(65h) A Microfluidic Device for Electrophoretic Trapping and Irreversible Electroporation of Bacterial Cells

Cell lysis or cellular disruption is a process by which cellular membrane integrity is disrupted by inducing small pores in order to release the internal contents of the cell such as DNA, RNA, and proteins. Various techniques that can be categorized as mechanical, chemical, thermal and electrical have been used for lysing cells [1]. In the electrical method, cells are lysed by exposure to an external electric field. The advantage of this method over others is that it allows lysis without the introduction of any chemical and biological reagents and also allows rapid recovery of inter-cellular organelles. When an electric field is applied, the cell membrane can act as a capacitor and a potential difference is established between intercellular and extracellular regions. This difference is known as transmembrane potential (TMP) [2] and for a spherical cell can be determined by-

TMP:  

Where: a= radius of cell, E= external electric field, t_m= charging time of membrane.

When this potential is about 1V, the cell membrane becomes permeable as small pores are created on its surface. This process is called electroporation [2]. Depending on the field strength and duration of the field, these pores might be transient or permanent. Permanent pores can be created by increasing the intensity and exposure to the electric field. This process is known as irreversible electroporation and can be used for cell lysis. Depending on the size of the cells, electric field strength of 1000V/cm to 2000 V/cm is required to lyse bacterial cells [3].

Such high electric fields can be obtained either by applying a high potential or by reducing the gap between the electrodes through microfabrication [4]. However, microfabrication of the electrodes is expensive. In this paper, we demonstrate a method to obtain localized high electric field without the use of microfabricated electrodes. We also demonstrate eletrophoretic trapping and accumulation of cells at the localized region and subsequent lysis of those cells. The device consists of a nanoporous polycarbonate membrane sandwiched between two microchannels with electrodes embedded at the reservoirs of the microchannels. Since the resistances of the nanopores are significantly larger than that of the microchannels and the thickness of membrane is very small, any potential applied at the electrodes generates a localized high electric field. For instance, this device can generate approximately 1.8 KV into the intersection of two channels using an operating voltage of 300V. We demonstrate that there is a threshold electric field below which the device operates predominantly as an accumulator of cells suspended in the microchannel and above which it starts to lyse the accumulated cells.  For instance, application of 50 V (electric field 308 V/cm) to the device with a flow rate in the channel of 100uL/hr leads to rapid accumulation of cells on top of the membrane.  We use GFP expressed E. coli as well as normal E. coli with propidium iodide dye to visualize the accumulation and lysis. When GFP expressed E. coli is used, the fluorescence burst is observed when the electric field is higher than a threshold value confirming lysis. Lysis or electroporation creates pores in the cells allowing transfer of propidium iodide from the outside into the cell which then attaches to the DNA and makes the cell red. Subsequent to the accumulation when normal E. coli with propidium iodide dye is used, application of 300 V (electric field 1850 V/cm) leads to the change in color to red indicating that the cells are lysed. Cell lysis can be completed within 2 to 5 min. Apart from rapid accumulation and low voltage lysis of cells another advantage of this device may be handling of intercellular contents. Since the pore size of the polycarbonate membrane used is 400nm, intercellular organelles are retained while DNA can easily pass through them and be collected downstream. This device is useful for sample pretreatment in a micro total analysis system. As it can easily lyse bacteria, lysis of mammalian cells as well as reversible electroporation for transfection of molecules into the cell are possible by using this device.

References:

1.   Ying Huang, Elizabeth L. Mather, Janice L. Bell, Marc Madou, MEMS-based Sample preparation for Molecular diagnostics, Anal Bioanal Chem, 2002, 372: 49-65.

2.   Neumann, E., Sowers, A.E.,C.A.E., Electroporation and Electrofusion in Cell Biology, 1999.

3. Hsiang-Yu Wang, Arun K. Bhunia, Chang Lu, A microfluidic flow-through device for high throughput electrical lysis of bacterial cells based on continuous dc voltage, BIOSENSORS & BIOELECTRONICS, 2006.

4.    Hang L U, Martin A. Schmidt and Klavs F. Jensen, A microfluidc electroporation device for cell lysis, Lab on a Chip, 2004.