Droplets are generated by merging oil with an aqueous stream. During the hybrid Flow-Based/DMF manipulation processes, first sets of droplets with samples (e.g., cells) are sequentially immobilized at the trapping sites by the flow through the bypass channels. This sequential trapping are realized by blocking the bypass channel by trapped droplets to force the following droplets to the adjacent chamber, which is visualized in the result from numerical simulations. Next, immobilized droplets are transported to the storage with the DMF transportation. Second set of droplets (e.g., reagents) can be arrayed at the empty trapping chambers and merged with the first set of droplets. Merged droplets are released into the main flow after incubation. We experimentally verified our system with prototyped device with 150 µm channel height and 300 µm square chambers and electrodes. Droplets with fluorescein were formed in fluorinated oil and successfully demonstrated trapping, transporting, and merging processes at the hybrid DMF part.

An Automated Flow-Based/Digital Microfluidic Platform with On-Chip Optical Detection System for High-Throughput Gene Editing Processes

Abstract In recent years, synthetic biology has drawn significant interest for both scientific research and industrial applications such as biofuel and pharmaceutical production. Synthetic biology process requires multiple molecular biology steps making it a very time-consuming and labor-intensive effort. Using droplets as reaction chambers can be a powerful approach to improve the process with benefits including faster reactions because of small dimensions, small volume of reagent consumption required (to bring down the cost), and better control of experimental environment. We are developing a hybrid platform of flow-based droplet microfluidics and digital microfluidics (DMF), integrated with on-chip optical detection. The system can handle large numbers of droplets at once as well as actively manipulate target droplets in a programmable manner. We use electric-field driven manipulation of droplets to merge and sort droplets. In addition, we integrate optical fibers in the microchannels to add on-chip capability for fluorescence-based detection of encapsulated cells and enzymatic activities in discrete droplets, and for triggering sorting of droplets. Unlike conventional microtiter plate based reactions, the analog-digital optofluidic device allows completely automated genetic engineering steps using drastically smaller amounts of reagents and can be useful for application requiring high throughput screening and reactions.

Kosuke Iwai, Philip C. Gach, Peter W. Kim, Manasi Raje, Joshua V. Heinemann, Todd A. Duncombe, Kai Deng, Trent R. Northen, Nathan J. Hillson, Paul D. Adams, and Anup K. Singh Technology Division Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA

Acknowledgments The authors greatly appreciate the help and support from Biomolecular Nanotechnology Center (BNC) and Marvell Nanofabrication Laboratory (Nanolab). An OAI Model 200 mask aligner was utilized for the device fabrication processes and the travel cost was partially supported by OAI Travel Grant Program. This work conducted by the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

References 1. S.C.C. Sigh, et al., “A Versatile Microfluidic Device for Automating Synthetic Biology,” ACS Synth. Biol., 2015, 4, 1151-1164. 2. S.C.C. Shih, et al., “A Droplet-to-Digital (D2D) Microfluidic Device for Single Cell Assays,” Lab Chip, 2015, 15, 225-236. 3. P.C. Gach, et al., “A Droplet Microfluidic Platform for Automating Genetic Engineering,” ACS Synth. Biol., 2016, 5, 426-433. 4. J. Heinemann, K. Deng, S.C.C. Shih, J. Gao, P.D. Adams, A.K. Singh and T.R. Northen, “On-Chip Integration of Droplet Microfluidics and Nanostructure-Initiator Mass Spectrometry for Enzyme Screening,” Lab Chip, 2017, 17, 323-331. 5. A.R. Apel, et al., “A Cas9-Based Toolkit to Program Gene Expression in Saccharomyces Cerevisiae,” Nucleic Acids Res., 2017, 45, 496-508.

Conceptual illustration of the digital microfluidic system, consisting of hybrid flow-based/droplet generator, multi-functional hybrid DMF and integrated detector/sorter.

Demonstrations of multi-functional hybrid DMF for (a) trapping, (b) transporting/storing, and (c) merging of droplets. Scale bars = 300 µm.

Fiber-Based Optical Detection and Sorting of Droplets we optically detected the droplets with 488nm laser excitation and an avalanche photodiode detector at the optical detector/sorter, and initiated DMF captured the fluorescent droplets.

CRISPR-Cas9 Gene Expression of Saccharomyces Cerevisiae

(a) On-chip electroporation of yeast cells in droplets, and transformed yeast with GFP (b) in droplets and (c) on dish. Scale bars = 300 µm.

(a) (b) (c)

(a) DMF sorter integrated with optical fiber. Optical detection with optical fiber for (b) counting droplets and (c) detecting GFP. (d) DMF capturing of fluorescent droplets. Scale bars = 300 µm.

Hybrid Flow-Based/Digital Microfluidic Manipulation of Large Set of Droplets

Device Structure and Fabrication

(a) Numerical Simulation (b) Sequential Trapping and Arraying Procedure (c) DMF Manipulation for Merging and Releasing

(a) Prototype with Optical Fiber

(d) DMF Capturing of Droplets

(b) Droplet Count

(c) GFP Detection

Manipulation procedure of hybrid flow-based/digital microfluidic part to trap, array, transport, merge and release of a set of droplets.