We describe a microfluidic genetic analysis system that represents a previously undescribed integrated microfluidic device capable of accepting whole blood as a crude biological sample with the endpoint generation of a genetic profile. Upon loading the sample, the glass microfluidic genetic analysis system device carries out on-chip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with sample-inanswer-out results in <30 min. A single syringe pump delivers sample/reagents to the chip for nucleic acid purification from a biological sample. Elastomeric membrane valving isolates each distinct functional region of the device and, together with resistive flow, directs purified DNA and PCR reagents from the extraction domain into a 550-nl chamber for rapid target sequence PCR amplification. Repeated pressure-based injections of nanoliter aliquots of amplicon (along with the DNA sizing standard) allow electrophoretic separation and detection to provide DNA fragment size information. The presence of Bacillus anthracis (anthrax) in 750 nl of whole blood from living asymptomatic infected mice and of Bordetella pertussis in 1 l of nasal aspirate from a patient suspected of having whooping cough are confirmed by the resultant genetic profile.full integration ͉ micro total analysis system ͉ microdevice ͉ pumping ͉ valving T he next revolution in personalized medicine, forensic science, and biowarfare defense will be impelled by analysis systems that provide a quantum leap in terms of functionality, time to result, and cost effectiveness. These systems need to meet several requirements, including a design conducive with low-cost manufacturing, turn-key operation with fast analysis times, and the ability to manipulate small volumes from crude samples. One example is the micrototal analysis system (-TAS) described conceptually more than a decade ago by Manz et al. (1). Prophetically, they stated that, ''. . . the detector or sensor in a TAS does not need high selectivity, because the sample pretreatment serves to eliminate most of the interfering chemical compounds.'' There are multiple examples in the literature of steps taken toward the advancement of integrated microfluidic genetic analysis (MGA) systems (refs. 2-4; also see ref. 5 for a comprehensive review); however, after a decade and a half, no bona fide microfluidic device has been presented that is capable of nanoliter flow control and integration of an electrophoretic separation with comprehensive sample pretreatment (DNA purification and PCR amplification).The MGA system described in this report brings together many advances in microfluidics over the last decade, exploiting differential channel flow resistances (6), elastomeric valves (7, 8), laminar flow (9), and electrophoretic mobility within the device, in concert with external fluid flow control from a syringe pump for sample and reagent delivery. Nucleic acid purification through solid-phase e...
Poly(dimethylsiloxane) (PDMS) membrane valves were utilized for diaphragm pumping on a PDMS-glass hybrid microdevice in order to couple infrared-mediated DNA amplification with electrophoretic separation of the products in a single device. Specific amplification products created during non-contact, infrared (IR) mediated polymerase chain reaction (PCR) were injected via chip-based diaphragm pumping into an electrophoretic separation channel. Channel dimensions were designed for injection plug shaping via preferential flow paths, which aided in minimizing the plug widths. Unbiased injection of sample could be achieved in as little as 190 ms, decreasing the time required with electrokinetic injection by two orders of magnitude. Additionally, sample stacking was promoted using laminar or biased-laminar loading to co-inject either water or low ionic strength DNA marker solution along with the PCR-amplified sample. Complete baseline resolution (Res = 2.11) of the 80- and 102-bp fragments of pUC-18 DNA marker solution was achieved, with partially resolved 257- and 267-bp fragments (Res = 0.56), in a separation channel having an effective length of only 3.0 cm. This resolution was deemed adequate for many PCR amplicon separations, with the added advantage of short separation time-typically complete in <120 s. Decreasing the amount of glass surrounding the PCR chamber reduced the DNA amplification time, yielding a further enhancement in analysis speed, with heating and cooling rates as high as 13.4 and -6.4 degrees C s(-1), respectively. With the time requirements greatly reduced for each step, it was possible to seamlessly couple IR-mediated amplification, sample injection, and separation/detection of a 278-bp fragment from the invA gene of <1000 starting copies of Salmonella typhimurium DNA in approximately 12 min on a single device, representing the fastest PCR-ME integration achieved to date.
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