Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells

Abstract

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.

Keywords: cell printing; droplet array; droplet microfluidics; fluorescence-activated droplet sorting; single-cell analysis.

Fig. 1.

The printer consists of a sorter-based print head that selectively dispenses microfluidic droplets to a substrate under a cover of oil. The printing process is automated by coupling the print head to a motorized stage. Deterministic combinations of cells and reagents can be formed at each location through additive droplet printing.

Fig. S1.

Components of the PMD system. (A) Droplets flow out of the nozzle in carrier oil to nanowell features on the substrate, where they are immobilized. (B) The print head consists of a dielectrophoretic droplet sorter fitted with fiber optics. Four-color excitation and detection sort droplets of interest to the print nozzle, whereas all other droplets flow into the waste stream. Droplet printing can use on-chip drop making or the reinjection of preformed emulsions. (C) The printing substrate consists of a nanowell array fabricated on top of a grid of dielectrophoretic traps. Typical nanowells in this work have a diameter of 150 µm and a depth of 75 µm and are fabricated on a 400-µm pitch. (D) Wells are photo-patterned from SU8 on top of a laser etched ITO-coated glass slide.

Fig. S2.

Fundamental droplet printer operations performed on a flat substrate. (A) Print. Deposition of a single droplet at an array position, after which the printer rasters to the next position. (B) Add. Successive printing and merging of three consecutive droplets to the same position. (C) Recover. Removal of a single droplet from the substrate via suction.

Fig. 2.

Fluorescent dye printing. (A) Flow cytometry data for print head using a dual drop maker to produce emulsions on chip. (B) A 100-position array printed with four droplets of FITC at each position from the emulsion shown in A. (C) A 100-position array with alternating positions printed with four droplets of either FITC or TRITC. (D) Flow cytometry data from a reinjected emulsion containing four dyed droplet types [empty, FITC, TRITC, and Cy5 (Alexa 647)] (E) A 100-position array printed with alternating positions containing four droplets of FITC, TRITC, and Cy5. (F) Position well gradient array printed with eight droplets containing combinations of FITC, Cy5, and empty droplets. (G) Construction of a binary image by printing FITC droplets to a 10,000-position array.

Fig. S3.

High-resolution image of the printed 10,000-position array shown in Fig. 2G.

Fig. 3.

Cell printing. (A) Flow cytometry data for an emulsion formed from a mix of calcein green- and calcein red-stained PC3 cells. (B) A 25-position array where every position is printed with alternating green- and red-stained single cells. (C) A 25-position array where every well is printed with both a single green- and red-stained cell. (D) A 25-position array printed with one to five green-stained cells on the vertical axis and one to five red-stained cells on the horizontal axis.

Fig. S4.

High-resolution image of single-cell gradient array in Fig. 3D.

Fig. 4.

Intracellular calcium release assay. (A) Assay results with and without the addition of 200 mM KCl to induce Ca²⁺ release. (B) Box plots of bulk cell assay data. Boxes represent first–third quartiles, and whiskers give 5–95% values. (C) Assembled GFP channel imaging data from 50 of 100 array positions from single-cell experiments. (D) Box plots of single-cell assay data. Boxes represent first–third quartiles, and whiskers give 5–95% values.