Flexible ssDNA–DNA Tetrahedron FRET Sensor for Tumor Cell Discrimination via Intracellular Crowding

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.