# Carbon Nanodots as Peroxidase Nanozymes for Biosensing
## 3 Carbon Nanodots (C-Dots) as POD Nanozymes  
In pioneering work, [PERSON], [PERSON] and co-workers [36] discovered that C-dots from candle soot can exhibit POD-like catalytic activity. In particular, in the presence of H\({}_{2}\)O\({}_{2}\) in sodium acetate buffer, C-dots could catalyze the three POD substrates, namely, TMB, OPD and THB, producing typical color reactions (Figure 3). It was concluded that the catalytic mechanism is probably due to an increase in the electron density and mobility in the C-dots due to the electron transfer from TMB to C-dots. This results in an acceleration of electron transfer from C-dots to H\({}_{2}\)O\({}_{2}\) and thus increase the rate of TMB oxidation. Under the optimized conditions (pH 3.5, 35 \({}^{\circ}\)C, and 300 mM H\({}_{2}\)O\({}_{2}\)), C-dots exhibited maximum catalytic activity, which was strongly dependent on different conditions such as pH, temperature, and H\({}_{2}\)O\({}_{2}\)/C-dots concentration, similar to that of HRP. Nevertheless, in contrast to HRP, the catalytic activity of C-dots remained stable over a wide pH range (2-12) as well as temperature (0-90 \({}^{\circ}\)C). The kinetic data revealed that C-dots has higher affinities to H\({}_{2}\)O\({}_{2}\) and POD substrates and display a ping-pong mechanism, similar to HRP. Glucose oxidase (GOx) also known as notatin (EC 1.1.3.4) is a dimeric oxido-reductase protein, widely used in POD reactions in the determination of free glucose in blood plasma for diagnostics. Figure 3: **(A)** Schematic illustration of oxidation color reaction of TMB by H\({}_{2}\)O\({}_{2}\) catalyzed by C-dots; **(B)** Time-dependent absorbance changes at 652 nm of TMB in different reaction systems: (a) C-dots + TMB, (b) TMB + H\({}_{2}\)O\({}_{2}\) and (c) TMB + C-dots + H\({}_{2}\)O\({}_{2}\) in sodium acetate buffer (0.2 M) at pH 3.5 and a temperature of 35 \({}^{\circ}\)C. Reproduced with permission from [36]. Copyright (2011) Royal Society of Chemistry. By combining the POD-like activity of C-dots with GOx, a simple, cheap, selective, and sensitive colorimetric assay was developed to detect glucose in serum samples [36]. In parallel with this work, [PERSON] and co-workers [37] also prepared C-dots from candle soot and examined their POD-like activity. In contrast to HRP, CNTs, graphene oxide (GO) and Fe\({}_{3}\)O\({}_{4}\) NPs, the catalytic activity of C-dots was found to increase with increasing temperature with activation energy of ca. 59.3 kJ/mol. Based on the intrinsic enzyme activity of C-dots, a quantitative assay was developed for colorimetric detection of glucose in real samples, such as diluted blood and fruit juice. Aside from their intrinsic POD-like activity, C-dots can act as electron donors and transporters, and were successfully used to form C-dots-porphyrin supramolecular composites by making the use of electrostatic and \(\pi\)-stacking interactions. These two initial studies set the new directions and opened the door for the development of a variety of C-dots as well as their composites as POD nanzymes. [PERSON], [PERSON] and co-workers [38] relied on the use of an eco-friendly, cost-effective, and additive-free one step electrochemical approach for the preparation of C-dots (3-6 nm), without the assistance of any chemicals but only ultra-pure water in the reaction system (Figure 4). The as-prepared C-dots exhibited remarkable PL properties and mimicked POD functions in the degradation of methyl orange (MO). Upon mixing H\({}_{2}\)O\({}_{2}\) and MO (pH 7.0) with C-dots, only a moderate degradation of MO (23.6%, 160 min) was recognized. However, a better catalytic activity was achieved in the presence of visible light and MO could be photo-decomposed completely within 80 min. Following a hydrothermal route, the protocol was further extended to fabricate a hybrid photocatalyst (TiO\({}_{2}\)/C-dots) with an excellent visible-light-driven photocatalytic activity. [PERSON] and co-workers [39] introduced a MW-assisted ionic liquid (MW-IL) method to generate C-dots with narrow size distribution.