Peyvan Systems

An Automated Cartridge System and Instrument for the Detection of Bio threat Agents Using Multiplex PCR Amplification and Electrochemical Detection.

We developed a comprehensive system for detecting a broad range of bio threat agents using broad-scan microarray analysis via on-chip electrochemical detection. Important features of the system include (1) a parallel multiplexed PCR that was implemented upstream of the microarray to increase the concentration of detectable targets in the sample a parallel multiplexed PCR was used upstream of the microarray. A microfluidic cartridge and processing instrument were developed to demonstrate field portability of the process. This cartridge contains all the reagents necessary to perform the PCR, microarray hybridization, and electrochemical detection; it also stores all of the waste thereby preventing accidental contamination of the surrounding environment. A computer controlled processing instrument automates the entire workflow, including data analysis. The user simply pipettes the sample into the cartridge, places the cartridge into the instrument and hits the start button. After approximately 3 hours the computer provides the user with analytical graphs indicating the possible presence of pathogens in the sample

Microfluidics Cartridge:

The microfluidic cartridge was designed to be a sealed consumable, which contains all of the reagents and a microarray to conduct a multiplex PCR genotyping assay. It is 8.9 x 7.4cm and is comprised of three major polymer layers (see figure to the right): the top layer is rigid polycarbonate, the middle layer is polydimethylsiloxane (PDMS), and the bottom layer is rigid polypropylene. All layers were molded using a combination of in-house equipment and outside vendors. When these layers are sandwiched together with a microarray, they form reagent blisters, fluidics channels and valves, a multiplex PCR chamber, a hybridization chamber, and a waste chamber. Reagent blisters were designed in the shape of a dome. A companion instrument would selectively press on these flexible domes to dispense or draw the reagents in or out. The resolution of dispensing fluids was roughly 1µL.

Multiplexed PCR Chamber:

We designed probes to detect more than 20 bacteria, viruses and their virulence factors on the microarray. Since the microarrays are inherently insensitive we needed to selectively increase the concentration of the target in the sample via PCR amplification. Multiplexing more than eight targets in one tube proved to be prone to false priming, so we grouped our primers into compatible pools that resulted in approximately ten primer sets. A serpentine channel with four sets of alternating “reaction” and “isolation” channels was developed to facilitate the PCR process. The geometry and size of the channels were designed to prevent cross talk between the reaction chambers, thereby creating a virtually contained PCR cavity. Furthermore, the primers were immobilized inside corresponding chambers, such that they would not wash away during the sample introduction.