A trifunctional bio-inspired hydrogel-integrated biosensor: Capture substrate, sensing interface, and quasi-solid-state electrolyte for tumor-derived extracellular vesicles detection.

Extracellular vesicles (EVs) are key mediators of intercellular communication and promising biomarkers for cancer metastasis. However, the clinical EVs detection is limited by the poor isolation efficiency and insufficient specificity. To overcome these critical challenges, this work presented a mussel-inspired PAGTA-Ca2+ hydrogel-integrated electrochemiluminescence (ECL) biosensor. Notably, this PAGTA-Ca2+ hydrogel integrated the roles of EVs capture substrate, sensing interface and quasi-solid-state electrolyte in the biosensor. The pore size of the biomimetic PAGTA-Ca2+ hydrogel was precisely engineered to match the physical dimensions of EVs, which can gently enrich gastric cancer EVs from clinical ascites samples via microporous confinement and reversible polyphenol binding. And luminescent covalent organic framework (TFPB-COF-Bpy) was synthesized as cathodic ECL nano-emitters. Furthermore, the PAGTA-Ca2+ hydrogel-based sensing interface enabled an orthogonal dual-protein (MUC1 and PD-L1) recognition strategy via proximity ligation assay, ensured highly specific identification of tumor-derived EVs. Moreover, the hydrogel with a reinforced hydrogen bond network can suppress hydrogen evolution and ensured stable ECL generation as a quasi-solid-state electrolyte. As a result, this PAGTA-Ca2+ hydrogel-integrated ECL biosensor enabled simplified the capture process and achieved detection with a limit of detection of 143 particles/mL. This novel ECL biosensor research not only provided a novel and cost-effective strategy for the online capture and in situ detection of tumor-derived EVs, but also offered a promising route for designing multifunctional hydrogel-integrated biosensor.