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This approach has been validated for paraoxon previously [19].
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Starting from AE, we then performed the reverse evolution to restore phosphotriesterase activity using an experimental setup equivalent to the forward process with the following modifications: the pre-screen was carried out using a fluorogenic phosphotriester as a surrogate for paraoxon (Supplementary file 1) and then validated in 96-well format as described above.
The limit of detection for paraoxon, based on absorbance detection, was calculated to be 6 nM.
The two analytes were determined with prediction errors of 0.4 μg/l for paraoxon and 0.5 μg/l for carbofuran.
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