Handheld Bioidentifier for Biodefense Applications

 

Christopher Cooney Ph.D.*, Phil Belgrader, Ph.D.*, Kristin McFarlane*, Robert Doebler, Ph.D.**, Andrew Miller, Ph.D.^†, Matthew Lesho, Ph.D.*

Detection of biological warfare agents with a handheld device is enabling for pathogen identification by first responders of civil support teams, medical diagnosis of military personnel in theater, and environmental sampling for strategic and tactical missions. This talk will summarize our work towards the development of a DNA detection device using isothermal reactions.  This device, supported by DARPA under the Handheld Isothermal Silver Standard Sensor (HISSS) program executed by the Defense Microelectronics Activity, Sacramento, CA, is designed to achieve lab quality results in a handheld, battery-powered format.  The device, shown in Figure 1, is designed to give rapid results from sample-to-answer (i.e, spore lysis, sample purification or enrichment, DNA amplification, and signal readout).

Figure 1.  Appropriately-sized mock-up device of the HISSS bioidentifier.

            Current methods of bead beating and ultrasonication are challenging to integrate into an inexpensive handheld device using disposable cartridges.  Thus, we explored the use of a rapid, compact, and low-power electrical method to disrupt spores.  This method implements dielectrophoresis to concentrate the spores on the electrodes and an abrupt electrical pulse to disrupt them.  The polymerase chain reaction (PCR) results, shown in Figures 2a and 2b, suggest that the electrical method is as good or better than the gold standard of bead beating.  The sample processing time was approximately one minute.  Sample preparation is achieved using a porous frit to trap large macromolecular inhibitors and a porous membrane filter to trap agents and flush out inhibitors.  This data suggests that back flushing the membranes improved the results further, compared to e-lysis and bead beating in the absence of filter trapping.  

Figure 2.  (a) Real-time PCR curves show electrical lysis (E-lysis) has improved efficiency compared to bead beating (BB) (b) Crossing threshold comparison of twelve replicates across four separate experiments.  The data compares unlysed, bead beating, e-lysis, and e-lysis with the use of a porous membrane filter as clean up.

Isothermal nucleic acid amplification is achieved with a nicking enzyme and a strand displacing polymerase, similar to work described elsewhere¹.  Using a similar approach we show in Figure 3 detection of B. subtilis genomic DNA down to 100 cfu.

Figure 3.  Real-time amplification curve of B. subtilis genomic DNA following lysis by ultrasonication. 

            This work suggests that rapid biological identification can be realized in a hand-held, battery-powered device for biodefense applications.

¹ Van Ness, Van Ness, and Galas (2003) Isothermal reactions for the amplification of oligonucleotides, PNAS 100 (8): 4504-4509.