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

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 × 10(6) 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.