Electrochemical cell lysis device for DNA extraction.

We present a novel electrochemical cell lysis device to prepare DNA samples for lab-on-a-chip (LOC) applications. It utilizes the electrolysis of saline solution to generate hydroxide ions (OH(-)) at the cathode as alkaline lytic agents. Cathode and anode chambers are separated by a negatively-charged ion exchangeable polymer diaphragm to maintain the high pH level for efficient cell lysis in the cathode chamber, to prevent inflow of PCR-amplification inhibitors from the anode chamber, and to minimize binding of DNA molecules. Electric current flow and pH maintenance, which depended on the device design, were two important parameters of the device performance. After optimizing the design and visually confirming cell lysis of Chinese hamster ovary (CHO) cells in a very short amount of time, we directly electrolyzed four bacterial cell types suspended in saline solution. Real-time PCR (qPCR) analysis showed that our device could lyse both gram-positive and gram-negative bacterial cells with higher efficiency than other common methods and could detect DNA on the microlitre scale. Our data demonstrate several advantages of the proposed device: absence of cell lysis chemicals and heating; no adverse effects on PCR amplification; low DNA loss; low voltage and power consumption; and rapid processing. The device could potentially be applied as an on-chip DNA extraction component.

[1]  A. Giuffrida,et al.  Method for the lysis of Gram-positive, asporogenous bacteria with lysozyme , 1980, Applied and environmental microbiology.

[2]  Bo Huang,et al.  Counting Low-Copy Number Proteins in a Single Cell , 2007, Science.

[3]  Culp,et al.  Handbook of public water systems , 1986 .

[4]  G. Węgrzyn,et al.  Studies on recovery plasmid DNA from Echerichia coli by heat treatment , 2002 .

[5]  Mark Bachman,et al.  Fast electrical lysis of cells for capillary electrophoresis. , 2003, Analytical chemistry.

[6]  Y. Arata,et al.  The Efficiency of Disinfection of Acidic Electrolyzed Water in the Presence of Organic Materials , 2000 .

[7]  T. Park,et al.  Microfluidic cell disruption system employing a magnetically actuated diaphragm , 2008, Electrophoresis.

[8]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.

[9]  J. K. Hurst,et al.  Biological reactivity of hypochlorous acid: implications for microbicidal mechanisms of leukocyte myeloperoxidase. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Paul Yager,et al.  Diffusion-based analysis of molecular interactions in microfluidic devices , 2004, Proceedings of the IEEE.

[11]  Richard Smith,et al.  /spl beta/-galactosidase assays of single-cell lysates on a microchip: a complementary method for enzymatic analysis of single cells , 2004, Proceedings of the IEEE.

[12]  Marc Madou,et al.  MEMS-based sample preparation for molecular diagnostics , 2002, Analytical and bioanalytical chemistry.

[13]  G. Whitesides The 'right' size in nanobiotechnology , 2003, Nature Biotechnology.

[14]  Zhao-Lun Fang,et al.  Integration of single cell injection, cell lysis, separation and detection of intracellular constituents on a microfluidic chip. , 2004, Lab on a chip.

[15]  K. Woo,et al.  New hydrolysis method for extremely small amount of lipids and capillary gas chromatographic analysis as n(O)-tert.-butyldimethylsilyl fatty acid derivatives compared with methyl ester derivatives. , 1999, Journal of chromatography. A.

[16]  P. Höllsberg,et al.  A Sensitive Quantification of HHV-6B by Real-time PCR , 2002, Biological Procedures Online.

[17]  T. Kaneko,et al.  Electrically triggered insertion of single-stranded DNA into single-walled carbon nanotubes , 2006 .

[18]  Nigel Beard,et al.  Dealing with real samples: sample pre-treatment in microfluidic systems. , 2003, Lab on a chip.

[19]  Paul Yager,et al.  Cell lysis and protein extraction in a microfluidic device with detection by a fluorogenic enzyme assay. , 2002, Analytical chemistry.

[20]  Manuel Miró,et al.  Miniaturization of environmental chemical assays in flowing systems: the lab-on-a-valve approach vis-à-vis lab-on-a-chip microfluidic devices. , 2007, Analytica chimica acta.

[21]  Paul C. H. Li,et al.  Transport, manipulation, and reaction of biological cells on-chip using electrokinetic effects. , 1997, Analytical chemistry.

[22]  Brian N. Johnson,et al.  An integrated nanoliter DNA analysis device. , 1998, Science.

[23]  M. McClain,et al.  Microfluidic devices for the high-throughput chemical analysis of cells. , 2003, Analytical chemistry.

[24]  Luke P. Lee,et al.  Integrated microfluidic cell culture and lysis on a chip. , 2007, Lab on a chip.

[25]  Y. Tai,et al.  A micro cell lysis device , 1999 .

[26]  Dino Di Carlo,et al.  On-chip cell lysis by local hydroxide generation. , 2005, Lab on a chip.

[27]  K. Davies,et al.  The inhibition of bacterial growth by hypochlorous acid. Possible role in the bactericidal activity of phagocytes. , 1988, The Biochemical journal.

[28]  R. Levin,et al.  Effect of lysing methods and their variables on the yield of Escherichia coli O157: H7 DNA and its PCR amplification , 1998 .

[29]  Peter R. C. Gascoyne,et al.  Dielectrophoresis-based sample handling in general-purpose programmable diagnostic instruments , 2004, Proceedings of the IEEE.

[30]  K. Voelkerding,et al.  A single-tube nucleic acid extraction, amplification, and detection method using aluminum oxide. , 2006, The Journal of molecular diagnostics : JMD.

[31]  Stephen R Quake,et al.  Solving the "world-to-chip" interface problem with a microfluidic matrix. , 2003, Analytical chemistry.

[32]  P. Yager,et al.  Microfluidic Diffusion-Based Separation and Detection , 1999, Science.

[33]  Jae-Chan Park,et al.  Bacterial DNA sample preparation from whole blood using surface-modified Si pillar arrays. , 2008, Analytical chemistry.

[34]  M. Lappalainen,et al.  Real‐time PCR for detection and quantitation of hepatitis B virus DNA , 2001, Journal of medical virology.

[35]  Michael G. Roper,et al.  A fully integrated microfluidic genetic analysis system with sample-in–answer-out capability , 2006, Proceedings of the National Academy of Sciences.

[36]  James P Landers,et al.  A microchip-based proteolytic digestion system driven by electroosmotic pumping. , 2003, Lab on a chip.

[37]  Yau-Huei Wei,et al.  Detection of DNA mutations associated with mitochondrial diseases by Agilent 2100 bioanalyzer. , 2002, Clinica chimica acta; international journal of clinical chemistry.

[38]  Axel Günther,et al.  Cell stimulus and lysis in a microfluidic device with segmented gas-liquid flow. , 2005, Analytical chemistry.

[39]  Jong Rak Choi,et al.  Clinical evaluation of micro-scale chip-based PCR system for rapid detection of hepatitis B virus. , 2006, Biosensors & bioelectronics.

[40]  N J Titchener-Hooker,et al.  Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations , 2004, Biotechnology and bioengineering.

[41]  Jan Lichtenberg,et al.  Sample pretreatment on microfabricated devices. , 2002, Talanta.