions appear to be largely homogeneous solutions of unassembled monomer. These results indicate that the simplest explanations for kinetics of acid-mediated assembly-protonation-induced nucleation by monomeric addition followed by subsequent stages of aggregation and elongation-are inappropriate in this system. In fact, the system only exists as pure monomer in very low concentrations, nucleation actually occurs in the absence of protonating elements at concentrations typically used for experiments, and pH triggered assembly proceeds from these preassembled aggregates. Accordingly, triggered assembly must be considered to operate outside the domain of nucleation-dependent models.

Macroscopic Alignment and Assembly of π-Conjugated Oligopeptides Using Colloidal Microchannels.

One-dimensional (1-D) supramolecular self-assembly offers a powerful strategy to achieve long-range unidirectional ordering of organic semiconducting materials via noncovalent interactions. Using a hierarchical assembly, electronic and optoelectronic materials can be constructed for applications including organic conducting nanowires, organic field-effect transistors (OFETs), and organic light-emitting devices (OLEDs). Despite recent progress, it remains challenging to precisely align and assemble 1-D structures over large areas in a rapid and straightforward manner. In this work, we demonstrate a facile strategy to macroscopically align supramolecular fibers using a templating method based on sacrificial colloidal microchannels. Through use of this approach, colloidal microchannels are generated on a solid surface using a simple fabrication method, followed by the spontaneous self-assembly of π-conjugated oligopeptides inside large arrays of microchannels triggered by solvent evaporation. Following oligopeptide assembly and removal of sacrificial microchannels, the structural properties of oligopeptide fibers were characterized using atomic force microscopy (AFM), atomic force microscope-infrared spectroscopy (AFM-IR), photoinduced force microscopy (PiFM), fluorescence polarization microscopy, and electron microscopy. These results reveal the macroscopic alignment of oligopeptide fibers into ordered structures over millimeter length scales, facilitated by colloidal microchannel templating. In addition, the charge transport properties (I-V curves) of π-conjugated oligopeptides assembled using this method were determined under a wide range of applied voltages using interdigitated array electrodes and conductive AFM. Overall, this work illustrates a simple yet robust strategy to pattern 1-D supramolecular fibers over large areas, thereby offering new routes for assembling materials for organic electronics.

Nonequilibrium Self-Assembly of π-Conjugated Oligopeptides in Solution.

Supramolecular assembly is a powerful method that can be used to generate materials with well-defined structures across multiple length scales. Supramolecular assemblies consisting of biopolymer-synthetic polymer subunits are specifically known to exhibit exceptional structural and functional diversity as well as programmable control of noncovalent interactions through hydrogen bonding in biopolymer subunits. Despite recent progress, there is a need to control and quantitatively understand assembly under nonequilibrium conditions. In this work, we study the nonequilibrium self-assembly of π-conjugated synthetic oligopeptides using a combination of experiments and analytical modeling. By isolating an aqueous peptide solution droplet within an immiscible organic layer, the rate of peptide assembly in the aqueous solution can be controlled by tuning the transport rate of acid that is used to trigger assembly. Using this approach, peptides are guided to assemble under reaction-dominated and diffusion-dominated conditions, with results showing a transition from a diffusion-limited reaction front to spatially homogeneous assembly as the transport rate of acid decreases. Interestingly, our results show that the morphology of self-assembled peptide fibers is controlled by the assembly kinetics such that increasingly homogeneous structures of self-assembled synthetic oligopeptides were generally obtained using slower rates of assembly. We further developed an analytical reaction-diffusion model to describe oligopeptide assembly, and experimental results are compared to the reaction-diffusion model across a range of parameters. Overall, this work highlights the importance of molecular self-assembly under nonequilibrium conditions, specifically showing that oligopeptide assembly is governed by a delicate balance between reaction kinetics and transport processes.