Flexible ssDNA–DNA Tetrahedron FRET Sensor for Tumor Cell Discrimination via Intracellular Crowding
Junjie Yuan, Yating Zhang, Ao Sun, Ting Xu, Li Jiang, Chuming Zhang, Yuting Hou, Haoyang Han, Bingzhe Xu, Huanhua Xu, Xiangmeng QuABSTRACT
Intracellular crowding, a crucial biophysical parameter linked to tumor progression, can serve as a potential marker for phenotyping. Existing sensors rely on transfection, which disrupts the intracellular microenvironment and reduces accuracy. Herein, we design a DNA biosensor with a DNA tetrahedron scaffold and a flexible ssDNA‐FRET element, achieving near‐physiological crowding sensing via a chemical‐driven soft‐matter response. Cy3/Cy5‐conjugated ssDNA aggregates more tightly with increased crowding, shortening the interfluorophore distance to trigger quantifiable FRET signals, validating the structure‐dependent correlation between flexible soft‐matter agglomeration and crowding degree via simulations, PEG experiments, and FRET characterization. The tetrahedron enables transfection‐agent‐free delivery, avoiding reagent drawbacks. We achieved multivariate statistical discrimination of six cell lines based on osmotic‐stress‐induced FRET fingerprints via osmotic pressure‐induced crowding variations, confirming cellular crowding as a hallmark of functional heterogeneity. This work advances soft matter‐based biosensing and tumor biophysics.