Double-enzymes-mediated Fe2+/Fe3+ conversion as magnetic relaxation switch for pesticide residues sensing

Highlights: • A double-enzymes-mediated Fe²⁺/Fe³⁺ conversion strategy was developed for acetamiprid residue detection (2.66 ng mL⁻¹). • Fe²⁺ as T₂ signal transformation and amplification can show stable and sensitive signal out-put. • Fe²⁺, ACh, AChE and CHO are optimized to obtain the best reaction system with one-step mixing assay. • The sensor showed a 335-fold improvement in the sensitivity than that of TEIA method (0.89 μg mL⁻¹). It is a great challenge to develop a newly rapid and accurate detection method for pesticide residues. In this work, based on acetylcholinesterase (AChE) and choline oxidase (CHO), a double-enzymes-mediated Fe²⁺/Fe³⁺ conversion as magnetic relaxation switch was explored for the measurement of acetamiprid residue. In the double-enzymes reactions, acetylcholine chloride (ACh) can be catalyzed to produce choline by AChE, which is successively hydrolyzed to betaine and hydrogen peroxide (H₂O₂) by CHO. According to the enzyme inhibition principle, AChE activity will be inactivated in the presence of acetamiprid, thus leading to the less production of H₂O₂. Wherein, Fe²⁺, ACh, AChE and CHO were optimized as the reaction substrates. In the reaction system, acetamiprid can be reflected by the transverse relaxation time (T₂) that related with H₂O₂ mediated Fe²⁺ variations, which was further developed as an enzyme cascade amplification method. The detection linear range is 0.01∼1000 μg mL⁻¹ (R² = 0.99), and the limit of detection (LOD) is 2.66 ng mL⁻¹ (S/N = 3, n = 3), behaving a 335-fold improvement in LOD than that of traditional enzyme inhibition method (0.89 μg mL⁻¹). This method can realize "one-step mixing" detection of acetamiprid, which makes it a promising analytical tool for monitoring pesticide residue in complicated samples.