Detection of Point Mutation in Individual Living Circulating Malignant Cells by Targeting Molecular Beacon Delivery

As a prevalent form of genetic alteration, single‐base substitution serves as a critical determinant of targeted cancer therapeutic strategies and a driver of acquired drug resistance. Despite its clinical significance, detecting these mutations at the single‐cell level remains challenging due to high costs, long analysis times, and difficulties in capturing real‐time cellular dynamics and tumor heterogeneity. Herein, we demonstrate that an aptamer‐functionalized triple‐targeting nanoprobe integrating AT11 and R13 aptamers, along with hyaluronic acid, enables efficient and specific delivery of molecular beacons into circulating malignant cells (CMCs) in whole blood from cancer patients, leading to the hybridization of the molecular beacons with their target mRNAs (reference GAPDH mRNA, EGFR mRNA lacking the L858R mutation, and EGFR mRNA harboring the L858R mutation) in living CMCs. By comparison of fluorescence intensities between the molecular beacons targeting the L858R‐lacking and L858R‐harboring EGFR mRNAs, this approach allows for the single‐cell‐resolution profiling of EGFR L858R point mutation status and patient‐specific tumor heterogeneity. Our study establishes a versatile, non‐invasive liquid biopsy strategy to achieve in situ tumor genotyping at single‐cell resolution and map tumor heterogeneity at the individual patient level, offering wide applicability for cancer monitoring and therapeutic guidance.