# Carbon Nano-Onion Peroxidase Composite Biosensor for Electrochemical Detection of 2,4-D and 2,4,5-T
## 3 Results and Discussion
### Characterization of Modified Electrodes  
Cyclodextrins possess the remarkable property to form inclusion complexes with a wide variety of hydrophobic guests [26]. Modified and polymeric CDs were widely used as a platform for enzyme immobilization [27] and electrode modification in biosensors [28], so we hypothesized that the combination of inclusion ability and immobilization platform might be beneficial in the construction of a biosensor for 2,4-D and 2,4,5-T based on inhibi  
Figure 2: **Left:** Calculated 3D structure for the interaction of 2,4-D (**a**) and 2,4,5-T (**c**) with the active site of HRP. **Right:** Tertiary structure of HRP with 2,4-D (**b**) and 2,4,5-T (**d**) bound to the active site. Inset: lateral view (rotated \(\sim\)90\({}^{\circ}\) from the above). tion of the enzymatic activity of HRP (Figure 3). To prepare the biosensor, a CNO/HRP conjugate was first prepared by covalent attachment of maleimide activated HRP to the carboxylic acid groups of ox-CNO previously modified with cysteamine. This conjugate contained an average of three HRP molecules per CNO as determined by atomic absorption spectroscopy [29] and was further cross-linked with a thiolated CD polymer to form a coating composite. Figure 4a shows an HRTEM image of the starting CNOs obtained from nanodiamond annealing in which the multilayered graphitic structure is clearly visible. TEM images of a thin film of the CD/CNO/HRP conjugate show a network-like distribution of the CNO/HRP aggregate nanoparticles in the organic polymer matrix (Figure 4b). The CD/CNO/HRP composite completely covers the electrode surface forming a thin film of \(\sim\)2 \(\upmu\)m thickness. The surface shows a markedly rough and filamented morphology as compared with the bare electrode due to the presence of the CNO/HRP conjugate (Figure 4c,d) as revealed by ESEM analysis. Figure 3: Preparation of SPE/CD/CNO/HRP electrodes. Figure 5a shows the cyclic voltammograms (CV) of bare SPE, SPE/CD/CNO/HRP and a control sensor without CNO (SPE/CD/HRP) using Fe[(CN)\({}_{6}\)]\({}^{3-}\) in KCl as an electroactive marker. SPE/CD/CNO/HRP showed a notably higher peak current signal as compared to SPE/CD/HRP and SPE. The electroactive area of the electrodes accessible to the redox probe was determined using the Randles-Sevcik equation: \(i_{p}\) = 2.69 \(\times\) 10\({}^{5}\)D\({}^{1/2}\)n\({}^{3/2}\)v\({}^{1/2}\)Ac, where \(i_{p}\) is the peak current (A), \(D\) is the diffusion coefficient of Fe[(CN)\({}_{6}\)]\({}^{3-}\) in aqueous solution (7.3 \(\times\) 10\({}^{-6}\) cm\({}^{2}\) s\({}^{-1}\)) [30], \(n\) is the number of transferred electrons by Fe[(CN)\({}_{6}\)]\({}^{3-}\) (\(n\) = 1), \(v\) is the scan rate (V s\({}^{-1}\)), \(A\) is the electroactive surface (cm\({}^{2}\)) and \(c\) is the concentration of the marker (mol cm\({}^{-3}\)). Based on this equation, by plotting the values of \(i_{p}\) versus \(v^{1/2}\), the calculated electroactive surface of were (0.69 \(\pm\) 0.03) cm\({}^{2}\), (0.59 \(\pm\) 0.04) and (0.51 \(\pm\) 0.01) cm\({}^{2}\) for SPE/CD/CNO/HRP, SPE/CD/HRP and SPE, respectively, which indicate that the composites deposited on SPE significantly increased their surface area. The presence of CNO also increased the peak current by 42% in comparison to SPE/CD/HRP and decreased the peak-to-peak separation from 183 mV in the SPE/CD/HRP electrode to 149 mV in SPE/CD/CNO/HRP. This indicates that the CNOs also play an important role in the composite by facilitating electron transfer across the solution/electrode interface. Figure 5b shows the CVs of bare and modified electrodes using TMB in PBS as an electroactive marker. In all cases, two current peaks appear in both oxidation and reduction sweeps, corresponding to the exchange of one electron in each case. At the bare electrode, these pairs of peaks are well resolved, while in the modified electrodes, the peaks are broad, and their intensity decreases in the order SPE/CD/CNO/HRP \(>\) SPE/CD/HRP \(>\) SPE, similar to what was observed for Fe[(CN)\({}_{6}\)]\({}^{3-}\).