Mi Ho Jeong ¹, Taehwang Son ¹, Yoo Keung Tae ², Chan Hee Park ², Hee Seung Lee ², Moon Jae Chung ², Jeong Youp Park ², Cesar M Castro ^1,3, Ralph Weissleder ^1,3,4,5, Jung Hyun Jo ², Seungmin Bang ², Hyungsoon Im ^1,4

¹Center for Systems Biology, Massachusetts General Hospital, Boston, MA, 02114, USA.
²Division of Gastroenterology, Department of Internal Medicine, Severance Hospital, Yonsei University College of Medicine, Seoul, 03722, Republic of Korea.
³Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA.
⁴Department of Radiology, Massachusetts General Hospital, Boston, MA, 02114, USA.
⁵Department of Systems Biology, Harvard Medical School, Boston, MA, 02115, USA.

M.H.J., T.S., and Y.K.T. contributed equally to this work.
CORRESPONDING AUTHORS: Jung Hyun Jo, Seungmin Bang, Hyungsoon Im

Cholangiocarcinoma (CCA) is a fatal disease often detected late in unresectable stages. Currently, there are no effective diagnostic methods or biomarkers to detect CCA early with high confidence. Analysis of tumor-derived extracellular vesicles (tEVs) harvested from liquid biopsies can provide a new opportunity to achieve this goal. Here, an advanced nanoplasmonic sensing technology is reported, termed FLEX (fluorescence-amplified extracellular vesicle sensing technology), for sensitive and robust single EV analysis. In the FLEX assay, EVs are captured on a plasmonic gold nanowell surface and immunolabeled for cancer-associated biomarkers to identify tEVs. The underlying plasmonic gold nanowell structures then amplify EVs' fluorescence signals, an effective amplification process at the single EV level. The FLEX EV analysis revealed a wide heterogeneity of tEVs and their marker levels. FLEX also detected small tEVs not detected by conventional EV fluorescence imaging due to weak signals. Tumor markers (MUC1, EGFR, and EPCAM) are identified in CCA, and this marker combination is applied to detect tEVs in clinical bile samples. The FLEX assay detected CCA with an area under the curve of 0.93, significantly better than current clinical markers. The sensitive and accurate nanoplasmonic EV sensing technology can aid in early CCA diagnosis.