A droplet-to-digital (D2D) microfluidic device for single cell assays

Abstract

We have developed a new hybrid droplet-to-digital microfluidic platform (D2D) that integrates droplet-in-channel microfluidics with digital microfluidics (DMF) for performing multi-step assays. This D2D platform combines the strengths of the two formats-droplets-in-channel for facile generation of droplets containing single cells, and DMF for on-demand manipulation of droplets including control of different droplet volumes (pL-μL), creation of a dilution series of ionic liquid (IL), and parallel single cell culturing and analysis for IL toxicity screening. This D2D device also allows for automated analysis that includes a feedback-controlled system for merging and splitting of droplets to add reagents, an integrated Peltier element for parallel cell culture at optimum temperature, and an impedance sensing mechanism to control the flow rate for droplet generation and preventing droplet evaporation. Droplet-in-channel is well-suited for encapsulation of single cells as it allows the careful manipulation of flow rates of aqueous phase containing cells and oil to optimize encapsulation. Once single cell containing droplets are generated, they are transferred to a DMF chip via a capillary where they are merged with droplets containing IL and cultured at 30 °C. The DMF chip, in addition to permitting cell culture and reagent (ionic liquid/salt) addition, also allows recovery of individual droplets for off-chip analysis such as further culturing and measurement of ethanol production. The D2D chip was used to evaluate the effect of IL/salt type (four types: NaOAc, NaCl, [C2mim] [OAc], [C2mim] [Cl]) and concentration (four concentrations: 0, 37.5, 75, 150 mM) on the growth kinetics and ethanol production of yeast and as expected, increasing IL concentration led to lower biomass and ethanol production. Specifically, [C2mim] [OAc] had inhibitory effects on yeast growth at concentrations 75 and 150 mM and significantly reduced their ethanol production compared to cells grown in other ILs/salts. The growth curve trends obtained by D2D matched conventional yeast culturing in microtiter wells, validating the D2D platform. We believe that our approach represents a generic platform for multi-step biochemical assays such as drug screening, digital PCR, enzyme assays, immunoassays and cell-based assays.

A Droplet Microfluidic Platform for Automating Genetic Engineering

“…A custom C++ program was written to control an Arduino board which provides electrode actuation employed for all droplet manipulations including droplet generation, merging, and movement. 19 Three Peltier modules attached to the bottom of the digital microfluidic chip provide three controllable thermal zones. The chip was designed such that the three temperature zones had minimal overlap and stable and discrete temperature regions could be maintained on the device for extended periods as confirmed by thermal imaging.…”

“…Two strategies were employed for reagent delivery to the reservoirs: pipetting prior to assembly or constant fluid addition during operation, as described previously. 19 Reagent storage on device consists of a reservoir, which supports up to 3 μL volume at a time. From these reservoirs, individual 235 ± 11 nL droplets were generated by actuation of the electrode at a rate up to 1 droplet/s.…”

“…For experiments requiring more reagents, the ITO glass slide was first bound to the device then ends of capillary tubes connected to a neMESYS syringe pump (Cetoni GmbH, Korbussen, Germany) were glued into channels associated with the device reservoirs using 5 min Epoxy Gel (Devcon, Danvers, MA). This configuration uses a neMESYS syringe pump to continuously inject reagents directly into the reservoirs through a capillary imbedded in a channel on the device (similar to what is described in Shih et al 19 ). Following assembly of either technique, a capillary was inserted into a fifth channel at the entrance of the culture chamber from which HFE 7500 with 0.5% v/v Picosurf (Dolomite Microfluidics, Charlestown, MA) was flowed continuously to remove droplets from the reaction chamber and replenish evaporated oil.…”

“…A customized C++ program controlled the driving potentials of the Arduino board to allow automation of droplet movement in the device. 19 Droplets were manipulated on the digital microfluidic device by applying a 100 V rms driving potential in the form of an 8 kHz sine wave to the designated actuation electrodes as described previously. 19 Microfluidic Transformation.…”

“…19 Droplets were manipulated on the digital microfluidic device by applying a 100 V rms driving potential in the form of an 8 kHz sine wave to the designated actuation electrodes as described previously. 19 Microfluidic Transformation. Information regarding the preparation of cells and plasmids are available in the Supporting Information.…”