lab-on-a-chip ͉ microfabrication ͉ RNAi ͉ stochastic gene expression

A novel injection method is developed that utilizes a thermally switchable oligonucleotide affinity capture gel to mediate the concentration, purification, and injection of dsDNA for quantitative microchip capillary electrophoresis analysis. The affinity capture matrix consists of a 20 base acrydite modified oligonucleotide copolymerized into a 6% linear polyacrylamide gel that captures ssDNA or dsDNA analyte including PCR amplicons and synthetic oligonucleotides. Double stranded PCR amplicons with complementarity to the capture probe up to 81 bases from their 5' terminus are reproducibly captured via helix invasion. By integrating the oligo capture matrix directly with the CE separation channel, the electrophoretically mobilized target fragments are quantitatively captured and injected after thermal release for unbiased, efficient, and quantitative analysis. The capture process exhibits optimal efficiency at 44 degrees C and 100 V/cm with a 20 microM affinity capture probe (TM = 57.7 degrees C). A dsDNA titration assay with 20 bp fragments validated that dsDNA is captured at the same efficiency as ssDNA. Dilution studies with a duplex 20mer show that targets can be successfully captured and analyzed with a limit of detection of 1 pM from 250 nL of solution (approximately 150,000 fluorescent molecules). Simultaneous capture and injection of amplicons from E. coli K12 and M13mp18 using a mixture of two different capture probes demonstrates the feasibility of multiplex target capture. Unlike the traditional cross-injector, this method enables efficient capture and injection of dsDNA amplicons which will facilitate the quantitative analysis of products from integrated nanoliter-scale PCR reactors.

An integrated polymerase chain reaction (PCR)-capillary electrophoresis (CE) microdevice with an efficient in-line affinity-based injector has been developed for genetic analysis. Double stranded DNA PCR amplicons generated in an integrated 250 nL PCR reactor are captured, purified, and preconcentrated by an oligonucleotide probe immobilized in an in situ polymerized gel matrix followed by thermal release and injection into the CE-separation channel. This in-column injector employs a photopolymerized oligonucleotide-modified acrylamide capture gel to eliminate band broadening and increase the injection efficiency to 100%. The on-chip generated PCR amplicons processed on this microdevice exhibit a 3-5 fold increase in signal intensities and improved resolution compared to our previous T-shaped injector. Multiplex analysis of 191-bp amplicons from Escherichia coli O157 and 256-bp amplicons from E. coli K12 is achieved with a 6-fold increase in resolution. These advances are exploited to successfully detect E. coli O157 in a 500-fold higher background of E. coli K12. This microdevice with in-line affinity capture gel injection provides an improved platform for low-volume, high sensitivity, fully integrated genetic analysis.

We developed a two-layer, four-channel PCR-capillary electrophoresis microdevice that integrates nucleic acid amplification, sample cleanup and concentration, capillary electrophoretic separation and detection for multiplex analysis of four human respiratory viral pathogens influenza A, influenza B, coronavirus OC43, and human metapneumovirus. Biotinylated and fluorescently labeled double-stranded (ds) DNA amplification products are generated in a 100-nL PCR reactor incorporating an integrated heater and a temperature sensor. After amplification, the products are captured and concentrated in a crosslinked acrylamide gel capture matrix copolymerized with acrydite-functionalized streptavidin-capture agents. Thermal dehybridization releases the fluorescently labeled DNA strand for capillary electrophoresis injection, separation and detection. Using plasmid standards containing the viral genes of interest, each target can be detected starting from as few as 10 copies/reactor. By employing one-step reverse transcription PCR amplification, the device can detect RNA analogues of all four viral targets with detection limits in the range of 25–100 copies/reactor. The utility of the microdevice for analyzing samples from nasopharyngeal swabs is demonstrated. Combining size-based separation with four-color detection, this platform provides excellent product discrimination, making it readily extendable to higher-order multiplex assays. This portable microsystem is also suitable for performing automated assays in point-of-care diagnostic applications.