800 Views
High-Definition Biotechnology
Podium
Chueh-Yu Wu, PhD
Postdoc Researcher
University of California, Los Angeles
The capability to partition a fluid sample into nanoliter-scale volumes of uniform size plays an important role to achieve uniform reactions enabling massive and high-throughput single-cell and single-molecule analysis, and leading to ultimate limits in nucleic acid sequencing, drug discovery, and personalized medicine. Microfluidics-based droplet technology has enabled a number of applications, including digital ELISA/PCR and InDrops/Drop-seq techniques for single-cell RNA sequencing. However, microfluidics requires expensive liquid handling equipment and Poisson encapsulation statistics limit the efficiency of approaches making use of encapsulated solid-phases, limiting adoption by non-centralized clinical labs and the efficiency of data acquisition in research settings respectively. We demonstrate an economic microfluidic-device-free droplet generation technology with high monodispersity using 3D-shaped amphiphilic particles that promote uniform aqueous drop formation through simple manual mixing and centrifuging steps. We engineered microscale particles, called drop-carrier particles, to be constructed with a void, hydrophilic inner layer, and hydrophobic shell, which thermodynamically stabilize a specific aqueous fluid volume to associate with each particle. The particles were manufactured using a modified high throughput optical Transient Liquid Molding (OTLM) setup, which photo-crosslinks microparticles by illuminating patterned UV light onto a pre-deformed flow stream of polymer precursor. We designed a microchannel to deform a co-flow of poly(ethylene glycol) diacrylate and hydrophobic external pre-cursor to a pattern with concentric layers and illuminated an array of UV patterns, simple rectangular slits here, onto the flow stream to reach a high production rate, up to ~36,000 particles/hour. We resuspended purified drop-carrier particles in oil, mix it with aqueous solutions, and centrifuged to generate particle-drops with identical size and shape across the population. We discovered that different from droplets stabilized by surfactants, the size and shape of the particle-drops can be determined by the 3D structure and interfacial tensions of the drop-carrier particles. The controlled shape was found to be non-spherical with circularity of ~0.4 and the size was narrowly distributed with a diameter of ~260 µm, allowing efficient image processing with a cut-off of shape or size. Importantly, the drops were observed to remain stable over more than 4 days without significant coalescence and size/shape change. Moreover, we found that the transportation of fluorescent molecules between the particle-drops can be permitted, slowed down, or prohibited depending on the surfactant used and molecular weight of the target molecule. Last but not least, we also demonstrated proof of concept of applications using particle-drops: solid-phase reactions and microgel encapsulation. The uniform reaction conditions in each particle-drop across the population was shown by incubating the particles with inner biotinylation and associated drops with fluorescent-labeled streptavidin while single microgels with various diameters ranging from 50 to 160 µm was shown to be successfully encapsulated in one particle-drop following a single Poisson distribution.