Use of surface enhanced blocking (SEB) electrodes for microbial cell lysis in flow-through devices.

Abdossamad Talebpour,Tino Alavie,Aye Aye Khine,Robert Maaskant,Lyle L Moldawer

doi:10.1371/journal.pone.0102707

Abstract

By simultaneously subjecting microbial cells to high amplitude pulsed electric fields and flash heating of the cell suspension fluid, effective release of intracellular contents was achieved. The synergistic effect of the applied electric field and elevated temperature on cell lysis in a flow-through device was demonstrated for Gram-negative and Gram-positive bacteria, and Mycobacterium species. The resulting lysate is suitable for downstream nucleic acid amplification and detection without requiring further preparation. The lysis chamber employs surface enhanced blocking electrodes which possess an etched micro-structured surface and a thin layer of dielectric metal oxide which provides a large effective area and blocks transmission of electrical current. The surface enhanced blocking electrodes enable simultaneous suppression of the rapid onset of electric field screening in the bulk of the cell suspension medium and avoidance of undesired electrochemical processes at the electrode-electrolyte interface. In addition the blocking layer ensures the robustness of the cell lysis device in applications involving prolonged flow-through processing of the microbial cells.

Highlights

The unprecedented advances in detecting and identifying microorganisms using nucleic acids have not been adequately matched with corresponding progress in pre-analytical sample preparation techniques required for efficiently and rapidly providing an inhibitor and contamination free nucleic acid suspension
Owing to the disadvantages of electrolysis from unprotected electrodes in microfluidic cell lysis devices, blocking electrodes are preferred for electrical lysis of microbial cells
This effect is largely eliminated by employing surface enhanced blocking electrodes with a finely micro-structured surface and a thin dielectric coating which increases the screening time to levels expected from bare electrodes

Summary

Introduction

The unprecedented advances in detecting and identifying microorganisms using nucleic acids have not been adequately matched with corresponding progress in pre-analytical sample preparation techniques required for efficiently and rapidly providing an inhibitor and contamination free nucleic acid suspension. Seemingly inspired by the extensive studies on microorganism inactivation by pulsed electric fields (PEF) [1], irreversible electroporation was suggested as a convenient method of microbial cell lysis for molecular assay chips [2,3]. This approach, involving the formation of electrically induced nanoscale pores in the cell membrane, has been successfully demonstrated for the lysis of mammalian cells and a range of microbial species [4,5,6]. The use of indirect metrics for inferring microbial cell lysis can lead to misinterpretation of the effect of the electric fields on the microbial cells because, even though a microbial cell may be rendered inactive by the application of an electric field, it may not be sufficiently lysed to achieve release of nucleic acids

Methods

Results

Conclusion