Integrating polymerase chain reaction, valving, and electrophoresis in a plastic device for bacterial detection.
An integrated plastic microfluidic device was designed and fabricated for bacterial detection and identification. The device, made from poly(cyclic olefin) with integrated graphite ink electrodes and photopatterned gel domains, accomplishes DNA amplification, microfluidic valving, sample injection, on-column labeling, and separation. Polymerase chain reaction (PCR) is conducted in a channel reactor containing a volume as small as 29 nL; thermal cycling utilizes screen-printed graphite ink resistors. In situ gel polymerization was employed to form local microfluidic valves that minimize convective flow of the PCR mixture into other regions. After PCR, amplicons (products) are electrokinetically injected through the gel valve, followed by on-chip electrophoretic separation. An intercalating dye is admixed to label the amplicons; they are detected using laser-induced fluorescence. Two model bacteria, Escherichia coli O157 and Salmonella typhimurium, were chosen to demonstrate bacterial detection and identification based on amplification of several of their unique DNA sequences. The limit of detection is about six copies of target DNA.
Koh
Chee G
CG
ACLARA BioSciences, Inc., 1288 Pear Avenue, Mountain View, California 94043, USA.
Tan
Woei
W
Zhao
Ming-qi
MQ
Ricco
Antonio J
AJ
Fan
Z Hugh
ZH
D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis; D004586:Electrophoresis / Q000295:instrumentation* / Q000379:methods*; D019453:Escherichia coli O157 / Q000235:genetics / Q000302:isolation & purification; D005453:Fluorescence; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods; D010969:Plastics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012486:Salmonella typhimurium / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature; D013997:Time Factors
D004269:DNA, Bacterial; D010969:Plastics