High-sensitive detection of organophosphate and carbamate pesticide residues in milk based on bioluminescence method

Organophosphorus pesticides (OPs) and carbamate pesticides (CBs) are the most commonly used pesticides in agricultural cultivation. The pesticide residues in plant products can easily enter dairy cows through feed, resulting in the pesticide low-concentration residues in milk. Traditional acetylcholinesterase (AChE) inhibition-based colorimetric methods have low sensitivity and could not satisfy the detection of low-concentrations of OPs and CBs residues in dairy products. In this study, we combined firefly luciferase (FLuc)-mediated bioluminescence with the inhibition of AChE-catalyzed substrate activity by pesticides to develop a highly sensitive and rapid method for detecting OPs and CBs residues in milk. AChE breaks down D-luciferin acetate to produce D-luciferin, which is recognized by the FLuc and emits luminescence in the presence of ATP. However, the presence of OPs and CBs inhibits AChE, causing the reduction or disappearance of the luminescence signal. The luminescence signal can be detected using a hand-held luminescence photometer, eliminating the need for large instrumentation. The AChE-FLuc system accurately detected OPs and CBs in milk within 30 min, with detection limits of 7.89 ng/mL and 1.75 ng/mL, respectively. The sensitivity of this method is approximately ten times higher than that of traditional AChE inhibition methods, meeting the pesticide residue limits in milk set by China and the European Union.