DNA-mediated synergistic proximity ligation for ultrasensitive electrochemiluminescence detection of microcystin-LR

Abstract

A straightforward and practicable method to identify and detect low concentrations of small molecule microcystin-LR (MC-LR) is essential to clinical diagnostics, drug development, and environmental and food analysis. Here, we developed a novel electrochemiluminescence (ECL) aptasensor for sensitive detection of MC-LR based on DNA-mediated synergistic proximity ligation assay (PLA). The capture DNA was immobilized on the modified glassy carbon electrode with Ru(bpy)₃²⁺ (as the ECL signal), Nafion and gold nanoparticles (AuNPs) on its surface via a Au–S bond to obtain the ECL aptasensor. DNA1 was strategically designed with dual ferrocene (Fc) groups as quenchers at both its 5′ and 3′ ends, enabling simultaneous hybridization with capture DNA, MC-LR aptamer, and DNA2 which also contained a Fc group. In the absence of MC-LR, a proximate cooperative complex was formed among capture DNA, MC-LR aptamer, DNA1, and DNA2, drawing the Fc groups near the electrode surface and leading to a lower ECL signal. Conversely, in the presence of MC-LR, the target molecule MC-LR competed with capture DNA, DNA1, and DNA2 for the aptamer via a competitive reaction mode in a homogeneous solution, impeding the formation of a proximate cooperative complex and keeping the Fc groups far away from the electrode surface with a higher ECL intensity. The recovered ECL intensity rose in accordance with the MC-LR concentration, enabling the detection of MC-LR as low as 1.4 pg mL⁻¹ with a dynamic range of 0.005 ng mL⁻¹ to 5.0 ng mL⁻¹. Importantly, this method's synergistic proximity ligation effect was validated by comparing the ECL signals of the “4-DNA cooperative complex” versus “3-DNA cooperative complex” under identical conditions. The stronger ECL intensity observed in the “4-DNA cooperative complex” confirmed the synergistic proximity ligation effect, highlighting its critical role in facilitating the DNA hybridization. The developed PLA-based ECL aptasensor demonstrated high sensitivity and selectivity and quick response and it was successfully applied to the detection of MC-LR in tap water. The novelty of this work lies in the strategic design of a dual-quencher DNA probe and the implementation of a competitive, synergistic PLA, which together enable ultrasensitive detection of small molecules like MC-LR, a challenge for traditional PLA methods.