# Low-Cost Nanocarbon-Based Peroxidases from Graphite and Carbon Fibers
## 2 Results and Discussion
### The Peroxidase Catalytic Activities of Carbon Nanomaterials  
It has been reported that some carbon nanomaterials have the peroxidase-like activity, which can catalyze the oxidation reaction between H\({}_{2}\)O\({}_{2}\) and 3,3',5,5'-tetramethylbenzidine (TMB), resulting in a blue colored product [16]. In current system, after addition of both H\({}_{2}\)O\({}_{2}\) and TMB into the Carbon nanomaterials-2 solution, the solution can change to blue (the tube 4 in Figure 4a). While only TMB solution or the TMB and H\({}_{2}\)O\({}_{2}\) solution shows colorless (the tubes 1 and 2 in Figure 4a), and when adding TMB to the Carbon nanomaterials-2 solution, the solution shows brown due to the intrinsic color of Carbon nanomaterials-2 (the tube 3 in Figure 4a). Meanwhile, Carbon nanomaterials-1 and Carbon nanomaterials-3 have the same results in the color reaction. We noticed that there was no metal catalyst in these carbon nanomaterials from the XPS spectrum as shown in Figure 2. These result indicated that three carbon nanomaterials in our case possessed peroxidase-like catalytic activity. In addition, the profile of the time-dependent absorbance change at 652 nm (Figure 4b) indicated that the reaction rate and absorbance change greatly increased with increaseing of the Carbon nanomaterials-2 concentration. When the concentration of Carbon nanomaterials-2 is up to 40 \(\mathrm{\SIUnitSymbolMicro m}\mathrm{\SIUnitSymbolMicro m}^{-1}\), the reaction rate reaches the maximum value. Typically, the catalytic activity of peroxidase-like carbon nanomaterials mainly depends on the pH value, temperature and concentration of H\({}_{2}\)O\({}_{2}\). Figure S4a shows that the reaction is pH dependent, and the optimum pH value is 4.0, and the catalytic activity can be terminated by the addition of excess acid. As shown in Figure S4b, the temperature could consistantly accelarate the reaction in the tested range. Therefore the optimal temperature is kept at 35 \(\mathrm{\SIUnitSymbolCelsius}\), which is very beneficial for practical detection. Figure S4c shows the kinetic behaviors of H\({}_{2}\)O\({}_{2}\) function system. In the presence of  
Figure 4: (**a**) Typical photographs of 1 mL of 800 \(\mathrm{\SIUnitSymbolMicro m}\) 3,3’,5,5’-tetramethylbenzidine (TMB) reaction solutions oxidized by Carbon nanomaterials-2 in the presence of H\({}_{2}\)O\({}_{2}\) after 5 min incubation at 35 \(\mathrm{\SIUnitSymbolCelsius}\) in 25 mM phosphate buffer (pH 4.0): (1) 800 \(\mathrm{\SIUnitSymbolMicro m}\) TMB; (2) 50 mM H\({}_{2}\)O\({}_{2}\) and 800 \(\mathrm{\SIUnitSymbolMicro m}\) TMB; (3) 800 \(\mathrm{\SIUnitSymbolMicro m}\) TMB and 40 \(\mathrm{\SI  
fixed carbon nanomaterials, absorbance at 652 nm increases rapidly before 150 mM of H\({}_{2}\)O\({}_{2}\) and then increases slightly after 150 mM of H\({}_{2}\)O\({}_{2}\). The results demonstrate that the catalytic reaction is a H\({}_{2}\)O\({}_{2}\) concentration-dependent process, and the system could be applied in the determination of H\({}_{2}\)O\({}_{2}\). The peroxidase catalytic activities of different carbon nanomaterials were compared in the oxidation reaction between H\({}_{2}\)O\({}_{2}\) and TMB (Figure S5). The results show that the three kinds of carbon nanomaterials can significantly accelerate the reaction, of which Carbon nanomaterials-2 and Carbon nanomaterials-3 have better effects relative to Carbon nanomaterials-1. To further compare the catalytic activities of different carbon nanomaterials, state kinetic parameters were examined within an appropriate concentration range of TMB and H\({}_{2}\)O\({}_{2}\). In the experiment, the concentration of one substrate is fixed, and the concentration of the other substrate is variable. The initial velocity of the reaction can be obtained by measuring the relationship curve of absorbance at 652 nm and time. Lineweaver-Burk plot is used to calculate Michaelis Menten constant (\(K_{\text{m}}\)) and maximum reaction rate (\(V_{\text{max}}\)) of the three kinds of carbon nanomaterials (Figure S6).