A rapid, instrument-free, sample-to-result nucleic acid amplification test.

The prototype demonstrated here is the first fully integrated sample-to-result diagnostic platform for performing nucleic acid amplification tests that requires no permanent instrument or manual sample processing. The multiplexable autonomous disposable nucleic acid amplification test (MAD NAAT) is based on two-dimensional paper networks, which enable sensitive chemical detection normally reserved for laboratories to be carried out anywhere by untrained users. All reagents are stored dry in the disposable test device and are rehydrated by stored buffer. The paper network is physically multiplexed to allow independent isothermal amplification of multiple targets; each amplification reaction is also chemically multiplexed with an internal amplification control. The total test time is less than one hour. The MAD NAAT prototype was used to characterize a set of human nasal swab specimens pre-screened for methicillin-resistant Staphylococcus aureus (MRSA) bacteria. With qPCR as the quantitative reference method, the lowest input copy number in the range where the MAD NAAT prototype consistently detected MRSA in these specimens was ∼5 × 10(3) genomic copies (∼600 genomic copies per biplexed amplification reaction).

A versatile valving toolkit for automating fluidic operations in paper microfluidic devices.

Failure to utilize valving and automation techniques has restricted the complexity of fluidic operations that can be performed in paper microfluidic devices. We developed a toolkit of paper microfluidic valves and methods for automatic valve actuation using movable paper strips and fluid-triggered expanding elements. To the best of our knowledge, this is the first functional demonstration of this valving strategy in paper microfluidics. After introduction of fluids on devices, valves can actuate automatically after a) a certain period of time, or b) the passage of a certain volume of fluid. Timing of valve actuation can be tuned with greater than 8.5% accuracy by changing lengths of timing wicks, and we present timed on-valves, off-valves, and diversion (channel-switching) valves. The actuators require ~30 µl fluid to actuate and the time required to switch from one state to another ranges from ~5 s for short to ~50 s for longer wicks. For volume-metered actuation, the size of a metering pad can be adjusted to tune actuation volume, and we present two methods - both methods can achieve greater than 9% accuracy. Finally, we demonstrate the use of these valves in a device that conducts a multi-step assay for the detection of the malaria protein PfHRP2. Although slightly more complex than devices that do not have moving parts, this valving and automation toolkit considerably expands the capabilities of paper microfluidic devices. Components of this toolkit can be used to conduct arbitrarily complex, multi-step fluidic operations on paper-based devices, as demonstrated in the malaria assay device.

Rapid protein depletion from complex samples using a bead-based microfluidic device for the point of care.

Translation of sample preparation methods to point-of-care formats has remained a challenge. We present a plastic laminate microfluidic device for protein depletion from human plasma using ligand immobilized porous beads stored dry within a novel, pneumatically-driven mixer. The card design accelerated the protein depletion process from hours to minutes. Using immunoglobulin G as a model protein, we have successfully shown protein removal efficiency from spiked buffer between 70-80% and from diluted human plasma samples between 66-77%. Low non-specific binding of our downstream target ligand, immunoglobulin M, was observed with the spiked buffer and diluted human plasma samples. For future device optimization, the physical limitations to rapid protein removal on card were also explored. Bench-top experiments with improved mixing efficiency and a lower sample dilution factor achieved 99% IgG removal using the same amount of mixing time. This design can easily be adapted for depletion of other high abundance or interfering proteins by inclusion of other ligand immobilized beads.