A novel muscarinic receptor-based cellular bioanalytical method for sensitive detection and differentiation of highly toxic organophosphorus compounds

Highly toxic organophosphorus compounds (OPs), including pesticides and nerve agents, pose a significant threat to food security and public health. The emergence of novel nerve agents, Novichoks, coupled with artificial intelligence (AI)-driven rapid creation of potentially unknown toxic OPs, has created unprecedented challenges for detection and identification. Traditional methods have limitations for screening and identifying these emerging or unknown OPs, highlighting the urgent need for sensitive, high-throughput, and non-targeted screening strategies. Here, we developed a novel muscarinic receptor-based cellular bioanalytical method using recombinant U2OS cells expressing the M3 receptor and the enhanced green fluorescent protein (EGFP)-labeled nuclear factor of activated T-cells c1 (NFATc1). Normally, acetylcholinesterase (AChE) hydrolyzes acetylcholine (ACh), thus preventing ACh-induced EGFP-NFATc1 nuclear translocation in U2OS cells. However, when exposed to OPs, AChE activity is inhibited, leading to an accumulation of ACh that activates the M3 receptor and triggers a noticeable EGFP-NFATc1 nuclear translocation. This method achieves low detection limits: 30 pM for Novichok A230, A232, and venomous agent X (VX); 0.1 nM for soman (GD) and A234; 0.3 nM for tabun (GA) and sarin (GB); 1 nM for paraoxon. It also offers multi-parameter cytological information post-OPs exposure, enabling the differentiation of various AChE inhibitors and exploration of novel toxicological pathways. Moreover, this method has been successfully applied to detect highly toxic OPs, such as VX and paraoxon, in spiked food matrices. This new method offers a reliable and efficient tool for the sensitive, high-throughput screening and identification of both known and unknown toxic substances with AChE inhibitory activity.