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.

A jet injector platform technology that provides improved performance over existing jet injectors through the use of a controllable linear Lorentz-force actuator and software-based control system has been developed. Injectors designed on this platform are capable of delivering injections using arbitrary pressure pulse shaping. Pulse shaping has been shown to allow a wide degree of control over the depth to which the injection is delivered. A software-based injector control system improves repeatability and allows for automatic reloading of the injector, a task that would be difficult to implement using existing jet injector platforms. A design for a prototype autoloading controllable jet injector (cJI) based on this platform is detailed. The injection capability of this cJI was evaluated both in-vitro and in-vivo using a tissue analog, excised porcine tissue, and ovine tissue. An analysis of the cJI’s performance indicates that this design is capable of delivering a controllable volume of fluid to a controllable depth based entirely on the parameter’s input into the control software.

To improve the speed and efficiency of genetic screening, we are developing an instrument to detect mutations via a high throughput, automated system. Detection is based on changes in the melting temperature induced by single point mutations as in Denaturing Gradient Gel Electrophoresis (DGGE). This instrument measures the migrating position of test and wild type molecules in a spatial thermal gradient during capillary electrophoresis. In this thesis, the concept is described. A thorough design process is conducted focusing on controlling thermal expansion, creating a stable, predictable temperature gradient, and optimizing software and hardware. Finally, the concept is proved in a series of experiments successfully identifying the presence of a mutation in the test sample.

Real-time polymerase chain reaction (PCR) is the gold-standard for quantitation in both mutation and gene expression analyses. Already this technique has found valuable clinical application in disease diagnosis and progression evaluation. As the number of known gene-disease correlations continues to rise, there will be increased demand for higher throughput and decreased cost for these analyses. Present real-time PCR measurement is based upon the fluorescent intensity of either intercalating dyes or oligonucleotide probes. Intercalating dye methods suffer from a lack of binding specificity, while probe methods are expensive and require increased assay optimization.In this thesis, a new method is presented for monitoring real-time PCR that utilizes the fluorescent anisotropy (FA) of labeled primers. FA, when measured at constant temperature, is indicative of the molecular mass to which the fluorophore is attached. Specificity is improved with the FA method over the use of intercalating dyes since the selective binding of primers is required for signal change. Assay complexity and cost are reduced compared to fluorogenic probe methods since the probes are eliminated.The design of a prototype instrument, which successfully implements this new method, is presented. Instrument and assay performance are compared to intercalating dye assays run in commercially available instrumentation. Theoretical limits on performance are also presented and compared to experimental results. Excellent repeatability and linearity are observed with respect to these benchmarks. This new method, having both high specificity and low optimization complexity, is expected to be particularly applicable to the demanding robustness requirements of nano-scale PCR.

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