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Issue 13, 2015
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Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA

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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.

Graphical abstract: Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA

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Publication details

The article was received on 11 Mar 2015, accepted on 21 Apr 2015 and first published on 23 Apr 2015


Article type: Paper
DOI: 10.1039/C5LC00291E
Citation: Lab Chip, 2015,15, 2759-2766
  • Open access: Creative Commons BY license
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    Centrifugal step emulsification applied for absolute quantification of nucleic acids by digital droplet RPA

    F. Schuler, F. Schwemmer, M. Trotter, S. Wadle, R. Zengerle, F. von Stetten and N. Paust, Lab Chip, 2015, 15, 2759
    DOI: 10.1039/C5LC00291E

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