An adaptive and label-free colorimetric assay for EDTA using copper(II)-aptamer complexes as soft nanozymes

Copper is a vital trace element that plays a crucial role in various physiological processes due to its ability to exist in multiple oxidation states. Inspired by natural enzymes, researchers have developed copper-based nanozymes that mimic enzyme functions, offering cost-effective and stable alternatives to traditional enzymes. Despite their promising properties, the design and synthesis of these nanozymes can be complex and challenging. To address this issue, this work explores an intuitive approach to construct "soft" nanozymes simply by binding Cu²⁺ with its aptamers to form Cu(II)-aptamer complexes.

Using a screening strategy, we identified a specific aptamer against Cu²⁺, designated P8, that significantly enhanced peroxidase-like activity when complexed with Cu²⁺, demonstrated by a notably smaller K_m value. Furthermore, we investigated the effects of various lengths of a "molecular clamp" on the conformational dynamics of the P8 aptamer, which perturbs the encapsulation of Cu²⁺ and modulated its peroxidase activity. Our findings indicate that optimizing the molecular clamp to a moderate length (N_d = 20) can further enhance the peroxidase activity of the Cu²⁺-P8 complex. The peroxidase-like soft nanozymes based on copper(II)-aptamer complexes were finally utilized for label-free detection of EDTA. The developed colorimetric sensing method exhibits outstanding detection capabilities for EDTA in Sprite and mineral water samples, achieving an extended dynamic range that accommodates higher concentrations through the incorporation of molecular clamps.

This work highlights a novel and straightforward approach to generate and improve the enzymatic properties of copper-based nanozymes. The strategy to regulate the enzymatic activity effectively addresses the limitations of narrow detection ranges commonly observed in traditional methods.