Jacquelyn A. DuVall

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. In order for an integrated DNA analysis system such as this to be realized, device operations must always be concluded by a rapid separation of short-tandem repeat (STR) DNA fragments. Contributing to this, we report the development of a unique, multi-level, centrifugal microdevice that can perform both reagent loading and DNA separation. The fabrication protocol was inspired by the print, cut and laminate (PCL) technique described previously by our group, and in accordance, offers a rapid and inexpensive option compared with existing approaches. The device comprises multiple polyester-toner fluidic layers, a cyclic olefin copolymer separation domain and integrated gold leaf electrodes. All materials are commercially-available and complement the PCL process in a way that permits fabrication of increasingly sought after single-use devices. All reagents, including a viscous sieving matrix, are loaded centrifugally, eliminating external pneumatic pumping, and the sample is separated in <5 minutes using an effective separation length of only 4 cm (reagent loading to completed separation, is <37 minutes). The protocol for gold leaf electrode manufacture yielded up to 30 electrodes for less than

A rotationally-driven polyethylene terephthalate microdevice with integrated reagent mixing for multiplexed PCR amplification of DNA

3 (cost of a 79 mm × 79 mm gold leaf sheet) and when using a device combining these electrodes and centrifugal reagent/polymer loading, the electrophoretic separation of STR fragments with two base resolution was demonstrated. This exemplary performance makes the device an ideal candidate for further integration and development of an inexpensive, portable and rapid forensic human identification system.

Optical Imaging of Paramagnetic Bead-DNA Aggregation Inhibition Allows for Low Copy Number Detection of Infectious Pathogens

We demonstrate the capabilities of a centrifugal polyethylene terephthalate toner (PeT) microdevice for integrated on-chip reagent mobilization, mixing, and PCR amplification for genetic analysis of short tandem repeats (STR). Fluid flow, including reagent mobilization and mixing, is achieved by centrifugal force, eliminating the need for bulky instrumentation. The use of a passive valve also eliminates the need for extra hardware and simplifies the chip and the device design. A custom-built system is capable of thermocycling through a dual Peltier clamping system, as well as variable rate spinning with a DC motor. A multiplex PCR amplification of alleles associated with 18 genomic loci was successfully performed on-chip, followed by capillary electrophoretic separation, which showed efficient amplification of DNA from multiple sources. The genetic profiles generated were 100% concordant with those obtained using conventional PCR methods. The resultant system represents a novel microfluidic PCR amplification platform that uses inexpensive PCR microdevices that are simple to fabricate, yet effective for complex, multiplexed PCR.

Rapid Fabrication of Electrophoretic Microfluidic Devices from Polyester, Adhesives and Gold Leaf

DNA-paramagnetic silica bead aggregation in a rotating magnetic field facilitates the quantification of DNA with femtogram sensitivity, but yields no sequence-specific information. Here we provide an original description of aggregation inhibition for the detection of DNA and RNA in a sequence-specific manner following loop-mediated isothermal amplification (LAMP). The fragments generated via LAMP fail to induce chaotrope-mediated bead aggregation; however, due to their ability to passivate the bead surface, they effectively inhibit bead aggregation by longer ‘trigger’ DNA. We demonstrate the utility of aggregation inhibition as a method for the detection of bacterial and viral pathogens with sensitivity that approaches single copies of the target. We successfully use this methodology for the detection of notable food-borne pathogens Escherichia coli O157:H7 and Salmonella enterica, as well as Rift Valley fever virus, a weaponizable virus of national security concern. We also show the concentration dependence of aggregation inhibition, suggesting the potential for quantification of target nucleic acid in clinical and environmental samples. Lastly, we demonstrate the ability to rapidly detect infectious pathogens by utilizing a cell phone and custom-written application (App), making this novel detection modality fully portable for point-of-care use.

Rapid detection of Clostridium difficile via magnetic bead aggregation in cost-effective polyester microdevices with cell phone image analysis

In the last decade, the microfluidic community has witnessed an evolution in fabrication methodologies that deviate from using conventional glass and polymer-based materials. A leading example within this group is the print, cut and laminate (PCL) approach, which entails the laser cutting of microfluidic architecture into ink toner-laden polyester sheets, followed by the lamination of these layers for device assembly. Recent success when applying this method to human genetic fingerprinting has highlighted that it is now ripe for the refinements necessary to render it amenable to mass-manufacture. In this communication, we detail those modifications by identifying and implementing a suitable heat-sensitive adhesive (HSA) material to equip the devices with the durability and resilience required for commercialization and fieldwork. Importantly, this augmentation is achieved without sacrificing any of the characteristics which make the PCL approach attractive for prototyping. Exemplary HSA-devices performed DNA extraction, amplification and separation which, when combined, constitute the complete sequence necessary for human profiling and other DNA-based analyses.