An APE1-assisted signal amplification strategy for highly sensitive and rapid analysis of mercury ions

Mercury (Hg²⁺) is a highly toxic heavy metal pollutant whose environmental persistence and biomagnification through the food chain pose serious risks to ecosystems and human health. Hence, there is considerable interest in developing sensitive, selective, and convenient methods for its detection. Herein, we report a novel fluorescent sensor for ultrasensitive and rapid analysis of Hg²⁺, based on apurinic/apyrimidinic endonuclease 1 (APE1)-assisted signal amplification. The sensor comprises a hairpin reporter probe (RP) containing an abasic (AP) site and dually labeled with CY3 and BHQ2, along with a T-rich helper probe (Helper). Upon addition of Hg²⁺, the Helper hybridizes to the T-rich loop of RP through T-Hg²⁺-T coordination, forming a duplex that enables APE1 to cleave the AP site efficiently. This cleavage releases a short CY3-labeled fragment, generating a strong fluorescence signal. Simultaneously, both Hg²⁺ and the Helper are regenerated, allowing successive cleavage cycles to occur and thereby achieving multiple signal amplification. The entire assay is performed in a homogeneous solution in a single step, with a total reaction time of 40 min. Under optimal conditions, the sensor achieves a detection limit as low as 2.7 pM and a linear dynamic range of 0.01-10.0 nM, and it exhibits excellent selectivity against common interfering metal ions. Spike-recovery tests in real water samples gave recoveries between 95.0% and 104.8%, showing no significant deviation from results obtained by ICP-MS. This strategy is operationally simple, cost-effective, and highly sensitive, offering a promising tool for rapid screening of trace Hg²⁺ in environmental and food safety monitoring.