DNA analysis using an integrated microchip for multiplex PCR amplification and electrophoresis for reference samples.

A system that automatically performs the PCR amplification and microchip electrophoretic (ME) separation for rapid forensic short tandem repeat (STR) forensic profiling in a single disposable plastic chip is demonstrated. The microchip subassays were optimized to deliver results comparable to conventional benchtop methods. The microchip process was accomplished in sub-90 min compared with >2.5 h for the conventional approach. An infrared laser with a noncontact temperature sensing system was optimized for a 45 min PCR compared with the conventional 90 min amplification time. The separation conditions were optimized using LPA-co-dihexylacrylamide block copolymers specifically designed for microchip separations to achieve accurate DNA size calling in an effective length of 7 cm in a plastic microchip. This effective separation length is less than half of other reports for integrated STR analysis and allows a compact, inexpensive microchip design. This separation quality was maintained when integrated with microchip PCR. Thirty samples were analyzed conventionally and then compared with data generated by the microfluidic chip system. The microfluidic system allele calling was 100% concordant with the conventional process. This study also investigated allelic ladder consistency over time. The PCR-ME genetic profiles were analyzed using binning palettes generated from two sets of allelic ladders run three and six months apart. Using these binning palettes, no allele calling errors were detected in the 30 samples demonstrating that a microfluidic platform can be highly consistent over long periods of time.

Ultra-rapid real-time microfluidic RT-PCR instrument for nucleic acid analysis.

The polymerase chain reaction (PCR) is paramount in nucleic acid amplification testing, and for many assays, the use of PCR or qPCR is considered the 'gold standard'. While instrumentation for executing PCR has advanced over the last two decades, a growing interest in point-of-need testing has highlighted the deficit that exists for 'rapid PCR' systems. Here, we describe a field-forward prototype instrument capable of ultra-fast thermal cycling for real-time PCR amplification of DNA and RNA. The custom-designed, injection-molded microfluidic chips interface with a novel mechatronic system to complete 40 cycles of real-time PCR in under 10 minutes, an 84% reduction in time compared to a standard 50 minute assay. Such rapid amplification is enabled by two thermoelectric Peltiers capable of efficiently heating and cooling the sample at 12 and 10 °C s-1, respectively. Judicious selection and strategic placement of the thermal cyclers and fluorescence detector relative to the microchip enable synchronized thermal cycling and fluorescence monitoring, further reducing time-to-result. Robust amplification and detection of DNA and RNA targets empowers laboratories to achieve rapid, actionable information in endless applications.

Acoustic trapping of sperm cells from mock sexual assault samples.

We report the successful separation of sperm cells from a relevant composition of mock sexual assault samples using a novel acoustic differential extraction (ADE) technology. A multi-layer microfluidic device fabricated in a non-photolithographic process from glass and polydimethylsiloxane (PDMS) was capable of interfacing with custom-built instrumentation to exploit a standing acoustic wave for the trapping of individual sperm cells in a sample containing an abundance of epithelial cells. Samples were generated from buccal and vaginal swabs to mimic post-coital vaginal swabs, and processed through the ADE system followed by DNA extraction of the captured cells with amplification of DNA using a custom short tandem repeat (STR) chemistry. The prototype acoustic trapping technology was fully capable of isolating intact sperm cells from mock samples with disparate masses of male and female DNA. Other biological components were evaluated for adverse effects on sperm cell trapping, including blood, yeast, and bacteria (E. coli), and these had negligible effects on observed sperm cell trapping. Finally, we demonstrate the successful capture of sperm cells from mock samples containing a 40-fold excess in female epithelial cells over sperm cells. The effectiveness of sperm cell purification was ascertained with polymerase chain reaction (PCR) amplification of STR loci from the male fraction post separation with an 18-plex amplification kit, which resulted in male-only profiles.

An integrated sample-in-answer-out microfluidic chip for rapid human identification by STR analysis.

A fully integrated microfluidic chip for human identification by short tandem repeat (STR) analysis that includes a unique enzymatic liquid preparation of the DNA, microliter non-contact PCR, and a polymer that allows a high-resolution separation within a compact microchip footprint has been developed. A heat-activated enzyme that digests biological materials is employed to generate the target yield of DNA from a buccal swab or FTA paper. The microfluidic architecture meters an aliquot of the liberated DNA and mixes it with the PCR reagents prior to non-contact IR-mediated PCR amplification. The products of PCR amplification are mixed with a sizing standard (ladder) and the 18-plex STR amplicons are separated in an effective length (Leff) of just 7 cm. The development, optimization and integration of each of these processes within the microfluidic chip are described. The device is able to generate genetic profiles in approximately 2 hours that match the profiles from the conventional processes performed using separate conventional instruments. Analysis is performed on a single plastic microchip with a size similar to that of a 96-well plate and only a few mm thick with no pretreatment of any of the functional domains. This is significant advancement in terms of ease of fabrication over glass microdevices or polymeric systems assembled from multiple components. Consequently, this fully integrated sample-in-answer-out microchip is an important step toward generation of a rapid micro-total analysis system for point-of-collection human identification based on genetic analysis.