We present a programmable microfluidic static droplet array (SDA) device that can perform user-defined multistep combinatorial protocols. It combines the passive storage of aqueous droplets without any external control with integrated microvalves for discrete sample dispensing and dispersion-free unit operation. The addressable picoliter-volume reaction is systematically achieved by consecutively merging programmable sequences of reagent droplets. The SDA device is remarkably reusable and able to perform identical enzyme kinetic experiments at least 30 times via automated cross-contamination-free removal of droplets from individual hydrodynamic traps. Taking all these features together, this programmable and reusable universal SDA device will be a general microfluidic platform that can be reprogrammed for multiple applications.

Quorum sensing (QS) is a type of cell-cell communication using signal molecules that are released and detected by cells, which respond to changes in their population density. A few studies explain that QS may operate in a density-dependent manner; however, due to experimental challenges, this fundamental hypothesis has never been investigated. Here, we present a microfluidic static droplet array (SDA) that combines a droplet generator with hydrodynamic traps to independently generate a bacterial population gradient into a parallel series of droplets under complete chemical and physical isolation. The SDA independently manipulates both a chemical concentration gradient and a bacterial population density. In addition, the bacterial population gradient in the SDA can be tuned by a simple change in the number of sample plug loading. Finally, the method allows the direct analysis of complicated biological events in an addressable droplet to enable the characterization of bacterial communication in response to the ratio of two microbial populations, including two genetically engineered QS circuits, such as the signal sender for acyl-homoserine lactone (AHL) production and the signal receiver bacteria for green fluorescent protein (GFP) expression induced by AHL. For the first time, we found that the population ratio of the signal sender and receiver indicates a significant and potentially interesting partnership between microbial communities. Therefore, we envision that this simple SDA could be a useful platform in various research fields, including analytical chemistry, combinatorial chemistry, synthetic biology, microbiology, and molecular biology.

Economic production of chemicals from microbes necessitates development of high-producing strains and an efficient screening technology is crucial to maximize the effect of the most popular strain improvement method, the combinatorial approach. However, high-throughput screening has been limited for assessment of diverse intracellular metabolites at the single-cell level. Herein, we established a screening platform that couples a microfluidic static droplet array (SDA) and an artificial riboswitch to analyse intracellular metabolite concentration from single microbial cells. Using this system, we entrapped single Escherichia coli cells in SDA to measure intracellular l-tryptophan concentrations. It was validated that intracellular l-tryptophan concentration can be evaluated by the fluorescence from the riboswitch. Moreover, high-producing strains were successfully screened from a mutagenized library, exhibiting up to 145% productivity compared to its parental strain. This platform will be widely applicable to strain improvement for diverse metabolites by developing new artificial riboswitches.

The synthesis of organic-inorganic hybrid particles with highly controlled particle sizes in the micrometer range is a major challenge in many areas of research. Conventional methods are limited for nanometer-scale fabrication because of the difficulty in controlling the size. In this study, we present a microfluidic method for the preparation of organic-inorganic hybrid microparticles with poly (1,10-decanediol dimethacrylate-co-trimethoxysillyl propyl methacrylate) (P (DDMA-co-TPM)) as the core and silica nanoparticles as the shell. In this approach, the droplet-based microfluidic method combined with in situ photopolymerization produces highly monodisperse organic microparticles of P (DDMA-co-TPM) in a simple manner, and the silica nanoparticles gradually grow on the surface of the microparticles prepared via hydrolysis and condensation of tetraethoxysilane (TEOS) in a basic ammonium hydroxide medium without additional surface treatment. This approach leads to a reduction in the number of processes and allows drastically improved size uniformity compared to conventional methods. The morphology, composition, and structure of the hybrid microparticles are analyzed by SEM, TEM, FT-IR, EDS, and XPS, respectively. The results indicate the inorganic shell of the hybrid particles consists of SiO2 nanoparticles of approximately 60 nm. Finally, we experimentally describe the formation mechanism of a silica-coating layer on the organic surface of polymeric core particles.