Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA.

Friedrich Schuler,Simon Wadle,Roland Zengerle,Martin Trotter,Frank Schwemmer,Nils Paust,Felix Von Stetten

doi:10.1039/c5lc00291e

Abstract

Aqueous microdroplets provide miniaturized reaction compartments for numerous chemical, biochemical or pharmaceutical applications. We introduce centrifugal step emulsification for the fast and easy production of monodisperse droplets. Homogenous droplets with pre-selectable diameters in a range from 120 μm to 170 μm were generated with coefficients of variation of 2-4% and zero run-in time or dead volume. The droplet diameter depends on the nozzle geometry (depth, width, and step size) and interfacial tensions only. Droplet size is demonstrated to be independent of the dispersed phase flow rate between 0.01 and 1 μl s(-1), proving the robustness of the centrifugal approach. Centrifugal step emulsification can easily be combined with existing centrifugal microfluidic unit operations, is compatible to scalable manufacturing technologies such as thermoforming or injection moulding and enables fast emulsification (>500 droplets per second and nozzle) with minimal handling effort (2-3 pipetting steps). The centrifugal microfluidic droplet generation was used to perform the first digital droplet recombinase polymerase amplification (ddRPA). It was used for absolute quantification of Listeria monocytogenes DNA concentration standards with a total analysis time below 30 min. Compared to digital droplet polymerase chain reaction (ddPCR), with processing times of about 2 hours, the overall processing time of digital analysis was reduced by more than a factor of 4.

Highlights

One of the most important applications of aqueous droplets is the use in digital amplification techniques such as digital PCR.[1]
We present centrifugal step emulsification featuring dead volume free production of monodisperse droplets combined with adjustable and high droplet generation frequencies and robust and easy handling
The diameter of the produced droplets had a coefficient of variation (CV) of 2–4%, independent of the diameters generated by the different channel depths

Summary

Introduction

One of the most important applications of aqueous droplets is the use in digital amplification techniques such as digital PCR (dPCR).[1]. The design, production and operation of step emulsification systems are simple and robust since the size of only one microchannel and the flow of only one liquid need to be controlled[26,27] as opposed to T-junction and co-flow systems.[28] For a wide range of flow rates, the diameter of droplets is independent of pressure and flow rate. We present centrifugal step emulsification featuring dead volume free production of monodisperse droplets combined with adjustable and high droplet generation frequencies and robust and easy handling.

Results

Conclusion