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

Lab on a chip (2011-01-21)
Robert H Meltzer, Jeffrey R Krogmeier, Lisa W Kwok, Richard Allen, Bryan Crane, Joshua W Griffis, Linda Knaian, Nanor Kojanian, Gene Malkin, Michelle K Nahas, Vyacheslav Papkov, Saad Shaikh, Kedar Vyavahare, Qun Zhong, Yi Zhou, Jonathan W Larson, Rudolf Gilmanshin
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 550, for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Atto 550 NHS ester, BioReagent, suitable for fluorescence, ≥80% (coupling to amines)
Sigma-Aldrich
Atto 550-Biotin, BioReagent, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Atto 550 azide, BioReagent, suitable for fluorescence, ≥80.0% (HPCE)
Sigma-Aldrich
Atto 550 amine, BioReagent, suitable for fluorescence

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