Robert H. Meltzer

A lab-on-chip for biothreat detection using single-molecule DNA mapping

Rapid, specific, and sensitive detection of airborne bacteria, viruses, and toxins is critical for biodefense, yet the diverse nature of the threats poses a challenge for integrated surveillance, as each class of pathogens typically requires different detection strategies. Here, we present a laboratory-on-a-chip microfluidic device (LOC-DLA) that integrates two unique assays for the detection of airborne pathogens: direct linear analysis (DLA) with unsurpassed specificity for bacterial threats and Digital DNA for toxins and viruses. The LOC-DLA device also prepares samples for analysis, incorporating upstream functions for concentrating and fractionating DNA. Both DLA and Digital DNA assays are single molecule detection technologies, therefore the assay sensitivities depend on the throughput of individual molecules. The microfluidic device and its accompanying operation protocols have been heavily optimized to maximize throughput and minimize the loss of analyzable DNA. We present here the design and operation of the LOC-DLA device, demonstrate multiplex detection of rare bacterial targets in the presence of 100-fold excess complex bacterial mixture, and demonstrate detection of picogram quantities of botulinum toxoid.

Simultaneous DNA Stretching and Intercalation in Continuous Elongational Flow

Fluorescent microscopic observation of DNA stretching in homogenous elongational flow has previously been used to characterize biophysical properties of the polymer. Typically, individual molecules are trapped at the stagnation point of opposed fluidic flows in a cross-slot microfluidic structure. Such observations intrinsically have a low throughput of analyzable molecules. We present a continuous-flow microfluidic funnel that replicates the intramolecular tension distribution established in cross-slot DNA stretching experiments, but achieves greater than 10 megabase per second analyzable DNA throughput.Intercalating fluorescent dyes are required for single-molecule visualization of stretched DNA. DNA elasticity under tension however is affected by bound intercalator. Careful experimental control of the relative concentration of intercalator and DNA is therefore required for reproducible DNA stretching. In the presented device, intercalator is introduced using convergent sheathing flows, which center DNA in the microfunnel over a series of confocal laser excitation spots. The intercalation reaction therefore proceeds one molecule of DNA at a time, thus eliminating dependence on DNA concentration. Using this experimental platform, we demonstrate differing effects on DNA elasticity for the monomeric dye POPRO-1 and its bis-intercalating analogue POPO-1. We also characterize the effect of intramolecular tension on the orientation of intercalating dye in its DNA binding site using single-molecule fluorescence anisotropy.Better understanding of stretching mechanics and tension distribution along extended DNA yields practical improvements in comparing fluorescence traces of site-specific probes to sequence-derived templates. This enhances genomic comparison and identification in continuous flow optical mapping applications.