Microfluidic Devices for Imaging and Biochemistry Analysis of Microbes Under Mechanical Pressure

Abstract

Growth-induced pressure arises when proliferating cell populations are confined within rigid microenvironments and is increasingly recognized as a determinant of microbial physiology in soils, biofilms, and host tissues. We present two complementary microfluidic devices that confine microbes within polydimethylsiloxane (PDMS) chambers and apply defined, optically read-out growth-induced pressure of up to 1.5 MPa. The first, the self-closing (SC) chip, confines cells in multiple small chambers accommodating hundreds of cells; it provides excellent nutrient supply and rapid medium exchange, supports single-cell imaging, and allows multiplexing of cellular or chemical conditions. The second, the pressure-recovery (PR) chip, confines cells in a single 5 cm- long chamber accommodating hundreds of thousands of cells; a scalpel-cut step recovers live cells from the channel within minutes for bulk biochemical assays. The two devices share a single two-layer soft-lithography fabrication process and a common brightfield wall-displacement pressure readout. Together, they enable single-cell imaging and bulk biochemical analysis under matched, defined pressure conditions. We illustrate the protocol with two representative validations: rapid β-estradiol-induced transcription in the SC chip, in which nascent transcription foci appear within 5 min independently of the applied pressure, and the PR chip, which produces uniform growth-induced pressure along the entire 5 cm channel and recovers between 0.1 × 106 and 1 × 106 cells per device in a pressure-tunable manner. SUMMARY This protocol describes two complementary polydimethylsiloxane (PDMS) microfluidic devices that apply defined growth-induced pressure to microbial populations while maintaining uniform nutrient supply. The self-closing chip supports single-cell imaging under multiplexed pressure conditions; the pressure-recovery chip yields up to approximately 1 × 106 cells per device for downstream biochemical analysis.