Nucleic acid purification using microfabricated silicon structures.
A microfluidic device has been designed, fabricated and tested for its ability to purify bacteriophage lambda DNA and bacterial chromosomal DNA, a necessary prerequisite for its incorporation into a biosensor. This device consists of a microfabricated channel in which silica-coated pillars were etched to increase the surface area within the channel by 300-600%, when the etch depth is varied from 20 to 50 microm. DNA was selectively bound to these pillars in the presence of the chaotropic salt guanidinium isothiocyanate, followed by washing with ethanol and elution with low-ionic strength buffer. Positive pressure was used to move solutions through the device, removing the need for centrifugation steps. The binding capacity for DNA in the device was approximately 82 ng/cm2 and on average, 10% of the bound DNA could be purified and recovered in the first 50 microl of elution buffer. Additionally, the device removed approximately 87% of the protein from a cell lysate. Nucleic acids recovered from the device were efficiently amplified by the polymerase chain reaction suggesting the utility of these components in an integrated, DNA amplification-based biosensor. The miniaturized format of this purification device, along with its excellent purification characteristics make it an ideal component for nucleic acid-based biosensors, especially those in which nucleic acid amplification is a critical step.
Cady
Nathaniel C
NC
Department of Microbiology, Cornell University, 317 Stocking Hall, Ithaca, NY 14853, USA. ncc4@cornell.edu
Stelick
Scott
S
Batt
Carl A
CA
D000327:Adsorption; D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis / Q000737:chemistry; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006146:Guanidines / Q000737:chemistry; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods; D008904:Miniaturization; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012825:Silicon / Q000737:chemistry*; D013861:Thiocyanates / Q000737:chemistry
D020099:Coated Materials, Biocompatible; D004269:DNA, Bacterial; D006146:Guanidines; D013861:Thiocyanates; C054436:guanidine thiocyanate; D004247:DNA; D012825:Silicon