Dr. Shakila Peli Thanthri

Breast cancer (BC) standard-of-care depends on the tumor receptor status and molecular subtype of the patient. Nanostring nCounter instrument with Prosigna-PAM50 assay has been successfully used for BC molecular subtyping with high multiplexing capabilities. However, nCounter technique has few limitations such as high mass input requirement, low sample efficiency and necessity of extensive optical instruments for single molecule imaging with fluorescence. In this project, we are working on developing an innovative single molecule mRNA assay without using reporter probe which requires fluorescent detection inside a thermoplastic microfluidic device. Here, one of the main goals is to generate a coding system to detect and identify single mRNAs using resistive pulse sensing (RPS) in a multiplexed format without using any fluorescent detection method. Initially, tumor derived extra cellular vesicles will be isolated from plasma samples and BC related mRNA will be isolated inside a microfluidic chip. In a different microfluidic chip, a capture probe will be immobilized to capture target mRNAs from the sample using a solid-phase isolation method. Then, the reporter probe will be introduced to the system which can also hybridize with the target mRNA. The reporter probes will selectively hybridize with each different mRNA type which can accommodate multiplexing. The capture probe + target mRNA + reporter probe complex will be sent through an RPS nanopore sensing platform with a pair of in-plane nanopores. The time of flight, signal peak shape and dwell time will be used to characterize each different probe + mRNA complex.