# Carbon Nano-Onion Peroxidase Composite Biosensor for Electrochemical Detection of 2,4-D and 2,4,5-T
## 3 Results and Discussion  
### Inhibition Mechanism of 2,4-D and 2,4,5-T on the Activity of HRP  
The determination of the inhibition mechanism is an important step in the design of sensors based on the inhibition of enzyme activity. The dependence of the enzymatic activity versus HRP concentration in the presence of increasing concentrations of the herbicides is presented in Figure 1a,b. The obtained family of straight lines with intercepts essentially equal to zero indicate a reversible inhibition since increasing inhibitor concentrations provoked a decrease in the slopes of the lines.  
Since in our sensor HRP is part of a composite deposited on the electrode surface, we also studied the inhibition of HRP immobilized on maleimide plates using a thiolated cyclodextrin polymer as a cross-linker. The maximum enzyme activity inhibition (\(\sim\)60%) was attained after 15 min of the interaction of the herbicides with immobilized HRP (Figure 1a,b, inset). The kinetic behavior of HRP in the presence of 2,4-D and 2,4,5-T was also studied using the Michaelis-Menten formalism by constructing double-reciprocal Lineweaver-Burk plots (Figure 1c,d). As can be seen, both herbicides gave a family of straight lines with different slopes and the same intercept located on the \(1/V_{0}\) (vertical) axis, indicating a competitive inhibition. This means that they are bound to the free enzyme but not to the enzyme-substrate complex. This results in an increase of \(K_{m}\) of HRP without  
Figure 1: Top: Effect of 2,4-D (**a**) and 2,4,5-T (**b**) on the activity of HRP at different enzyme concentrations. Inset: Residual activity vs. time plots. Bottom: Lineweaver–Burk plots for the inhibition of 2,4-D (**c**) and 2,4,5-T (**d**) on the activity of immobilized HRP at different concentrations of pesticide. Inset: Secondary plots (slope vs. concentration and intercept vs. concentration) used for the determination of inhibition constants. Conditions: 10 mM phosphate-buffered saline pH 6.5, 25 \({}^{\circ}\)C.  
changing \(V_{max}\). The inhibitor constant (\(K_{l}\)) for the binding of these herbicides with the enzyme was estimated from the slope of the apparent Michaelis-Menten constant (\(K_{m\,app}\)) versus inhibitor concentration to be (210 \(\pm\) 20) and (560 \(\pm\) 40) \(\upmu\)M for 2,4,5-T and 2,4-D, respectively (Figure 1c,d, inset).  
To further understand the inhibition mechanism of HRP by 2,4-D and 2,4,5-T, their docking geometries were examined based on the X-ray crystal structure of HRP (PDB code 1 HCH) and fully deprotonated ligands (Figure 2). In both cases, the ligand is oriented with the carboxylate group pointing toward the inside of the cavity interacting with guanidine residue of Arg-38. The interactions are stabilized by a set of hydrophobic and \(\pi\) interactions. In 2,4-D (Figure 2a), the 2-Cl atom interacts with Leu-138 via \(\pi\)-\(\pi\) interaction, while the 4-Cl atom and the aromatic ring of Phe-68 form a halogen-\(\pi\) bridge. There is also a favorable \(\pi\)-\(\sigma\) interaction between the aromatic ring of 2,4-D and Ala-140, and the 4-Cl atom is exposed to the solvent (Figure 2b). Meanwhile, 2,4,5-T has a more complex interaction pattern with the active site of HRP (Figure 2c). The hydrophobic interaction involving the 4-Cl atom is similar to 2,4-D, but the interactions involving the other two chlorine atoms are different, with the 2-Cl atom halogen bound simultaneously to Phe-142 and Phe-179 and the 5-Cl atom to Leu-138, resulting in 2,4,5-T being rotated \(\sim\)180\({}^{\circ}\) as compared to 2,4-D. This more complex interaction pattern results in a deeper penetration in the active site (compare lateral views in Figure 2b,d) and is consistent with the lower \(K_{l}\) of 2,4,5-T with respect to 2,4-D. This means that a lower concentration of 2,4,5-T is required to produce half-maximum inhibition.