Sample Preparation on-chip: Accumulation, Lysis of and DNA Extraction from Bacteria

摘要

We present a microfluidic chip that collects and accumulates bacteria from a sample by positive dielectrophoresis and subsequently lyses cells to retrieve DNA / RNA. Biological samples containing Escherichia coli are transferred to a suitable medium (low conductivity) by on-chip dialysis. This sample is pumped through a microchannel comprising an array of interdigitated electrodes at the bottom. An AC-voltage is applied to the electrodes resulting in an inhomogeneous electric field that extends into the channel and induces dielectropho-retic forces acting on the bacteria. Herringbone- mixer structures between the electrodes induce vortices in the flow and thus rotate the fluid volume from top to bottom. This assures that all bacteria are brought under the influence of the electric field and thus may be trapped. In addition, the herringbone structures induce further inhomogeneities in the electric field. Due to this combined effect, the system allows for the trapping of bacteria even at high flow rates which results in high throughput. Bacteria from several mL of sample are concentrated into 20 μL inside the chamber. This is monitored on-line by measuring the impedance between the electrodes.In a second step the flow is stopped and the frequency of the electric field is lowered. This induces lysis of the bacteria as a result of a high voltage drop over their cell membrane. The lysis of E. coli is monitored using a live-dead staining kit that changes the fluorescence color of bacteria.The bacteria release DNA which is collected and analyzed further using real time PCR. PCR of the lysate retrieved from the chip showed a signal about 12 PCR cycles earlier than in case of the original E. coli suspension. This confirms the accumulation and lysis of bacteria in the chip, the release of DNA and indicates an approximately 4000-fold increase in DNA concentration.