Microfluidic Aqueous Two-Phase Focusing of Chemical Species for In Situ Subcellular Stimulation

Confinement of chemical species in a controllable micrometer-level (several to a dozen micrometers) space in an aqueous environment is essential for precisely manipulating chemical events in subcellular regions. However, rapid diffusion and hard-to-control micrometer-level fluids make it a tough challenge. Here, a versatile open microfluidic method based on an aqueous two-phase system (ATPS) is developed to restrict species inside an open space with micron-level width. Unequal standard chemical potentials of the chemical species in two phases and space-time correspondence in the microfluidic system prevent outward diffusion across the phase interface, retaining the target species inside its preferred phase flow and creating a sharp boundary with a dramatic concentration change. Then, the chemical flow (the preferred phase with target chemical species) is precisely manipulated by a microfluidic probe, which can be compressed to a micron-level width and aimed at an arbitrary position of the sample. As a demonstration of the feasibility and versatility of the strategy, chemical flow is successfully applied to subcellular regions of various kinds of living single cells. Subcellular regions are successfully labeled (cytomembrane and mitochondria) and damaged. Healing-regeneration behaviors of living single cells are triggered by subcellular damage and analyzed. The method is relatively general regarding the species of chemicals and biosamples, which could promote deeper cell research.