A portable electrochemiluminescence bipolar electrode array for the visualized sensing of Cas9 activity

CRISPR/Cas9 has become a powerful tool for genomic manipulation, and the evaluation of the Cas9 activity is essential for the precise control of the CRISPR system. Herein, we develop a point-of-care platform for the rapid and visible determination of the Cas9 activity. A bipolar electrochemiluminescence (ECL) biosensor platform comprises an Au electric circuit fabricated on a glass substrate and the PDMS layer acts as a reservoir. DNA probes are then modified on electrodes and hybridization chain reaction (HCR) is applied to introduce ferrocene-labeled DNA. The CRISPR/Cas9 system can recognize the immobilized DNA probes and cleave them from the electrode surface as well as the labeled H1 and H2. Therefore, the cleavage activity is closely related to the labeled ferrocene. The modified electrode is then embodied in a closed bipolar system such that the oxidation reaction on sensing poles could be detected by the anodic ECL reactions on isolated luminescent poles due to charge balance, leading to the changes in ECL signals. This portable, integrated and convenient platform may become a meaningful tool for the discovery of a new CRISPR/Cas9 system or additional Cas9 inhibitors. A new type of closed BPE-ECL system based on an Au electric circuit at a portable substrate is designed for the visualized sensing of Cas9 Activity in the CRISPR/Cas9 system.