A Novel Multi-Layer Microfluidic Pipette Aspiration Device for Studying Mechanosensitive Vesicles

Abstract

Mechanosensitive channel of large conductance (MscL) is a prokaryotic channel that opens due to increased membrane tension. It has been reconstituted in vesicles to study how various biophysical factors influence their mechanical gating properties. In recent years, several theoretical and computational models based on molecular dynamics and continuum mechanics have been developed to understand the underlying mechanisms for the activation of MscLs in vesicles. Experimental approaches for MscL gating studies have focused on cell-by-cell techniques, such as pressure patch clamp, on reconstituted MscL vesicles. Due to the limitation of throughput, such studies are time intensive with low sample numbers. Recently, a microfluidic pipette aspiration array device, based on PDMS multi-layer soft-lithography technique, has been developed in our group to trap and apply mechanical perturbation to single cells in a parallel manner1. This device was used to study the stiffness of human breast cancer cell lines and mechanical gating threshold of reconstituted MscL on infected mammalian cell lines. In this work, we have developed a new device that incorporates a pressure valve and a smaller micropipette dimension to increase trapping efficiency of vesicles. Using this device, we demonstrate stable trapping of single vesicles and expand our efforts to study mechanical gating threshold of reconstituted MscL in vesicles formed by electroformation. Development of novel microtechnology tools that can trap single vesicles and exert tension will have numerous applications to the study of other mechanosensitive channels.[1] Lee & Liu, Lab Chip, 2015, 15, 264-273.