Miniature PCR (polymerase chain reaction) device for detecting biological warfare agents

PI: Dr. Greg Hampikian, Biology Department, Boise State University.

Early detection of biological agents such as those which cause anthrax, small pox, tularemia, foot and mouth and other diseases is critical to homeland defense. Biological agents pose a special challenge because very small amounts of the agent can be amplified by human or animal infection.
Low Temperature Co-fired Ceramic (LTCC) is an ideal material for developing new pathogen sensors because the robust ceramic/glass material system is resistant to most chemical and biological compounds, can be sterilized by autoclaving or baking, and can be manufactured with embedded electrical components. As a result, sensors fabricated out of LTCC could operate in environments that would incapacitate other sensor devices, while remaining easy to decontaminate and reuse.

We propose to develop a new sensor fabricated out of LTCC to detect biological hazards. The sensor will employ polymerase chain reaction (PCR), the standard method used to amplify very low concentrations of DNA for biohazard detection, forensic identification, and bioengineering applications. PCR requires a thermocycler that is a simple heating and cooling chamber with three temperature settings, typically 94^o C, 52-62^o C, and 72^o C. Aqueous samples containing target DNA molecules are inserted into the chamber for amplification. The DNA samples are amplified over specific regions that bind small target-matched DNA primers. The reaction tube is heated and cooled in the three part temperature cycle between 20 and 32 times, and the result is up to a billion-fold increase in DNA from the specifically amplified sequence (Figure 1).
 

We will construct prototypes of this sensor using two approaches to microfluid thermal cycling in LTCC, which are distinguished by the type of movement of fluid within the device and by how the amplification function integrates with subsequent detectors for mass determination, such as the miniature IMS currently being developed. A description of each prototype is given below (Figure 2).

Continuous flow LTCC PCR. In this approach, the fluid moves through a serpentine microtube which is heated in specific segments along its length. The continuous flow method has the advantage that it could be integrated with coupled mass analysis so that a single liquid velocity is used throughout the amplification and analysis processes.

Static LTCC PCR. This approach is similar to methods developed for prototypical micro-PCR PDMS devices. In this embodiment of LTCC PCR, the liquid sample is injected into the thermal cycler channel, and subjected to several rounds of heating and cooling in situ. The limitation of this approach in previous studies is the lack of direct coupling to mass determination, which necessitates operator involvement. Our integrated device overcomes this roadblock.