Bo Li

Redox-active colloids (RACs) represent a novel class of energy carriers that exchange electrical energy upon contact. Understanding contact-mediated electron transfer dynamics in RACs offers insights into physical contact events in colloidal suspensions and enables quantification of electrical energy transport in nonconjugated polymers. Redox-based electron transport was directly observed in monolayers of micron-sized RACs containing ethyl-viologen side groups via fluorescence microscopy through an unexpected nonlinear electrofluorochromism that is quantitatively coupled to the redox state of the colloid. Via imaging studies, using this electrofluorochromism, the apparent charge transfer diffusion coefficient D _CT of the RAC was easily determined. The visualization of energy transport within suspensions of redox-active colloids was also demonstrated. Our work elucidates fundamental mechanisms of energy transport in colloidal systems, informs the development of next-generation redox flow batteries, and may inspire new designs of smart active soft matter including conductive polymers for applications ranging from electrochemical sensors and organic electronics to colloidal robotics.

Biomolecular condensates are dynamic cellular compartments that concentrate proteins and enzymes to regulate biochemical reactions in time and space. While these condensates can enhance enzyme activity, how this function changes as condensates age remains poorly understood. Here, we design synthetic catalytic condensates that selectively recruit enzymes to investigate this temporal evolution. We show that catalytic condensates exhibit time-dependent activity: they initially accelerate enzymatic reactions but gradually lose efficiency due to the transition from liquid-like to solid-like states. This aging process, characterized by protein aggregation and loss of selective barriers, impairs enzyme function both in vitro and living cells. We further demonstrate that small molecules which influence aging dynamics can modulate catalytic efficiency of condensates. Our findings show that condensate aging as a key regulator of enzymatic activity and provide crucial insights for designing functional synthetic condensates.

Self-assembled protein cages are attractive scaffolds for organizing various proteins of interest (POIs) toward applications in synthetic biology and medical science. However, specifically attaching multiple POIs to a single protein cage remains challenging, resulting in diversity among the functionalized particles. Here, we present the engineering of a self-assembled protein cage, _DTMi3_ST, capable of independently recruiting two different POIs using SpyCatcher (SC)/SpyTag (ST) and DogCatcher (DC)/DogTag (DT) chemistries, thereby reducing variability between assemblies. Using fluorescent proteins as models, we demonstrate controlled targeting of two different POIs onto _DTMi3_ST protein cages both in vitro and inside living cells. Furthermore, dual functionalization of the _DTMi3_ST protein cage with a membrane-targeting peptide and β-galactosidase resulted in the construction of membrane-bound enzyme assemblies in Escherichia coli, leading to a 69.6% enhancement in substrate utilization across the membrane. This versatile protein cage platform provides dual functional nanotools for biological and biomedical applications.

Naturally evolved metabolons have the ability to assemble and disassemble in response to environmental stimuli, allowing for the rapid reorganization of chemical reactions in living cells to meet changing cellular needs. However, replicating such capability in synthetic metabolons remains a challenge due to our limited understanding of the mechanisms by which the assembly and disassembly of such naturally occurring multienzyme complexes are controlled. Here, we report the synthesis of chemical- and light-responsive protein cages for assembling synthetic metabolons, enabling the dynamic regulation of enzymatic reactions in living cells. Particularly, a chemically responsive domain was fused to a self-assembled protein cage subunit, generating engineered protein cages capable of displaying proteins containing cognate interaction domains on their surfaces in response to small molecular cues. Chemical-induced colocalization of sequential enzymes on protein cages enhances the specificity of the branched deoxyviolacein biosynthetic reactions by 2.6-fold. Further, by replacing the chemical-inducible domain with a light-inducible dimerization domain, we created an optogenetic protein cage capable of reversibly recruiting and releasing targeted proteins onto and from the exterior of the protein cages in tens of seconds by on-off of blue light. Tethering the optogenetic protein cages to membranes enables the formation of light-switchable, membrane-bound metabolons, which can repeatably recruit-release enzymes, leading to the manipulation of substrate utilization across membranes on demand. Our work demonstrates a powerful and versatile strategy for constructing dynamic metabolons in engineered living cells for efficient and controllable biocatalysis.

In order to address the challenges presented by future electronics with complex functionality and architecture, new materials and fabrication methods must be developed. In contrast to traditional top-down approaches, bottom-up self-assembly has emerged as a robust means of organizing nanoscopic building blocks into highly ordered structures and materials. In particular, topographical micropatterns are typically fabricated using complicated lithographic methods that are expensive and time-consuming and not suitable for high throughput manufacturing. Colloidal cracks, also known as colloidal microchannels, have been identified as a simple and robust strategy for the fabrication of scalable microchannels. Despite extensive theoretical modeling to understand the mechanism of crack formation, the influence of the “coffee-ring” effect on crack formation remains largely unexplored. Herein, we explore how the “coffee-ring” effect determines the colloidal microchannel formation by leveraging the kinetic control of the non-equilibrium deposition process of PS latex nanoparticles. Surprisingly, by tuning the particle size, we found the “coffeering” effect plays a dual role in determining the colloidal microchannel formation. In addition, temperature-concentration phase diagrams of colloidal microchannel formation are also provided in this work. Overall, the study presented offers crucial insights into controlling the self-assembly of colloidal particles to form microchannels, which is essential for the low-cost and large-scale fabrication of topographical templates. This study has important applications in microfluidics, geometry confinement for functional materials deposition, and alignment.

Crafting integrated biomolecular circuits (IbC) via self-assembly of electronically active molecules using a “bottom-up” paradigm provides a potential solution to the technological challenges of the miniaturization of silicon-based electronics.