# Carbon Nanodots as Peroxidase Nanozymes for Biosensing
## 6 GQDs/C-Dots Conjugates and/or Nanocomposites as POD Nanozymes  
In pioneering work, [PERSON], [PERSON] and co-workers [53] prepared GQDs by irradiating a mixture of H\({}_{2}\)O\({}_{2}\) and GO's suspension using a mercury lamp (365 nm, 1000 W). The as-generated GQDs with an average size of 30 nm produced a blue color of oxidized TMB in the presence of H\({}_{2}\)O\({}_{2}\). Taking advantage of their structural, electric and excellent POD-like activities (over large-sized GO nanosheets), GQDs were covalently assembled on the surface of gold (Au) to develop a stable enzyme-like electrode. An illustrative fabrication process of the GQDs/Au electrode is shown in Figure 5. The electrochemical measurements revealed that the GQDs/Au electrode can display a fast amperometric response to H\({}_{2}\)O\({}_{2}\) with a wide linear range (0.002 to 8 mM) and a detection limit down to 0.7 uM. The detection limit was found comparable or even better than many HRP immobilized Au electrodes. Owing to its good electrocatalytic activity, facile preparation, and high stability, GQDs/Au electrode holds great potential as an enzyme-free electrode for sensing assays in pathology and physiology. Very recently, [PERSON] and co-workers [54] have reported a straightforward tactic to boost the low POD-like activity of cytochrome \(c\) (cyt _c_), an electron transfer protein in the respiratory chain, following its electrostatic assembly onto the C-dots surface. Specifically, citric acid (CA) as a carbon source was thermally carbonized alone as well as in the presence of various dopants/passivating agents such as urea (U), thiourea (T) and mercaptouscinic acid (M), to generate four different CA, CA-U, CA-T, and CA-M-derived C-dots. Figure 6 highlights the schematic postulated interactions between CA-derived C-dots and cyt \(c\). Specifically, either only CA-derived C-dots or bare cyt \(c\) exhibited minimal POD activity as observed by no (Figure 6 Ai) or little appearance (Figure 6 Aii) of green color  
Figure 5: Illustrative fabrication process of the GQDs/Au electrode. Reproduced with permission from [53]. Copyright (2013) Royal Society of Chemistry. Abbreviations: EDC, 1-Ethyl-3-(3-dimethylaminopropyl) carbodimide; NHS, \(N\)-hydroxysulfosuccinimide. in the H\({}_{2}\)O\({}_{2}\)-assisted oxidation of ABTS. However, in the presence of C-dots, the POD activity of cyt \(c\) enhanced markedly, manifested in a color change from colorless to green (Figure 6 Aiii) due to the oxidation of ABTS. The POD activity modulation of cyt \(c\) (which possesses a largely cationic surface with much of that positive charge residing proximal to the active heme site (Figure 6B)) in the presence of C-dots (having a negatively charged surface due to the presence of a complex cocktail of oxygen-containing functional groups) was attributed to the electrostatic-driven perturbation of heme microenvironment of cyt \(c\). The protein conformational changes (i.e., perturbation of secondary structure of cyt _c_) was evidenced using circular dichroism (CD) measurements, which indicate that an approximate \(\alpha\)-helical content of the cyt \(c\) decreased from an initial value of 24% to just 2.3% upon the addition of CA-derived C-dots. Meanwhile, the random content rose from 34% to 41%. These results indicate that the global protein fold is seriously altered and, in concert with changes local to the heme group, is responsible for the large catalytic enhancement. An exceptional catalytic efficiency (\(K_{\mathrm{cat}}/K_{\mathrm{M}}\)) of 8.04 \(\pm\) 1.74 \(\times\) 10\({}^{7}\) M\({}^{-1}\cdot\)s\({}^{-1}\) was determined for co-assemblies of C-dots/cyt \(c\), which were found to be close to the theoretical diffusion-controlled limit. Furthermore, the activity of the C-dots/cyt \(c\) assembly can be switched off simply by increasing the ionic strength, resulting in dissociation into non-catalytic components. This work suggests the appealing prospects for nanocarbon-based modulation of other potential Fe-centered POD mimics.