PROJECT SUMMARY The circulating population of microRNAs in biofluids are ideal biomarkers for various diseases. Point-of-care profiling of circulating microRNAs is in insatiable demand, but typical approaches, e.g., immunoassays and microRNA assays are lab-based/centralized, expensive ($400–1,000/test), and time-consuming (>6 hours). We will develop a highly integrated, all-nanobioelectronic platform technology for multiplex, high-accuracy circulating-microRNA analysis that is capable of profiling circulating microRNAs in a 50-μL plasma with ultra- high sensitivity (sub-fM) and efficiencies in time (<40 minutes) and cost (<$10/test), thereby enabling high- performance circulating-microRNA analysis at the point of test. The novelty of the program is to harness graphene-based bioelectronics to integrate circulating microRNA isolation, concentration, amplification, and quantification into a self-contained device. In order to proof the concept of this technology, the program will include the development and validation of two generations of graphene-based analytical platforms, GAP1 and GAP2. Three specific aims with measurable milestones will be pursued. (1) We will demonstrate that multiple microRNA analytes can be amplified via hybridization chain reaction on a probe-functionalized graphene sensor array and the analyte concentrations can be readily interrogated by the graphene sensor array and translated into electrical signals. We will develop GAP1 to selectively quantify eight pre-selected target microRNAs (MDCIS8) spiked in 5-μL buffer. The detection limit of the specific microRNAs is expected to be at fM level. (2) We will demonstrate that target circulating microRNAs can be isolated from plasma by immobilizing them on a DNA-functionalized graphene electrode and releasing them into a small-volume simple cargo solution upon the generation of pH gradient by applying voltage bias between the graphene-DNA electrode with a bare graphene electrode. We will develop a graphene-based circulating-microRNA isolation module, combine the module with GAP1 to form GAP2, and use GAP2 to profile circulating MDCIS8 in lysed samples of 50-μL plasma from NSG mice. The GAP2 is expected to concentrate the microRNAs by >5× and deliver sub-fM level sensitivity. (3) We will demonstrate the feasibility of using this platform technology for diagnostic applications. We will use GAP2 to quantify circulating MDCIS8, whose expression levels are indicative to pre-invasive breast cancer, in 50-μL plasma samples from a user blinded cohort of the MIND murine model. The profiling result will be analyzed to predict the progression of pre-invasive breast cancer whose rapid, inexpensive diagnosis remains a challenge. The GAP2 prediction outcome will be combined with that based on surgical biopsy to establish the accuracy of the technology for progression prediction. The expected prediction accuracy is >96%. If successful, the technology will offer a new pathway to next-generation poi...