Cu²⁺-quenched polyfluorene nanoparticles coupling tetra-way strand displacement amplification-activated CRISPR-Cas12a system for coreactant-free electrochemiluminescence analysis

Coreactant-free polyfluorene-based electrochemiluminescence (ECL) systems have attracted much attention and shown a good application prospect in various analyses. However, the signal regulators currently used are generally limited to hydrogen peroxide (H2O2). In order to expand the application of coreactant-free polyfluorene-based ECL systems, it is necessary to develop more signal regulators that are stable, cheap, efficient and easy to be immobilized. This work exploited copper ions (Cu2+) as highly effective ECL quenchers for coreactant-free poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1',3}-thiadazole)] nanoparticles (PFBT NPs). Cu2+-quenched PFBT NPs coupled with tetra-way strand displacement amplification (TW-SDA)-activated CRISPR-Cas12a system for detecting microRNA-132 (miRNA-132). In the absence of miRNA-132, bidirectional SDA was triggered, producing only one functional strand S5. In this case, the trans-cleavage activity of CRISPR-Cas12a system could not be activated to cut S5. As a consequence, the hybrid chain reaction (HCR) on the electrode was triggered by S5 and Cu2+ was enriched on the electrode surface via A-Cu2+-T bond, thus obtaining "signal off" state. When miRNA-132 was present, TW-SDA was triggered to produce a single-strand S5 and target strand (TS). TS activated trans-cleavage activity of CRISPR-Cas12a system for indiscriminately cutting S5. As a result, HCR on the electrode cannot be triggered and the quencher Cu2+ cannot be brought to the electrode surface via A-Cu2+-T bond, thus recovering the ECL for miRNA-132 detection. The integration of Cu2+-quenched PFBT NPs and TW-SDA strategy endowed the ECL biosensor wide linear range (1.0 fM similar to 1.0 nM) and low limit of detection (147 aM). Cu2+-quenched PFBT NPs created a very promising ECL platform for bioassay.