Activity monitoring of functional OprM using a biomimetic microfluidic device

Analyst. 2012 Feb 21;137(4):847-52. doi: 10.1039/c2an16007b. Epub 2012 Jan 4.

Abstract

This paper describes the fabrication and use of a biomimetic microfluidic device for the monitoring of a functional porin reconstituted within a miniaturized suspended artificial bilayer lipid membrane (BLM). Such a microfluidic device allows for (1) fluidic and electrical access to both sides of the BLM and (2) reproducible membrane protein insertion and long-term electrical monitoring of its conductance (G(i)), thanks to the miniaturization of the BLM. We demonstrate here for the first time the feasibility to insert a large trans-membrane protein through its β-barrel, and monitor its functional activity for more than 1 hour (limited by buffer evaporation). In this paper, we specifically used our device for the monitoring of OprM, a bacterial efflux channel involved in the multidrug resistance of the bacteria Pseudomonas aeruginosa. Sub-steps of the OprM channel conductance were detected during the electrical recordings within our device, which might be due to oscillations between several structural conformations (sub-states) adopted by the protein, as part of its opening mechanism. This work is a first step towards the establishment of a genuine platform dedicated to the investigation of bacterial proteins under reconstituted conditions, a very promising tool for the screening of new inhibitors against bacterial channels involved in drug resistance.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Outer Membrane Proteins / metabolism*
  • Biological Transport
  • Electric Conductivity
  • Lipid Bilayers*
  • Membrane Transport Proteins / metabolism*
  • Microfluidic Analytical Techniques*
  • Pseudomonas aeruginosa / metabolism*

Substances

  • Bacterial Outer Membrane Proteins
  • Lipid Bilayers
  • Membrane Transport Proteins
  • OprM protein, Pseudomonas aeruginosa