Performing a Digital Nucleic Acid Test in a Nanofluidic Device with Pneumatic Confinement Control

Nano/microfluidic-based nucleic acid tests have been proposed as a rapid and reliable diagnostic technology. The two key steps for many of these tests are target nucleic acid (NA) surface immobilization followed by an enzymatic reaction on the captured NAs to detect the presence of a disease associated sequence. NA capture within a geometrically confined volume is an attractive alternative to NA surface immobilization that eliminates the need for sample pre-treatment (e.g. label-based methods such as lateral flow assays) or use of external actuators (e.g dielectrophoresis) that are required for most nano/microfluidic-based NA tests. However, geometrically confined spaces hinder sample loading while making it challenging to capture, subsequently retain and simultaneously expose target NAs to required enzymes. Here, using a nanofluidic device that features real-time confinement control via pneumatic actuation of a thin membrane lid, we demonstrate loading of digital nanocavities by target NAs and exposure of target NAs to required enzymes/co-factors while the NAs are retained. In particular, as proof of principle, we amplified single-stranded DNAs (M13mp18 plasmid vector) in an array of nanocavities via two isothermal amplification approaches (loop-mediated isothermal amplification and rolling cyclic amplification).