Membrane-anchored catalytic hairpin assembly on exosomes: A fluorescent aptasensor for highly sensitive cancer diagnosis.

Tumor exosomes serve as ideal biomarkers for the early diagnosis of cancer. However, the existing methods are characterized by low sensitivity and are prone to interference from free proteins. In this study, a fluorescent strategy was developed, which combines the enrichment of aptamer-functionalized microspheres with the exosome membrane-anchored catalytic hairpin assembly (CHA). Firstly, SYL3C (EpCAM aptamer) was employed to functionalize microspheres for the specific enrichment of exosomes. Subsequently, the CHA reaction trigger was conjugated with a bivalent cholesterol probe and anchored to the exosome lipid bilayer membrane. The addition of hairpin probes H1 and H2 initiated the CHA reaction. H1 is a signal probe modified with ROX and BHQ-2. In the absence of the CHA reaction, H1 maintains its hairpin structure, facilitating fluorescence resonance energy transfer (FRET) between ROX and BHQ-2, which results in the quenching of ROX fluorescence. Upon encountering the CHA reaction trigger, the H1 hairpin undergoes unfolding, which disrupts FRET and restores ROX fluorescence. The intensity of ROX fluorescence exhibits a positive correlation with the exosome concentration. This work significantly reduces the interference from cellular debris, free proteins, and other vesicles during exosome detection and simultaneously achieves signal amplification. The detection limit of this method is 2.78 × 103 particles/μL. It has successfully differentiated serum samples from 6 breast cancer patients and 5 healthy individuals. This study offers a novel, highly sensitive, and specific strategy for exosome-based liquid biopsy.