Split G-Quadruplex Programmed Recyclable AIE-Biosensor for Label-Free Detection of miRNA in Acute Kidney Injury

We herein rationally designed a target-recyclable AIE-biosensor based on a split G-quadruplex for label-free detection of miRNA in acute kidney injury. Initially, the PG was in an "OFF" state, and the two split segments (G4-a and G4-b) of G4 were tethered at the two terminals of P1 and far away from each other due to the rigid duplex structure formed by the partially complementary intermediate sequences of P2 and P1, bringing MG with quenched fluorescence. In the presence of target, the 5'-PO₄ P2 was displaced from PG probe and competitively hybridized with target, which led to G4-a and G4-b tending to form an intact intermolecular G-quadruplex, providing sites for MG intercalation, thus generating an activated "ON" fluorescence signal due to the restriction of intermolecular motion. Successively, relying on the λ-exonuclease (λ-Exo) cleavage reaction-assisted target recycling, more amounts of targets will be liberated, accompanied by forming more G-quadruplex and binding more MG, resulting in a strong fluorescence signal, further realizing the sensitive detection of the targets. As a proof of concept, miRNA-21 was chosen as the model target. Endowing with the precise target recognition and efficient cleavage activity of λ-Exo, the AIE-biosensor exhibited excellent detection sensitivity and specificity, which could quantitatively detect miRNA-21 down to 10.36 fM with a single mismatch specificity. The results revealed that the distinctive attributes of noninvasive, simple, and efficient in this G-quadruplex-based AIE-biosensor offered promising prospects for extensive applications in AKI screening and early clinical diagnosis.