## 3 Results and Discussion
### AChE Assay Optimization
#### 3.1.2 Studied CMs and OPs Inhibitory Strength and AChE Assay Specificity  
The inhibition potency of the studied pesticides largely differed, with carbofuran being the strongest inhibitor followed by chlorpyrifos-oxon and malaoxon (Table 2). Worthy to notice that the tested oxon forms (OPs with a P=O moiety) were much stronger inhibitors compared to the thiono analogues (OPs with a P=S moiety). This fact was expected due to the low sulfur electronegativity of the P=S moiety resulting in low reactivity with the serine hydroxyl group at the active site of AChE. On the other hand, oxon forms (P=O) have relatively higher affinity for AChE active site compared to acetylthiocholine ([PERSON]'s substrate), thus they undergo nucleophilic substitution reaction by serine hydroxyl group resulting in a covalent binding of a pesticide to AChE [30]. Regarding the oxidation, this physiologically takes part during the metabolism, probably by the mixed function oxidases (MFO), resulting in a stronger AChE inhibitor [31]. Except the oxidation, which can occur at the P=S bond, also hydrolysis is possible [32], for example under acidic induced conditions resulting in phosphate ester hydrolysis and phosphoryl transfer [33]. Enzymatic hydrolysis of organophosphates also acts as a detoxification process exploiting various esterases and amidases depending the organism [34]. To assess AChE specificity towards carbofuran (the strongest AChE inhibitor), the cross-reactivity was calculated (Table 2). In the case of CM compounds, the cross-reactivity with aldicarb and carbaryl was less than 2% whilst with carbofuran-3-hydroxy (a carbofuran metabolite) was about 14% meaning that about 10-times higher concentration of carbofuran-3-hydroxy is necessary to achieve the same inhibition as in the case of carbofuran. Regarding chlorpyrifos, a widely used insecticide occasionally exceeding the EU maximum residue limits (MRLs) in various matrices, a very low cross-reactivity was calculated (about 0.024%). Nevertheless, chlorpyrifos-oxon and malaoxon showed a significant cross-reactivity (about 45% and 25%, respectively), which cannot be considered a problem for carbofuran detection in real food matrices because the P=S bond is metabolically oxidized to P=O. In other words, chlorpyrifos-oxon and malaoxon forms cannot occur in plant crops. In terms of dichlorvos, paraoxon, malathion and parathion, the cross-reactivity was negligible (<3% in all cases). All in all, a minor cross reactivity was noticed only for carbofuran-3-hydroxy. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Assay Characteristic** & **Ellman’s** & **IDA** & \(\alpha\)**-NAc** \\ \hline product color & yellow (412 nm) & blue (670 nm) & purple (525 nm) \\ AChE activity, U well\({}^{-1}\) & 0.009 & 0.75 & 1.1 \\ total assay time & 15 min & 45 min & 15 min \\ \({}_{\text{IC50}}\) for dichlorvos, \(\mu\)M, \(n=9\)1  & 3.5 (1.7–7.0) & 13 (8.3–22) & 11 (7.4–19) \\ IC\({}_{50}\) for carbofuran, \(\mu\)M, \(n=9\)1  & 0.099 (0.092–0.11) & 0.27 (0.22–0.36) & 0.23 (0.18–0.31) \\ cost, euro/plate & 0.16 & 14 & 22 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Critical comparison of Ellman’s, IDA and \(\alpha\)-NAc assays. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Pesticide** & **Structure** & **IC\({}_{20}\), \(\mu\)M 1** & **IC\({}_{50}\), \(\mu\)M 1** & **Cross-Reactivity \%** \\ \hline carbofuran & 0.037 (0.033–0.041) & 0.099 (0.092–0.11) & 100 \\ aldicarb & 2.1 (1.1–4.0) & 6.1 (4.4–8.5) & 1.6 \\ carbofuran-3-hydroxy & 0.25 (0.20–0.30) & 0.72 (0.62–0.83) & 14 \\ carbaryl & 2.1 (1.1–3.7) & 6.2 (4.5–8.5) & 1.6 \\ chlorryprifos & 251 (157–401) & 420 (298–594) & 0.024 \\ dichlorvos & 0.081 (0.050–0.13) & 0.22 (0.16–0.30) & 45 \\ dichlorvos & 1.2 (0.80–1.9) & 3.5 (1.7–7.0) & 2.8 \\ malaoxon & 0.92 (0.49–1.8) & 0.40 (0.25–0.62) & 25 \\ malathion & 85 (72–96) & 294 (220–305) & 0.034 \\ paraoxon & 0.92 (0.49–1.8) & 2.9 (1.7–4.8) & 3.4 \\ paraathon & 125 (102–145) & 357 (330–395) & 0.028 \\ \hline \hline \end{tabular}
\end{table}