Performing Isothermal Molecular Amplification in Nanocavities with Tunable Confinement*

Nanofluidic devices featuring real time-confinement control enable capture, isolation and biochemical processing of single analytes in nanoscale cavities for highly parallel and individualized single-particle/single-molecule analysis.  This technology may find a range of applications ranging from analysis of single extracellular vesicles to digital nucleic acid tests. Here, using a device approach based on pneumatic actuation of a thin membrane lid to seal an embedded nanocavity in a nanoslit, we demonstrate key steps necessary for single cavity molecular amplification.  These steps include cavity loading by target nucleic acids, exposure of the loaded cavity to required enzymes/co-factors and then implementation of an isothermal amplification reaction via Loop-mediated Isothermal AMPlification (LAMP) and rolling circle (RCA) approaches.