# Low-Cost Nanocarbon-Based Peroxidases from Graphite and Carbon Fibers
## 2 Results and Discussion
### H\({}_{2}\)O\({}_{2}\) Detection Utilizing Carbon Nanomaterials  
Our system could be applied in the determination of H\({}_{2}\)O\({}_{2}\) because the peroxidase-like activity of carbon nanomaterials is dependent on the concentration of H\({}_{2}\)O\({}_{2}\). As shown in Figure 5a,c,e, the absorbance keep increasing with the increase of H\({}_{2}\)O\({}_{2}\) concentration and the color reaction is obvious, which may be observed by naked eyes. As can be seen in Figure 5b,d,f, the absorbance at 652 nm shows a good linear relationship with the concentration of H\({}_{2}\)O\({}_{2}\) in the range of 0.02-0.2 mM when Carbon nanomaterials-1 and Carbon nanomaterials-2 are used. In the case of Carbon nanomaterials-1, the linear regression equation is \(A=0.34C+0.043\) with a correlation coefficient  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Catalyst** & **Substrate Varied** & **Substrate Fixed** & \(K_{\text{m}}\) & \(V_{\text{max}}\) \\  & & **(mM)** & **(mM)** & **(10\({}^{-8}\) M\(\cdot\)s\({}^{-1}\))** \\ \hline Carbon nanomaterials-1 (this work) & TMB (3,3’,5,5’- & \\  & Tetramethylbenzidine) & H\({}_{2}\)O\({}_{2}\) (25) & 2.45 & 155.9 \\ Carbon nanomaterials-1 (this work) & H\({}_{2}\)O\({}_{2}\) & TMB (0.2) & 0.01 & 10.31 \\ Carbon nanomaterials-2 (this work) & TMB & H\({}_{2}\)O\({}_{2}\) (5) & 1.06 & 337.7 \\ Carbon nanomaterials-2 (this work) & H\({}_{2}\)O\({}_{2}\) & TMB (0.2) & 0.001 & 55.13 \\ Carbon nanomaterials-3 (this work) & TMB & H\({}_{2}\)O\({}_{2}\) (5) & 0.97 & 328.5 \\ Carbon nanomaterials-3 (this work) & H\({}_{2}\)O\({}_{2}\) & TMB (0.2) & 0.0006 & 27.69 \\ \(N\)-GODs (\(N\)-doped graphene & TMB & H\({}_{2}\)O\({}_{2}\) (3.3) & 11.2 & 0.38 \\ \(N\)-GODs (Ref. [27]) & H\({}_{2}\)O\({}_{2}\) & TMB (0.3) & 0.10 & 0.14 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Michaelis-Menten constant (\(K_{\text{m}}\)) and maximum reaction rate (\(V_{\text{max}}\)) of different carbon nanomaterials in this work and references.  
of 0.9937; in the case of Carbon nanomaterials-2, the linear regression equation is \(A=1.484C+0.065\) with a correlation coefficient of 0.9943. In the case of Carbon nanomaterials-3, as the H\({}_{2}\)O\({}_{2}\) concentration changes from 0.02 to 0.4 mM, the linear regression equation is \(A=0.637C+0.032\) with a correlation coefficient of 0.9884. The detection limit of the method using Carbon nanomaterials-1, Carbon nanomaterials-2, Carbon nanomaterials-3 are 0.024 mM, 0.0042 mM and 0.014 mM, respectively. It has been reported that the presence of functional group could affect peroxidase-like activity of carbon nanomaterials [30]. The ketonic carbonyl groups act as the catalytically active sites and the carboxylic groups serve as the substrate-binding sites. The existence of phenol and alcohol groups decreases the catalytic activity of carbon nanomaterials. The Carbon nanomaterials-2 prepared from natural graphite has better catalytic activity than Carbon nanomaterials-1 prepared from carbon fibers. The possible reason is that the surface of the Carbon nanomaterials-2 contains more ketonic carbonyl groups and carboxylic groups than that of Carbon nanomaterials-1 (Figure 3).  
Figure 5: UV-vis absorption spectra and linear calibration plot for H\({}_{2}\)O\({}_{2}\) detection using (**a**,**b**): Carbon nanomaterials-1; (**c**,**d**): Carbon nanomaterials-2; (**e**,**f**): Carbon nanomaterials-3. The solution of 800 \(\upmu\)M TMB was treated with 40 \(\upmu\)g\(\cdot\)mL\({}^{-1}\) carbon nanomaterials in phosphate buffer (pH 4.0) at 35 \({}^{\circ}\)C. The concentration of H\({}_{2}\)O\({}_{2}\) was changed from 0 to 16 mM. The absorption spectra were acquired with an UV-3600 UV-vis-NIR spectrometer (Shimadzu, Kyoto, Japan).