# Carbon Nano-Onion Peroxidase Composite Biosensor for Electrochemical Detection of 2,4-D and 2,4,5-T
## 3 Results and Discussion
### Stability and Repeatability of the Developed Biosensors  
The long-term stability and repeatability of the biosensors were examined. Figure 8 shows the residual activities of immobilized HRP in the developed biosensors by monitoring their amperometric responses in 4-day intervals over a period of 4 weeks for electrodes stored in PBS buffer pH 6.5 at 4 \({}^{\circ}\)C. After the first week of storage, SPE/CD/CNO/HRP and SPE/CD/HRP maintain 97% and 85% of their initial activity, respectively, and then decreased to 77% and 58% after 4 weeks of storage. The presence of CNO covalently coupled to HRP, and the cyclodextrin matrix on the surface electrode improved the stability of SPE/CD/CNO/HRP compared to SPE/CD/HRP. This increased stability of the CNO biosensors might be attributed to Van der Waals interactions between the carbon nanoparticles and the enzyme resulting in a reduction in the enzyme mobility due to the anchorage to the support, thus shielding it from the denaturing effects of the envi  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Herbicide** & **Surface Chemistry** & \
\begin{tabular}{c} **Detection** \\ **Technique** \\ \end{tabular} &
\begin{tabular}{c} **Assay Time** \\ **(min)** \\ \end{tabular} & **LOD (\(\upmu\)M)** & **Reference** \\ \hline
2,4-D & \
\begin{tabular}{c} Catalase immobilized on porous \\ graphene \\ \end{tabular} &
\begin{tabular}{c} Amperometry, \\ enzyme inhibition \\ \end{tabular} & 10 & 0.15 & [16] \\
2,4-D & \
\begin{tabular}{c} Sulfatase based whole-cell \\ Agrobacteriumium biosensor \\ \end{tabular} &
\begin{tabular}{c} Fluorescence, \\ enzyme activity \\ \end{tabular} & overnight & 1.56 & [32] \\
2,4-D & \
\begin{tabular}{c} Iron (III) porphyrin on immobilized \\ on MWCNT \\ \end{tabular} &
\begin{tabular}{c} Amperometry, \\ electrocatalysis \\ \end{tabular} & 3 & 2.1 & [33] \\
2,4-D & \
\begin{tabular}{c} Quantum dot modified molecule \\ imprinting polymer \\ \end{tabular} &
\begin{tabular}{c} Fluorescence of \\ nitrobenzocadiazole \\ \end{tabular} & 5 & 0.14 & [34] \\
2,4-D & \
\begin{tabular}{c} Carbon nano-onion/HRP conjugate \\ on cyclodextrin polymer \\ \end{tabular} &
\begin{tabular}{c} Ampperometry, \\ enzyme inhibition \\ \end{tabular} & 5 & 0.023 & This work \\ \hline
2,4,5-T & \
\begin{tabular}{c} Alkaline phosphatase/sol-gel \\ chitosan film on carbon paste \\ electrode \\ \end{tabular} &
\begin{tabular}{c} Ampperometry, \\ enzyme inhibition \\ \end{tabular} & 15 & 1.9 & [15] \\
2,4,5-T & \
\begin{tabular}{c} Polyclonal antibody-based \\ immunoassay \\ \end{tabular} &
\begin{tabular}{c} Colorimetric, ELISA \\ \end{tabular} & 60 & 0.005 & [14] \\
2,4,5-T & \
\begin{tabular}{c} Polyclonal antibody on graphite \\ electrode \\ \end{tabular} &
\begin{tabular}{c} Potentiometry, \\ competitive assay \\ \end{tabular} & 10 & 200 & [35] \\
2,4,5-T & \
\begin{tabular}{c} Albumin-2,4,5-T immobilized in \\ membrane \\ \end{tabular} &
\begin{tabular}{c} Colorimetric, \\ competitive ELISA \\ \end{tabular} & 30 & 0.0028 & [36] \\
2,4,5-T & \
\begin{tabular}{c} Carbon nano-onion/HRP conjugate \\ on cyclodextrin polymer \\ \end{tabular} &
\begin{tabular}{c} Ampperometry, \\ enzyme inhibition \\ \end{tabular} & 5 & 0.010 & This work \\ \hline \hline \end{tabular}
\end{table}
Table 2: Comparison of reported detection methods for 2,4-D and 2,4,5-T.  
Figure 7: (a) Calibration curves of 2,4-D and 2,4,5-T obtained on SPE/CD/CNO/HRP and SPE/CD/HRP biosensors. (b) Detail of the curves below 1 \(\upmu\)M concentration and adjusted assuming a linear behavior.  
ronment, as observed recently for HRP and other enzymes immobilized on CNOs [21]. Regarding repeatability, SPE/CD/CNO/HRP is stable for 44 measurements as compared to SPE/CD/HRP (30 measurements), taken as a signal decrease of 10%. These results can also be explained based on the stability of the enzyme composites with CNO and the presence of the polymeric matrix.