Functional Molecule‐Integrated Nano‐Interfaces for Detection of Extracellular Vesicles

Abstract Functional molecule‐integrated polymer nanocavities for the detection of extracellular vesicles (EVs) are prepared via template polymerization and multistep post‐modification. A multifunctional polymer bearing a tertiary amino group for interaction with template silica nanoparticles (NPs) and a polymerizable methacryl group is prepared and complexed with 500‐nm silica NPs. The complex is monodispersedly immobilized on the carboxyl group and Br group introduced onto the Au‐coated glass substrate. Additionally, surface‐initiated atom transfer radical polymerization of 2‐methacryloyloxyethyl phosphorylcholine is conducted to form a biocompatible polymer layer. After the silica NPs are removed, the disulfide bond is cleaved using tris(2‐carboxyethyl)phosphine to remove the multifunctional polymer components, yielding a thiol group‐exposed nanocavity. The degree of functionalization is controlled by changing the mixing ratio of the fluorescent and non‐fluorescent dyes. A multifunctionalized nanocavity is prepared by introducing a fluorescent dye and nitrilotriacetic acid moiety, followed by His‐tagged protein G and an antibody for the CD9 protein, yielding EV‐sensing nano‐interfaces. This fluorescent signaling interface responds to EVs in a concentration‐dependent manner, with an estimated limit of detection of 0.1 fM. These responses are not observed when the reference antibody is introduced, confirming cavity‐selective functionalization. The proposed method is useful for fabricating multifunctional nano‐interfaces in the field of life sciences.