Chitosan-coated silica particles and chitosan-coated microchannels have been explored as an alternative to a standard silica phase for DNA extraction in a microdevice (Cao, W.; Easley, C. J.; Ferrance, J. P.; Landers, J. P. Anal. Chem. 2006, 78 (20), 7222-7228). A method that exploits the use of aqueous buffers for nucleic acid binding to and release from a solid phase is advantageous, avoiding the reagents used for conventional extraction (isopropanol and guanadinium hydrochloride), which are potent PCR inhibitors. The pH-controlled approach, which promotes nucleic acid binding to and release to the chitosan phase based on a change in buffer pH, is exploited here for RNA purification in a microfluidic device. The chitosan phase reproducibly allowed for higher RNA extraction efficiencies under aqueous conditions (71%) compared to that with a silica phase under chaotropic conditions (53%). The effectiveness of the chitosan phase was demonstrated with the successful purification of RNA from the alveolar rhabdomyosarcoma (ARMS) cancer cell line, with 3.5-fold greater extraction efficiencies than obtained when the same sample was purified using a silica phase: the resulting RNA was found to be amplifiable in reverse-transcription PCR. Low-molecular weight chitosan is also a proven inhibitor of RNases, further demonstrating the advantages of chitosan as a solid phase for RNA purification compared to silica. The chitosan phase is, therefore, a superior choice for extraction and purification of RNA in a microfluidic device and is compatible with biological samples found in a clinical or forensic setting.

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.

We describe the first miniaturized device capable of the front-end sample preparation essential for detecting RNA-based infectious agents. The microfluidic device integrates sample purification and reverse transcription PCR (RT-PCR) amplification for the identification and detection of influenza A. The device incorporates a chitosan-based RNA binding phase for the completely aqueous isolation of nucleic acids, avoiding the PCR inhibitory effects of guanidine and isopropanol used in silica-based extraction methods. The purified nucleic acids and the reagents needed for single-step RT-PCR amplification are fluidically mobilized simultaneously to a PCR chamber. Utilizing infrared (IR)-mediated heating allowed for a > 5-fold decrease in RT-PCR analysis time compared to a standard thermal cycling protocol used in a conventional thermal cycler. Influenza A virus [A/PR/8/34 (H1N1)] was used as a simulant in this study for virus-based infectious and biowarfare agents with RNA genomes, and was successfully detected in a mock nasal swab sample at clinically relevant concentrations. Following on-chip purification, a fragment specific to the influenza A nucleoprotein gene was first amplified via RT-PCR amplification using IR-mediated heating to achieve more rapid heating and cooling rates. This was initially accomplished on a two-chip system to optimize the SPE and RT-PCR, and then translated to an integrated SPE-RT-PCR device.

A microfluidic device was developed to carry out integrated volume reduction and purification of nucleic acids from dilute, large volume biological samples commonly encountered in forensic genetic analysis. The dual-phase device seamlessly integrates two orthogonal solid-phase extraction (SPE) processes, a silica solid phase using chaotrope-driven binding and an ion exchange phase using totally aqueous chemistry (chitosan phase), providing the unique capability of removing polymerase chain reaction (PCR) inhibitors used in silica-based extractions (guanidine and isopropanol). Nucleic acids from a large volume sample are shown to undergo a substantial volume reduction on the silica phase, followed by a more stringent extraction on the chitosan phase. The key to interfacing the two steps is mixing of the eluted nucleic acids from the first phase with loading buffer which is facilitated by flow-mediated mixing over a herringbone mixing region in the device. The complete aqueous chemistry associated with the second purification step yields a highly concentrated PCR-ready eluate of nucleic acids devoid of PCR inhibitors that are reagent-based (isopropanol) and sample-based (indigo dye), both of which are shown to be successfully removed using the dual-phase device but not by the traditional microfluidic SPE (muSPE). The utility of the device for purifying DNA was demonstrated with dilute whole blood, dilute semen, a semen stain, and a blood sample inhibited with indigo dye, with the resultant DNA from all shown to be PCR amplifiable. The same samples purified using muSPE were not all PCR amplifiable due to a smaller concentration of the DNA and the lack of PCR-compatible aqueous chemistry in the extraction method. The utility of the device for the purification of RNA was also demonstrated, by the extraction of RNA from a dilute semen sample, with the resulting RNA amplified using reverse transcription (RT)-PCR. The vrSPE-SPE device reliably yields a volume reduction for DNA and RNA purification on the order of 50- and 14-fold, respectively, both compatible with downstream PCR analysis. In addition, purification of all samples consumed less reagents (2.6-fold) than traditional purification methods, with the added advantage of being a "closed system" that eliminates sample transfer steps, thereby reducing the possible entrance points for contaminants.