Single molecule DNA intercalation in continuous homogenous elongational flow.

Sequence-nonspecific staining of DNA with intercalating fluorophores is required for fluorescence-based length estimation of elongated DNA in optical mapping techniques. However, the observed length of a DNA molecule is affected by the relative concentrations of DNA and dye. In some applications, predetermination of DNA concentration may not be possible. Here we present a microfluidic approach in which individual DNA molecules are entrained by converging laminar sheath flows containing the intercalating dye PO-PRO-1. This provides uniform staining regardless of DNA concentration, and uniform elastic stretching of DNA in continuous elongational flow. On-chip intercalation provides a unique process for concentration-independent staining of long DNA fragments for the optical mapping method Genome Sequence Scanning (GSS), and normalizes intramolecular elasticity across a broad range of molecule lengths. These advances permit accurate mapping of observed molecules to sequence derived templates, thus improving detection of complex bacterial mixtures using GSS.

High-throughput genome scanning in constant tension fluidic funnels.

Genome Sequence Scanning (GSS) is a bacterial identification technology that detects sparse sequence-specific fluorescent tags on long DNA molecules linearized in a continuous-flow microfunnel. The efficiency and sensitivity of GSS depends on the detection throughput of well-stretched molecules. Previous studies have investigated the fundamental roles of elongational and shear flow on DNA stretching in continuous flow devices. Here, we present a novel compound funnel design that significantly improves effective molecule throughput over previously described devices. First, exploring the relationship between fluid strain rate and molecule tension allows for design of funnel geometries that accommodate high fluid velocities without compromising molecules due to overstretching under high tension. Second, a constant-strain detection channel is utilized to significantly reduce the number of molecules lost to further analysis due to shear-induced molecular tumbling and relaxation. Finally, application of the constant-strain detection channel allows for a priori prediction of spatial resolution bias due to accelerating flow. In all, the refined funnel geometries presented here yield over thirty-fold increase in effective molecule throughput due to increased fluid flow and improved retention of stretched molecules, compared to previously described devices.

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.