Electrochemical cell lysis of Gram-positive and -negative bacteria for DNA extraction

Abstract

DNA extn. is an essential step for the nucleic acid-based microbial monitoring method. Chem. lysis is currently the most common technique for DNA extn. However, this method need time-consuming reagent adding process and residue removal step in the end to avoid the interference with the downstream detection. Elec. lysis based on irreversible electroporation has been reported for successful cell lysis with fast speed in several studies. But it requires a high voltage to achieve the crit. transmembrane potential and the joule heat due to the high voltage also affects the working fluid. In this study, a rapid, cost-effective and low power DNA extn. method without reagent adding process was developed based on electrochem. lysis. In the electrochem. reaction, hydroxide ions generated locally at cathode can break the fatty acid-glycerol ester bonds of phospholipid mols. within cell membrane and thereby release the intracellular materials. A cation exchange membrane was added between anodic and cathodic chambers for a fast accumulation of hydroxide ions. Fluorescent microscope and real-time PCR were used to measure the cell lysis efficiency and DNA extn. efficiency, resp. Both Gram-pos. bacteria (Enterococcus faecalis and Bacillus subtilis) and Gram-neg. bacteria (Escherichia coli, Salmonella typhi and Enterobacter) were successfully lysed within 1 min using 40 mA of DC (5 V). The DNA extn. efficiencies of Gram-neg. bacteria were over 80% and of Gram-pos. bacteria were 20%. A 3D-printed portable electrochem. cell lysis device was also developed for on-site field study and potentially could be a component for an integrated pathogen detection platform.