Fat mass and obesity-associated protein (FTO) is the first reported N⁶-methyladenosine (m⁶A) RNA demethylase. The dysregulation of FTO demethylation is strongly associated with various human cancers in a m⁶A-dependent manner. Herein, a homogeneous electrochemiluminescence (ECL) method for the determination of FTO was proposed based on the target-regulated DNAzyme cleavage. Moreover, the ECL signal was highly enhanced by host-guest interaction between β-cyclodextrin (β-CD) and tri-n-propylamine (TPrA). The m⁶A caged DNAzyme 17E-Me acted as a padlock, while the FTO served as the corresponding key. As the key, FTO could specifically remove m⁶A modification, restoring the cleavage activity of DNAzyme 17E. With the assistance of the Zn²⁺ cofactor, the substrate strand was cleaved at a specific site, and the ECL indicator of Ru(phen)₃²⁺ was discharged to produce an ECL signal. On the contrary, 17E-Me was blocked and no cleavage reaction occurred without the key. For the ECL detection, the electrode modification of β-CD@AuNPs concentrated Ru(phen)₃²⁺ species through electrostatic adsorption and gathered TPrA molecules through host-guest interaction with β-CD, which resulted in an intense ECL response. The results demonstrated the ECL intensity linearly correlated with the logarithm of the FTO concentration (from 0.0001 to 100 nM) with a low detection limit (30 fM). The IC₅₀ value for FTO inhibitors rhein and meclofenamic acid were 35.6 μM and 20.3 μM, respectively. The strategy was further validated for FTO detection in MCF-7 cell lysates and Hela cell lysates. This work reveals that this strategy is promising for developing homogeneous ECL method for detection of FTO and screening of the demethylase inhibitors.

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