Microfluidic devices have been downscaled to dimensions of 10–1000 nm. Manipulation methods for femtoliter samples are important for realizing novel analytical devices. In the present study, we developed a microfluidic device that utilizes two-step flow focusing by air flows to generate femtoliter liquid droplets that float in the gas phase with size and trajectory control. The device includes a branched and stepped hydrophobic microchannel with four air inlets to exploit the instability of the gas–liquid interface. We succeeded in the generation of 704 fL (11.0 ± 0.01 μm) droplets of pure water and a shooting frequency of 24 kHz at a sample flow rate of 1 μL/min. In addition to pure water, we succeeded in generating acetonitrile droplets. The device operation was stable even at a sample flow rate of 101 pL/s, which is similar to that used in recent nanofluidic analytical devices. The results confirm that the instability of the gas–liquid interface is the dominant factor in femtoliter droplet generation as designed. Based on the experimental results, our method has a potential to generate droplets with the minimum volume of 123 fL (6.1 μm) in case of pure water, which can be achieved by minimizing the channel size. The present study provides a method based on gas–liquid micro/nanofluidics for the generation of uniform femtoliter droplets with trajectory control. This method is suitable for various applications, such as a size interface for the transport of femtoliter samples from nanofluidic devices to analytical instruments.
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