Rapid, inexpensive fabrication of electrophoretic microdevices for fluorescence detection.

The laser print, cut, and laminate (PCL) method for microfluidic device fabrication can be leveraged for rapid and inexpensive prototyping of electrophoretic microchips useful for optimizing separation conditions. The rapid prototyping capability allows the evaluation of fluidic architecture, applied fields, reagent concentrations, and sieving matrix, all within the context of using fluorescence-compatible substrates. Cyclic olefin copolymer and toner-coated polyethylene terephthalate (tPeT) were utilized with the PCL technique and bonding methods optimized to improve device durability during electrophoresis.

Rapid multiplex DNA amplification on an inexpensive microdevice for human identification via short tandem repeat analysis.

Forensic DNA analysis requires several steps, including DNA extraction, PCR amplification, and separation of PCR fragments. Intuitively, there are numerous situations where it would be beneficial to speed up the overall DNA analysis process; in this work, we focus on the most time-consuming component in the analysis pipeline, namely the polymerase chain reaction (PCR). Primers were specially designed to target 10 human genomic loci, all yielding amplicons shorter than 350 bases, for ease of downstream integration with on-board microchip electrophoresis.

A centrifugal microfluidic device with integrated gold leaf electrodes for the electrophoretic separation of DNA.

Current conventional methods utilized for forensic DNA analysis are time consuming and labor-intensive requiring large and expensive equipment and instrumentation. While more portable Rapid DNA systems have been developed, introducing them to a working laboratory still necessitates a high cost of initiation followed by the recurrent cost of the devices. This has highlighted the need for an inexpensive, rapid and portable DNA analysis tool for human identification in a forensic setting.

Microfluidic enzymatic DNA extraction on a hybrid polyester-toner-PMMA device.

To date, the forensic community regards solid phase extraction (SPE) as the most effective methodology for the purification of DNA for use in short tandem repeat (STR) polymerase chain reaction (PCR) amplification. While a dominant methodology, SPE protocols generally necessitate the use of PCR inhibitors (guanidine, IPA) and, in addition, can demand timescales of up to 30 min due to the necessary load, wash and elution steps. The recent discovery and characterization of the EA1 protease has allowed the user to enzymatically extract (not purify) DNA, dramatically simplifying the task of producing a PCR-ready template.

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.

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.

Purification of HIV RNA from serum using a polymer capture matrix in a microfluidic device.

In this report, we demonstrate the purification of DNA and RNA from a 10% serum sample using an oligonucleotide capture matrix. This approach provides a one-stage, completely aqueous system capable of purifying both RNA and DNA for downstream PCR amplification. The advantages of utilizing the polymer capture matrix method in place of the solid-phase extraction method is that the capture matrix eliminates both guanidine and the 2-propanol wash that can inhibit downstream PCR and competition with proteins for the binding sites that can limit the capacity of the device.

Size-based protein separations by microchip electrophoresis using an acid-labile surfactant as a replacement for SDS.

We demonstrate the use of an acid-labile surfactant (ALS) as a replacement for SDS for size-based protein separations in a microfluidic device. ALS is of interest to the proteomic field as it degrades at low pH and hence can be removed to reduce surfactant interference with down-stream MS. A range of SDS and ALS concentrations were tested as denaturants for microchip electrophoresis to investigate their effects on the separation of proteins from 18 to 116 kDa and to provide a suitable comparison between the two surfactants.

Polymer systems designed specifically for DNA sequencing by microchip electrophoresis: a comparison with commercially available materials.

Electrophoresis-based DNA sequencing is the only proven technology for the de novo sequencing of large and complex genomes. Miniaturization of capillary array electrophoresis (CAE) instruments can increase sequencing throughput and decrease cost while maintaining the high quality and long read lengths that has made CAE so successful for de novo sequencing. The limited availability of high-performance polymer matrices and wall coatings designed specifically for microchip-sequencing platforms continues to be a major barrier to the successful development of a commercial microchip-sequencing instrument.

Thermoresponsive N-alkoxyalkylacrylamide polymers as a sieving matrix for high-resolution DNA separations on a microfluidic chip.

In recent years, there has been an increasing demand for a wide range of DNA separations that require the development of materials to meet the needs of high resolution and high throughput. Here, we demonstrate the use of thermoresponsive N-alkoxyalkylacrylamide polymers as a sieving matrix for DNA separations on a microfluidic chip. The viscosities of the N-alkoxyalkylacrylamide polymers are more than an order of magnitude lower than that of a linear polyacrylamide (LPA) of corresponding molecular weight, allowing rapid loading of the microchip.

Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes.

To realize the immense potential of large-scale genomic sequencing after the completion of the second human genome (Venter's), the costs for the complete sequencing of additional genomes must be dramatically reduced. Among the technologies being developed to reduce sequencing costs, microchip electrophoresis is the only new technology ready to produce the long reads most suitable for the de novo sequencing and assembly of large and complex genomes. Compared with the current paradigm of capillary electrophoresis, microchip systems promise to reduce sequencing costs dramatically by increasing throughput, reducing reagent consumption, and integrating the many steps of the sequencing pipeline onto a single platform.

Stochastic single-molecule videomicroscopy methods to measure electrophoretic DNA migration modalities in polymer solutions above and below entanglement.

We have studied the effects of polymer molar mass and concentration on the electrophoretic migration modalities of individual molecules of DNA in LPA, HEC, and PEO solutions via epifluorescent videomicroscopy. While both transient entanglement coupling (TEC) and reptation have been studied in the past, the transition between them has not. Understanding this transition will allow for polymer network properties to be optimized to enhance the speed and resolution of DNA separations in microfluidic devices.