Double-Sided Microwells with a Stepped Through-Hole Membrane for High-Throughput Microbial Assays

Abstract

To improve the throughput of microwell arrays for identifying immense cellular diversities even at a single-bacteria level, further miniaturization or densification of the microwells has been an obvious breakthrough. However, controlling millions of nanoliter samples or more at the microscale remains technologically difficult and has been spatially restricted to a single open side of the microwells. Here we employed a stepped through-hole membrane to utilize the bottom as well as top side of a high-density nanoliter microwell array, thus improving spatial efficiency. The stepped structure shows additional effectiveness for handling several millions of nanoliter bacterial samples in the overall perspectives of controllability, throughput, simplicity, versatility, and automation by using novel methods for three representative procedures in bacterial assays: partitioning cells, manipulating the chemical environment, and extracting selected cells. As a potential application, we show proof-of-concept isolation of rare cells in a mixed ratio of 1 to around 106 using a single chip. Our device can be further applied to various biological studies pertaining to synthetic biology, drug screening, mutagenesis, and single-cell heterogeneity.

Fabricating and Laminating Films with Through‐Holes and Engraved/Protruding Structures for 3D Micro/Nanofluidic Platforms

“…Reproduced with permission. [ 33 ] Copyright 2020, American Chemical Society. The scale bar is 100 µm.…”

“…To construct 3D multiscale micro/nanofluidic channel networks comprising mill-scale inlet/outlet ports and micro/nanoscale channels together, we laminated the prepared Reproduced with permission. [33] Copyright 2020, American Chemical Society. The scale bar is 100 μm.…”

“…The membrane structures were useful for biological applications such as organoid studies and functional microwells. [32,33] Given that the master mold can be prepared using standard photolithography, patterning of structures and their alignment could be achieved at a resolution of ≈2 μm.…”

“…The scale bar is 100 μm. C) shows stepped TIFs with two-layered through-holes.Reproduced with permission [33]. Copyright 2020, American Chemical Society.…”