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...

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A novel DNA solid-phase extraction protocol based on the pH-dependent charge of chitosan was developed specifically for low-volume DNA extraction on microchips. The method uses chitosan-coated beads to extract DNA at pH 5 and release it from the chitosan at pH 9. DNA extraction efficiency as high as 92% could be attained, even from complex samples such as human blood containing significant amounts of protein. Using this method, PCR inhibitors that are typically used in DNA extraction procedures (e.g., chaotropic salts, 2-propanol) can be avoided, making the method more conducive to downstream sample processing using PCR. A high-density multichannel microchip device was then fabricated and the microchannels coated with chitosan for DNA extraction in an open channel configuration without the need for an additional stationary phase. This design provided a relatively high surface area-to-volume ratio for extraction, while retaining the low flow resistance commensurate with open channels. With a flow rate of approximately 1 microL/min during the extraction, the total extraction time was less than 10 min, with most of the DNA recovered in the first 2 microL of elution buffer. Using the microchip device, extraction efficiencies for lambda-phage DNA and human genomic DNA were as high as 67 and 63%, respectively. Human genomic DNA from whole blood samples could be extracted in 10 min with an extraction efficiency of 75 +/- 4% (n = 3), and the purified DNA was suitable for PCR amplification of a fragment of the gelsolin gene. The combination of an entirely aqueous DNA extraction method with a high-density, low-flow resistance microchannel pattern sets the stage for future integration into microfluidic genomic analysis devices.

We have developed a separation-free, electrochemical assay format with direct readout that is amenable to highly sensitive and selective quantitation of a wide variety of target proteins. Our first generation of the electrochemical proximity assay (ECPA) is composed of two thrombin aptamers which form a cooperative complex only in the presence of target molecules, moving a methylene blue (MB)-conjugated oligonucleotide close to a gold electrode. Without washing steps, electrical current is increased in proportion to the concentration of a specific target protein. By employing a DNA-based experimental model with the aptamer system, we show that addition of a short DNA competitor can reduce background current of the MB peak to baseline levels. As such, the detection limit of aptamer-based ECPA for human thrombin was 50 pM via direct readout. The dual-probe nature of ECPA gave high selectivity and 93% recovery of signal from 2.5 nM thrombin in 2% bovine serum albumin (BSA). To greatly improve the flexibility of ECPA, we then proved the system functional with antibody-oligonucleotide conjugates as probes; the insulin detection limit was 128 fM with a dynamic range of over 4 orders of magnitude in concentration, again with high assay selectivity. ECPA thus allows separation-free, highly sensitive, and highly selective protein detection with a direct electrochemical readout. This method is extremely flexible, capable of detecting a wide variety of protein targets, and is amenable to point-of-care protein measurement, since any target with two aptamers or antibodies could be assayed via direct electrochemical readout.