Project Summary/Abstract Rapid and specific histopathologic diagnoses are critical for cancer treatment. Tumor tissue biopsy is routinely performed to detect and monitor cancer progression. Current test biopsies require surgically-collected tissue samples from detectable primary or metastatic tumors. Several difficulties, such as patient inconvenience, multistep complicated procedure, partial samplings, and non-specific findings, make this process slow, invasive, expensive, unfit for screening large sample sizes, and error-prone. Non-invasive selections of biomarkers in body fluids, known as liquid biopsy, offer great promise in complementing or even substituting surgical tissue biopsy in the diagnosis and prognosis of cancer patients. Recent studies have indicated exosomal microRNAs (exmiRs) as promising liquid biopsy biomarkers in detecting cancer progression and efficacy of therapy with high sensitivity and specificity. However, current technologies for ex-miR detection, such as qRT-PCR, and microarray screening tests, require high sample volume, are expensive, slow, tedious, requiring highly specialized skills and resources such as ultracentrifuge, expensive RNA extraction kits, etc. Single-exosome level studies can significantly circumvent these problems. However, the few single-molecule ex-miR quantification attempts lack amplification strategy, thus limiting their applications to resource-heavy research settings. To address these problems, we have developed a molecular beacon-based Transmembrane Nano-Sensor (TraNS) that inserts itself into the membrane of lipid vesicles and signals the presence of a DNA target by an increase in fluorescence. We have successfully demonstrated the ability of the TraNS device to spontaneously insert into the lipid membrane and sense membrane-enclosed nucleic acid biomarkers with high specificity. In this study, we propose to (1) optimize the TraNS device to sense cancer- specific ex-miRs from biofluids, (2) harness the transmembrane structural reconfiguration of TraNS to develop an isothermal signal amplification method to improve the sensitivity of detection significantly, and (3) integrate the TraNS device with our patented DNA origami-based biomarker detection array to improve the throughput, specificity, and sensitivity of digital quantification of ex-miR stoichiometry with low sample volume. We will use the platform’s sensitivity, specificity, and throughput on clinical samples from pancreatic cancer patients against their healthy controls. This effort’s potential impact can help physicians and clinicians with rapid, ultrasensitive, precise, and cost-effective cancer diagnostics without a surgical tissue biopsy.

Project Narrative/Relevance We propose to develop a transmembrane sensor nanoarray platform that, if successful, will allow rapid, ultrasensitive, and specific detection and quantification of exosomal micro-RNA at the level of single exosomes. We will design Aampanying isothermal signal amplification strategies to detect the low abundance of nucleic acid biomarkers in exosomes. If successful, the proposed work will provide scientists, physicians, and clinicians with a means for rapid, ultrasensitive, highly-specific, cost-effective prognosis and diagnosis of cancer patients without surgical tissue biopsies.