Label-free single-molecule apoptosis assay on optical fiber

Apoptosis detection is significant for understanding fundamental biological processes and is closely associated with numerous diseases, including cancer, cardiovascular disorders, and neurodegenerative conditions. However, limited by the intrinsic complexity of physiological environments, achieving highly sensitive detection of apoptotic signals at the single-molecule level remains a major challenge. Here, we propose a label-free single-molecule apoptosis assay based on fiber-optic interferometric imaging. The interference pattern generated by scattered light on the microfiber surface was utilized to resolve localized phase changes, enabling single-particle and single-molecule sensitivity. We found that the dynamic evolution of the interference patterns correlates with the physical heterogeneity among different molecular species, including the surface charge and molecular weight of individual molecules. We achieved single-molecule detection of small proteins down to 5.8 kDa, demonstrating the broad applicability of our method across a wide range of clinically relevant proteins and nucleic acids. After functionalizing the fiber surface with antibodies, the interference patterns exhibited excellent specificity toward the antigen molecules. The dynamic interference pattern was further employed to perform an apoptosis assay at the single-molecule level. Our method enables label-free quantification of biomarkers in complex physiological environments with ultrahigh sensitivity, opening new avenues for fundamental studies of cell-death pathways and early disease diagnosis.