Triple-helix as a target converter for trace pesticide detection based on CRISPR/Cas12a-based ECL biosensor

Developing a convenient and sensitive biosensor for trace pesticide detection is vital to guarantee food safety. Herein, we designed a triple-helix as a universal target converter coupling with CRISPR/Cas12a-based electrochemiluminescence (ECL) biosensor for the detection of trace pesticide pesticides. The aptamer with extending auxiliary sequences at both ends was designed and interacted with the primer to form the triple-helix DNA as the target converter, which exposed the aptamer sequence at the loop of the triple-helix. The binding event between the target and aptamer sequence would make the triple-helix deconstruct to expose the primer for the rolling circle amplification (RCA) initiation. With the template customization, the RCA product was encoded with a DNAzyme/substrate unit, which was cleaved into generous short DNA activators with the assistance of Zn2+ for efficient CRISPR/Cas12a activation. Finally, the activated CRISPR/Cas12a cleaved the quenching probes assembled on the electrode for signal output. Take acetamiprid as a model, the fabricated ECL biosensor based on zeolitic imidazolate framework-8 encapsulated perylene (ZIF-8@Pe) as ECL luminophore exhibited excellent selectivity and sensitivity with the linear of 1 pM ~ 100 μM and the limit of detection (LOD) of 394 fM. Furthermore, the universality of the target converter is realized by replacing the aptamer sequence of the triple-helix and the developed biosensor is anticipated to be applied in various pesticide molecule detection.