MEAM Ph.D. Thesis Defense: “Manually-Operated, Slider Cassette for Multiplexed Molecular Detection at the Point of Care”

Personalized medicine requires the identification of disease-causing agents prior to prescribing therapy. Currently, molecular detection requires shipping bio samples to centralized laboratories. The time delay between sample collection and test results prevents health care givers from making timely, informed decisions. Furthermore, there is a shortage in centralized laboratories and trained technicians in resource poor settings such as rural areas and developing countries. To address this need for cost-effective, reliable, user-friendly, real-time, multiplexed detection of co-endemic pathogens at the point of need by minimally trained personnel, I propose a 3D-printed, manually operated, slider cassette for multiplexed molecular detection. All needed reagents (in dry form) and buffers are pre-stored in the cassette, refrigeration-free. Once the user introduces a raw sample such as whole blood into the cassette, the user pushes a sliding bar that contains a nucleic acid isolation membrane, comprised of chitosan coated glass fiber, through a sequence of unit operations. The slider’s motion actuates blisters that discharge, in sequence, lysis buffer, wash solution, and elution solution. The lysis process is assisted with heat incubation, wherein heat is provided by an exothermic reaction. Nucleic acids are isolated from the lysate through binding to the chitosan-coated membrane under low pH conditions. At the end of the sliding process, the nucleic acids are eluted with a high pH buffer and aliquoted by capillary forces to individual reaction chambers. Each chamber stores a pre-dried LAMP reaction mix specific to a target. The chambers are then incubated at 65C, facilitating LAMP amplification. The reaction products are detectable either in real time with fluorescent dye and/or at the end point with color change (colorimetric dye). A two-dimensional finite element (COMSOL) simulation was carried out to test for potential crosstalk among the reaction chambers and a three-dimensional finite element (FLUENT) simulation was performed to evaluate the on-cassette chemical heater’s performance used in the lysis process. I demonstrate the entire molecular diagnostic process from sample (plasma and blood) to answer by testing contrived samples spiked with various concentrations of HIV, HCV, and HBV.