Lock-in Biosensing beyond the Analog Ensemble Detection Limits

Abstract Clinical assessment of biomarkers via “analog” ensemble detection of molecular signals is fundamentally limited by the background noise from a detector and nonspecific binding. Although digital assays have emerged as a solution, their clinical translation remains challenging due to their reliance on sophisticated single-molecule sensing technologies. Here, we demonstrate that these analog limitations can be overcome using lock-in detection of ensemble signals. By modulating endpoint molecular binding events into periodic optical fluctuations, our approach amplifies weak molecular signals above the background noise floor. Experimental validation demonstrates a three-fold increase in the limit of sensitivity for single-stranded DNA and a one-order-of-magnitude increase for interleukin-6 (IL-6), achieving a limit of detection at the femtomolar level. Compatible with existing assay platforms, this ensemble lock-in biosensing strategy offers a simple yet effective route for the clinical detection of low-abundance biomarkers.