Abstract Breast cancer (BC) continues to be a devastating disease representing 30% of newly diagnosed cancer cases amongst females in the US (276,480 newly diagnosed cases in 2020) and the second leading cancer cause of death in females. The challenge with BC is its heterogeneity in terms of molecular alterations. For example, Stage I/II BC patients are typically subjected to molecular subtyping using expression analysis of a mRNA gene panel, for example the PAM50 panel. The 4 major molecular subtypes include Luminal A, Luminal B, HER2- enriched, and Basal-like subtypes with each one associated with a certain treatment regimen to optimize clinical outcomes for those patients. These tests are performed from a solid tissue biopsy to satisfy the mass requirements of the molecular subtyping assay (100 ng of RNA). The assay to determine the molecular subtype of the BC patients uses reporter probes and fluorescent dyes to provide high multiplexing capabilities, but requires single-molecule fluorescence readout of mRNA/reporter probe assemblies stretched on a glass slide. Liquid biopsy samples, for example extracellular vesicles (EVs), are an attractive alternative to solid-tissue biopsies for managing cancer-related diseases. The attractive nature of liquid biopsies is the minimally invasive nature of their acquisition and that they can report on the status of the primary tumor as well as metastatic sites. However, a challenge with liquid biopsy samples is the limited mass of nucleic acid material they supply. For example, 108 EVs secured from a Stage I/II BC patient would provide ~1.5 ng of mRNA. In this R21 project, an innovative mRNA identification/quantification technology will be generated that can accommodate the mass limits associated with liquid biopsy samples. The technology consists of a dual in-plane nanopore sensor comprised of microfluidic and nanofluidic structures fabricated in a plastic, for example PMMA, using replication-based techniques such as injection molding. The sensor consists of properly engineered input microstructures to allow for high sampling efficiency to provide an exquisite limit-of-detection (<5 ng of RNA). The nanofluidic elements consist of a nanochannel (50 × 50 nm, width × depth, length >5 µm) flanked on either side by an in-plane pore (10 – 30 nm effective diameter). Using resistive pulse sensing (RPS), unique reporter probes can be identified by their characteristic current transient amplitudes, temporal profiles, and/or dwell times. In addition, because two pores are placed in series, the molecular-dependent electrophoretic mobility of the reporter probes can be deduced, which will add an additional layer of identification information to expand the multiplexing capability of the RPS readout (>27plex). The reporter probes consist of gene-specific sequences, and a DNA backbone to which is hybridized RNA segments bearing 1 of 3 different proteins (avidin, streptavidin, or neutravidin). The utility of the dual in-pl...