A Donor–Acceptor Covalent Triazine Framework-based “On–Off–On” Electrochemiluminescence Aptasensor for Detecting Aflatoxin B1 with the Assistance of Nicking Endonuclease-powered DNA Walking Machine

A designed "on-off-on" signal-switchable electrochemiluminescence (ECL) aptasensor based on a donor-acceptor (D-A) conjugated covalent triazine framework (CTF) has been constructed for the sensitive and accurate detection of aflatoxin B1 (AFB1) with the assistance of a nicking endonuclease (Nb.BbvCI)-powered DNA walking machine. The D-A conjugated CTF, generated from the reaction between 2,4,6-tris(4-formylphenyl)-1,3,5-triazine (TFPT) and tris(4-aminophenyl)methane (TAPM) (denoted as TFPT-TAPM-CTF), simultaneously serves as a superior ECL emitter and a platform for anchoring the bioprobe. The high ECL response of TFPT-TAPM-CTF can be quenched by the anchored Cy5-labeled single-strand DNA (Cy5-ssDNA) via ECL resonance energy transfer. Furthermore, the immobilization of the double-strand DNA generated between the AFB1-targeting aptamer and the DNA walker (Hp) reduces the ECL response of TFPT-TAPM-CTF. When detecting AFB1, the aptamer separates from the double-strand DNA to capture specific targets, resulting in the hybridization of the free Hp strand and Cy5-ssDNA. With the assistance of Nb.BbvCI, the released partial Cy5-ssDNA helps recover the ECL response of TFPT-TAPM-CTF to some extent, further liberating the Hp strand to autonomously bind to another Cy5-ssDNA and trigger a new cleavage process. In addition to its high selectivity and promising practicality, the constructed ECL aptasensor shows an ultralow detection limit of 0.59 pg·mL^-1 within a wide range from 1.0 pg·mL^-1 to 5.0 × 10⁴ pg·mL^-1. This work broadens the application of CTF in the food safety field and provides a new aptasensing strategy for the sensitive and precise inspection of mycotoxins in food products.