A Dual-Targeting Functionalized Graphene Film for Rapid and Highly Sensitive Fluorescence Imaging Detection of Hepatocellular Carcinoma Circulating Tumor Cells

High recurrence and metastasis rates are the major causes of the high mortality of hepatocellular carcinoma (HCC). Circulating tumor cells (CTCs) disseminating into the bloodstream play an essential role in cancer metastasis. However, since HCC-CTCs are extremely rare, limitations of current detection methods impede accurate discerning of HCC-CTCs under complicated biological context. Here, a dual-targeting functionalized reduced graphene oxide film (DTFGF) for specifically identifying HCC-CTCs was created via coinstantaneous targeting epithelial cell adhesion molecule (EpCAM) and HCC cell-specific asialoglycoprotein receptor (ASGPR). Anti-EpCAM antibodies and galactose-rhodamine-polyacrylamide nanoparticles (Gal-Rh-PAA NPs) specifically recognizing ASGPR are modified on the surface of a graphene film that quenches the rhodamine fluorescence. HCC-CTCs can be captured by anti-EpCAM antibodies and endocytose Gal-Rh-PAA NPs, recovering the rhodamine fluorescence. Profiting from the accuracy of dual-targeting, less handling steps, and high resolution of fluorescence imaging, a simple, rapid, and low-cost HCC-CTC enumeration method is established with excellent sensitivity and selectivity than conventional methods. Using DTFGFs, as low as five HCC-CTCs were detected in a 1 mL blood sample. Further results revealed that larger HCC-CTC quantities indicate more advanced stages of HCC in patients. Overall, this work holds great promise for the early diagnosis, prognosis, and therapeutic evaluation of HCC.