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Ultrarapid Generation of Femtoliter Microfluidic Droplets for Single-Molecule-Counting Immunoassays

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posted on 2016-02-19, 02:59 authored by Jung-uk Shim, Rohan T. Ranasinghe, Clive A. Smith, Shehu M. Ibrahim, Florian Hollfelder, Wilhelm T. S. Huck, David Klenerman, Chris Abell
We report a microfluidic droplet-based approach enabling the measurement of chemical reactions of individual enzyme molecules and its application to a single-molecule-counting immunoassay. A microfluidic device is used to generate and manipulate <10 fL droplets at rates of up to 1.3 × 106 per second, about 2 orders of magnitude faster than has previously been reported. The femtodroplets produced with this device can be used to encapsulate single biomolecular complexes tagged with a reporter enzyme; their small volume enables the fluorescent product of a single enzyme molecule to be detected within 10 min of on-chip incubation. Our prototype system is validated by detection of a biomarker for prostate cancer in buffer, down to a concentration of 46 fM. This work demonstrates a highly flexible and sensitive diagnostic platform that exploits extremely high-speed generation of monodisperse femtoliter droplets for the counting of individual analyte molecules.

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