Evaluating the thermostability of commercial fast real-time PCR master mixes.

There is an increasing need for affordable, thermostable PCR reagents that can be used for diagnostic testing in resource limited settings. The development of point-of-care devices in such settings is highly dependent on the availability and efficacy of thermostable reagents. Here, we assess the thermostability of commercial, intercalating dye-based real-time PCR master mixes. We show that several of these master mixes have thermostability and robust performance at 20°C, 40°C, and 45°C for 6, 4 and 2 weeks, respectively. However, none of the master mixes that we evaluated was able to withstand more than 1 month at 45°C incubation. Our findings have implications for clinical diagnostics in the developing world where cold-chain delivery of diagnostic assays is difficult to guarantee.

Microfluidic blood filtration device.

Rapid decentralized biomedical diagnostics have become increasingly necessary in a medical environment of growing costs and mounting demands on healthcare personnel and infrastructure. Such diagnostics require low-cost novel devices that can operate at bedside or in doctor offices using small amounts of sample that can be extracted and processed on the spot. Thus, point-of-care sample preparation is an important component of the necessary diagnostic paradigm shift. We therefore introduce a microfluidic device which produces plasma from whole blood. The device is inexpensive, reliable, easy to fabricate, and requires only 3.5 kPa pressure to operate. The device is fully compatible with microfluidic diagnostic chips. The output 23-gauge microtube of the former can be directly plugged into the input ports of the latter allowing immediate applicability in practice as a sample-prep pre-stage to a variety of emergent microfluidic diagnostic devices. In addition, the shown approach of filter encapsulation in elastomer has principle importance as it is compatible with and applicable to microfluidic sample-prep integration with analytical stages within the same elastomeric chip. This can eventually lead to finger-prick blood tests in point-of-care settings.

Exploring the limits of ultrafast polymerase chain reaction using liquid for thermal heat exchange: A proof of principle.

Thermal ramp rate is a major limiting factor in using real-time polymerase chain reaction (PCR) for routine diagnostics. We explored the limits of speed by using liquid for thermal exchange rather than metal as in traditional devices, and by testing different polymerases. In a clinical setting, our system equaled or surpassed state-of-the-art devices for accuracy in amplifying DNA∕RNA of avian influenza, cytomegalovirus, and human immunodeficiency virus. Using Thermococcus kodakaraensis polymerase and optimizing both electrical and chemical systems, we obtained an accurate, 35 cycle amplification of an 85-base pair fragment of E. coli O157:H7 Shiga toxin gene in as little as 94.1 s, a significant improvement over a typical 1 h PCR amplification.

Design and fabrication of chemically robust three-dimensional microfluidic valves.

A current problem in microfluidics is that poly(dimethylsiloxane) (PDMS), used to fabricate many microfluidic devices, is not compatible with most organic solvents. Fluorinated compounds are more chemically robust than PDMS but, historically, it has been nearly impossible to construct valves out of them by multilayer soft lithography (MSL) due to the difficulty of bonding layers made of "non-stick" fluoropolymers necessary to create traditional microfluidic valves. With our new three-dimensional (3D) valve design we can fabricate microfluidic devices from fluorinated compounds in a single monolithic layer that is resistant to most organic solvents with minimal swelling. This paper describes the design and development of 3D microfluidic valves by molding of a perfluoropolyether, termed Sifel, onto printed wax molds. The fabrication of Sifel-based microfluidic devices using this technique has great potential in chemical synthesis and analysis.

Electrical microfluidic pressure gauge for elastomer microelectromechanical systems.

We report on an electrical microfluidic pressure gauge. A polydimethylsiloxane microvalve closes at a characteristic applied pressure determined by the material's properties and the valve's dimensions. Hence, when the same pressure is applied to all valves of a heterogeneous valve array, some valves close while others remain open. The state of the array is combined with knowledge of the respective characteristic closing pressures of the individual valves to yield an estimate of the applied pressure. The state of each valve is obtained by electrical measurements, since the electrical resistance of the respective underlying fluid-filled channel increases by at least two orders of magnitude as the valve closes and its insulating elastomer material interrupts the electrical circuit. The overall system functions as a pressure gauge with electrical readout. This device would be a critical component in active pressure-regulation loops in future integrated microfluidic systems.