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...

A glass microdevice has been constructed for the on-line integration of solid-phase extraction (SPE) of DNA and polymerase chain reaction (PCR) on a single chip. The chromatography required for SPE in the microfluidic sample preparation device (muSPD) was carried out in a silica bead/sol-gel SPE bed, where the purified DNA was eluted directly into a downstream chamber where conventional thermocycling allowed for PCR amplification of specific DNA target sequences. Through rapid, simple passivation of the PCR chamber with a silanizing reagent, reproducible DNA extraction and amplification was demonstrated from complex biological matrixes in a manner amenable to any research laboratory, using only a syringe pump and a conventional thermocycler. The muSPD allowed for SPE concentration of DNA from 600 nL of blood coupled to subsequent on-chip amplification that yielded a detectable amplicon; this simple device can be applied to a variety of routine genetic analyses without the need for sophisticated instrumentation. In addition, the applicability of these developments to nonconventional thermocycling was demonstrated through the use of noncontact, IR-mediated heating. This was exemplified with the isolation of DNA from an anthrax spore-spiked nasal swab and the subsequent on-chip amplification of target DNA sequences in a total processing time of only 25 min.

The functionality of micropillars, microposts, silica beads, silica particles, sol-gels, and porous monoliths provides a framework for sample preparation and analysis for an integrated microfluidic system.

The advent of microfluidic technology for genetic analysis has begun to impact forensic science. Recent advances in microfluidic separation of short-tandem-repeat (STR) fragments has provided unprecedented potential for improving speed and efficiency of DNA typing. In addition, the analytical processes associated with sample preparation--which include cell sorting, DNA extraction, DNA quantitation, and DNA amplification--can all be integrated with the STR separation in a seamless manner. The current state of these microfluidic methods as well as their advantages and potential shortcomings are detailed. Recent advances in microfluidic device technology, as they pertain to forensic DNA typing, are discussed with a focus on the forensic community.

Effective microchip extraction of deoxyribonucleic acid (DNA) from crude biological matrixes has been demonstrated using silica beads or hybrid phases composed of beads and sol-gel. However, the use of monolithic sol-gels alone for extraction of human genomic DNA has been more difficult to define. Here we describe, for the first time, the successful use of monolithic tetramethyl orthosilicate-based sol-gels for effective micro-solid-phase extraction (muSPE) of DNA in a glass microchip format. A functional monolithic silica phase with micrometer-scale pores in the silica matrix resulted from addition of poly(ethylene glycol), a poragen, to the precursor mixture. This allowed a monolithic sol-gel bed to be established in a microchip channel that provided large surface area for DNA extraction with little flow-induced back pressure. DNA extraction efficiencies for simple systems (lambda-phage DNA) were approximately 85%, while efficiencies for the reproducible extraction of human genomic DNA from complex biological matrixes (human blood) were approximately 70%. Blockage of the sol-gel pores by components in the lysed blood was observed in repeat extraction on a single device as a decrease in the extraction efficiency. The developed muSPE protocol was further evaluated to show applicability to clinical samples and bacterial cultures, through extraction of PCR-amplifiable DNA.

Having previously detailed a method for chip-based extraction of DNA (Anal. Chem. 2003, 75, 1880-1886.), we describe here a microchip-based solid-phase extraction method for purification of RNA from biological samples is demonstrated. The method involves the use of silica beads as a solid phase, and the capacity of the device containing silica beads for RNA, RNA in the presence of protein, and DNA was determined. The capacity of the device for RNA binding in the presence of protein is 360 ng, which demonstrates sufficient capacity of the device for complete genetic analysis. An extraction of RNA can be performed on the device in as few as approximately 9 min (analytical time), a time comparable to that of a commercial extraction method, but with less reagent consumption. The microchip-based extraction is also performed in a closed system, unlike the commercial extraction method, which provides the advantage of decreased opportunity for the introduction of RNases and contaminants--essential for the sensitive RNA-based analyses presented in this work. RNA purified using the device was shown to be amplifiable using reverse transcription PCR (RT-PCR), allowing for translation of the method to the purification and subsequent amplification of biological samples. RNA was purified using the microchip-based method from neat semen, a mock semen stain, and cultured cells from a common pediatric cancer, alveolar rhabdomyosarcoma.

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The current backlog of casework is among the most significant challenges facing crime laboratories at this time. While the development of next-generation microchip-based technology for expedited forensic casework analysis offers one solution to this problem, this will require the adaptation of manual, large-volume, benchtop chemistry to small volume microfluidic devices. Analysis of evidentiary materials from rape kits where semen or sperm cells are commonly found represents a unique set of challenges for on-chip cell lysis and DNA extraction that must be addressed for successful application. The work presented here details the development of a microdevice capable of DNA extraction directly from sperm cells for application to the analysis of sexual assault evidence. A variety of chemical lysing agents are assessed for inclusion in the extraction protocol and a method for DNA purification from sperm cells is described. Suitability of the extracted DNA for short tandem repeat (STR) analysis is assessed and genetic profiles shown. Finally, on-chip cell lysis methods are evaluated, with results from fluorescence visualization of cell rupture and DNA extraction from an integrated cell lysis and purification with subsequent STR amplification presented. A method for on-chip cell lysis and DNA purification is described, with considerations toward inclusion in an integrated microdevice capable of both differential cell sorting and DNA extraction. The results of this work demonstrate the feasibility of incorporating microchip-based cell lysis and DNA extraction into forensic casework analysis.