Passive Micropump for Highly Stable, Long-Termed, and Large Volume of Droplet Generation/Transport Inside 3D Microchannels Capable of Surfactant-Free and Droplet-Based Thermocycled Reverse Transcription-Polymerase Chain Reactions Based on a Single Thermostatic Heater.

It is still one key challenge for traditional passive micropumps (e.g., surface tension micropump, hydrostatic micropump, enzymatic micropump, degassed-polydimethylsiloxane (PDMS) micropump, etc.) to transport a large volume of two-phased fluid for a long period. Herein we propose a user-friendly and passive approach to realize the microdroplet generation by waiving expensive or complex equipment. The automation principle is systemically studied in this paper. It is affirmed that this micropump can continuously transport over 2000 μL of two-phased aqueous/oil microdroplets over a 4 m long 3D microchannel for 8 h. In addition, variations in flow rate are little within each hour-period, and the evaporation bubbles can be well suppressed under high temperature (95 °C). As a proof of this concept, the novel micropump is applied to droplet-based continuous flow real-time polymerase chain reactions (PCRs), which only require several disposable syringes for oil/aqueous-phase storage, two 34 gauge needles for droplet generation, a Teflon tube for PCR amplification, and a single thermostatic heater for the thermal cycle. The results suggest this droplet generation method is acceptable for a house-made setup of microfluidic PCRs. Besides, the amplification efficiency of the droplet-based microcontinuous flow PCRs here is much higher than the plug-based microcontinuous flow PCRs in our previous work and reaches 91% of the commercial qPCR thermocycler for the target gene of Rubella virus (Rubv). Without expensive microfabrication instruments, this novel method is more accessible to nonprofessionals than previous reports and would extend the droplet-based applications to in-field and real-time analysis.