Rapid and automated preparation of PCR (polymerase chain reaction)-ready genomic DNA was demonstrated on a
multiplexed CD (compact disk) platform by using hard-to-lyse bacterial spores. Cell disruption is carried out while beadcell
suspensions are pushed back and forth in center-tapered lysing chambers by angular oscillation of the disk -
keystone effect. During this lysis period, the cell suspensions are securely held within the lysing chambers by heatactivated
wax valves. Upon application of a remote heat to the disk in motion, the wax valves release lysate solutions
into centrifuge chambers where cell debris are separated by an elevated rotation of the disk. Only debris-free DNA
extract is then transferred to collection chambers by capillary-assisted siphon and collected for heating that inactivates
PCR inhibitors. Lysing capacity was evaluated using a real-time PCR assay to monitor the efficiency of <i>Bacillus globigii </i>
spore lysis. PCR analysis showed that 5 minutes' CD lysis run gave spore lysis efficiency similar to that obtained with a
popular commercial DNA extraction kit (i.e., IDI-lysis kit from GeneOhm Sciences Inc.) which is highly efficient for
microbial cell and spore lysis. This work will contribute to the development of an integrated CD-based assay for rapid
diagnosis of infectious diseases.
A DNA hybridization and detection unit was developed for a compact disc (CD) platform. The compact disc was used as the fluidic platform for sample and reagent manipulation using centrifugal force. Chambers for reagent storage and conduits for fluidic functions were replicated from polydimethylsiloxane (PDMS) using an SU-8 master mold fabricated with a 2-level lithography process we developed specially for the microfluidic structures used in this work. For capture probes, we used self-assembled DNA oligonucleotide monolayers (SAMs) on gold pads patterned on glass slides. The PDMS flow cells were aligned with and sealed against glass slides to form the DNA hybridization detection units. Both an enzymatic-labeled fluorescence technique and a bioluminescent approach were used for hybridization detection. An analytical model was introduced to quantitatively predict the accumulation of hybridized targets. The flow-through hybridization units were tested using DNA samples (25-mers) of different concentrations down to 1 pM and passive assays (no flow), using samples of the same concentrations, were performed as controls. At low concentrations, with the same hybridization time, a significantly higher relative fluorescence intensity was observed in both enzymatic and bioluminescent flow-through assays compared to the corresponding passive hybridization assays. Besides the fast hybridization rate, the CD-based method has the potential for enabling highly automated, multiple and self-contained assays for DNA detection.
Reagentless mechanical cell lysis was demonstrated on a microfluidic CD (Compact Disc) microfabricated in PDMS (Polydimethylsiloxane). The motion of beads in a lysis chamber on the CD causes disruption of mammalian (CHO-K1), bacterial (<i>Escherichia coli</i>), and yeast (<i>Saccharomyces cerevisiae</i>) cells. Interactions between beads and cells are generated in the rimming flow established inside a partially filled annular chamber in the CD rotating around a horizontal axis. To maximize bead-cell interactions, the CD was spun forward and backwards around this axis, using high acceleration for up to 7 minutes. Based on our theoretical work, we investigated the following control parameters: bead density, angular velocity, acceleration rate, and solid volume fraction, all of which influence cell lysis efficiency. Cell disruption efficiency was verified either through direct microscopic viewing or measurement of DNA concentration after cell lysing. Lysis efficiency relative to a conventional lysis protocol was also determined. In the long term, this work is geared towards CD based sample-to-answer nucleic acid analysis which will include cell lysis, DNA purification, DNA amplification, and DNA hybridization detection.