# Carbon Nano-Onion Peroxidase Composite Biosensor for Electrochemical Detection of 2,4-D and 2,4,5-T
## 3 Results and Discussion
### _Amperometric Detection of 2,4-D and 2,4,5-T_  
As shown above, 2,4-D and 2,4,5-T behave as reversible competitive inhibitors; hence by measuring the amperometric response of the biosensors at increasing concentrations of the herbicides, it was possible to construct calibration plots (% inhibition vs. concentration) for both of them and estimate the analytical parameters based on a two-step assay protocol (Scheme 1a). To reach the highest sensitivity, various conditions such as substrate concentrations and incubation times were first optimized. These steps are necessary due to the heterogeneous nature of the biosensor interface given by the active membrane that has a finite permeability to ions and reagents. For this, the biosensors were incubated with the substrate for a given time, and the reduction current due to oxidized TMB was measured at a fixed potential (Scheme 1b). Hence, an important parameter to optimize in this type of system is the time the biosensors are kept in contact with the inhibitors (step 2 in Scheme 1a). In reversible competitive inhibition, the inhibitor and substrate compete for the active site of the enzyme, and for a constant substrate concentration, the activity decreases with increasing inhibitor concentration. In our case, this interaction requires some time to stabilize due to the heterogeneity of the thin film containing the enzyme, and, as seen in Figure 6a, the current decrease with respect to the non-inhibited system reached a maximum after 3 min of exposure to the herbicides. Another important parameter is the time needed to attain the maximum current response in steps 1 and 3 of Scheme 1a. Figure 6b shows the current progress vs. time of SPE/CD/CNO/HRP with and without the presence of a fixed concentration of the herbicides. As seen in the figure, the steady-state current is reached between 50 and 60 s after exposing the biosensors to the substrate solution, and the current response remained stable for several minutes. Hence, after measuring the initial substrate signal (step 1 in Scheme 1a), the biosensors were incubated for 3 min with the  
Figure 5: Cyclic voltammograms of 0.1 M KCl (**a**, **inset**), 1 mM K\({}_{3}\)[Fe(CN)\({}_{6}\)] in 0.1 M KCl (**a**) and 0.5 mM TMB hydrochloride in 0.1 M PBS pH 6.5 (**b**). Scan rate: 100 mV/s. herbicide (step 2 in Scheme 1a) before reading the amperometric signal for \(\sim\) 1 min (step 3 in Scheme 1a). The ability of many toxic substances to inhibit the catalytic activity of enzymes has contributed to the development of biosensors for the detection and quantification of pollutants such as herbicides and pesticides in general [13]. The relative inhibition of enzyme activity was correlated with the herbicide concentration by measuring the decrease in amperometric response of the biosensors in the absence and presence of 2,4-D and 2,4,5-T using the optimized conditions described above. Figure 7 shows the calibration curves of 2,4-D and 2,4,5-T for SPE/CD/CNO/HRP and control SPE/CD/HRP biosensors. The inhibition percent initially increases almost linearly with pesticide concentration then tends slowly to saturation, with a maximum inhibition percent of 44% for 2,4-D and 55% for 2,4,5-T. Table 1 shows the analytical parameters for the biosensors assuming a linear relationship below 1 uM. In the CNO-containing biosensor, the sensitivity was higher, and the LOD was lower than those obtained with the SPE/CD/HRP biosensors, similar to what was previously observed with other CNO-based transducer surfaces [2,4,5] and due to the enhancement of electron transfer provided by the CNO-nanoparticles. It should also be noted that the LODs obtained (<10 u/L) are well below the maximum contaminant level of 0.07 and 0.05 mg/L allowed in the United States for 2,4-D and 2,4,5-T herbicides and the value recommended by the World Health Organization for 2,4-D (30 ug/L).