# Carbon Nanodots as Peroxidase Nanozymes for Biosensing
## 2 Fundamentals of C-Dots: An Overview  
Given the size-controllable, handy, cost-effective, eco-friendly, and large scale synthesis of C-dots, a variety of synthetic routes have been developed during the last decade. Most of these routes can be broadly categorized into 'Top-down' and 'Bottom-up' methods. Typically, in the 'Top-down' approaches, C-dots are prepared by means of breaking down bulk carbon materials such as CNTs, graphene and suspended C-powders [29], to highlight a few. Laser irradiation, electrochemical carbonization, plasma treatment and arc discharge are the routinely employed 'Top-down' approaches. On another front, in the 'Bottom-up' approaches, an organic precursor or molecular carbon sources such as sucrose, citric acid and amino acids are used as seeds to generate C-dots under certain experimental conditions. Hydrothermal or solvothermal carbonization, ultrasonic synthesis and microwave (MW)-assisted synthesis of C-dots can be recapitulated under 'Bottom-up' approaches. Among these, MW-assisted synthesis of C-dots is considered as an energy efficient and eco-friendly process which can shorten the reaction time significantly, a factor than can dominate over ultrasonic synthesis. In their pioneering work, [PERSON] and co-workers [30] first reported the use of MW technology for the synthesis of C-dots from the ashes of egg-shell membrane (ESM), a protein-rich waste. The typical schematic illustration for the MW-assisted formation of C-dots is highlighted in Figure 2.  
C-dots are chiefly composed of carbon, oxygen, hydrogen and nitrogen atoms, and the ratio of these elements can vary significantly in raw and purified C-dots. For instance, the chemical composition of the raw candle soot (91.7% C, 1.8% H, 1.8% N, 4.4% O) has been found hugely different from that of purified C-dots (36.8% C, 5.9% H, 9.6% N, 44.7% O) [24]. Nevertheless, most of the purified C-dots contain oxygen as the abundant element due to the presence of oxygen-containing functional groups, rendering them hydrophilic in nature. Aside from that of elemental analysis, C-dots can be conveniently characterized by high resolution transmission electron microscopy (HRTEM), atomic force microscopy (AFM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), UV-visible and fluorescence spectroscopy, thermogravimetric analysis (TGA), and \({}^{13}\)C solid state NMR measurements [31]. Most C-dots usually exhibit a typical absorption band around 260-320 nm, which may shift to longer wavelengths after surface passivation with amino or silane compounds. It is worthwhile to mention here that despite the elusiveness of its mechanism, surface passivation significantly affects the photoluminescence (PL) of C-dots and appears to be associated with the synthetic method used. The PL emission of C-dots, including both the excitation wavelength-dependent and independent, is one of the most appealing features and can be used to manipulate the photoinduced electron transfer [32] and thus may pave the way for novel applications in energy conversion. Although the exact mechanism(s)  
Figure 2: Schematic illustration of a MW-assisted synthesis of C-dots. Adapted with permission from [30]. Copyright (2012) Royal Society of Chemistry. of the PL of C-dots are still a matter of debate, some aspects are well recognized. It is speculated that recombination of excitons, quantum effect (due to the different-sized NPs, and/or the different emissive traps on the surface of C-dots significantly contribute to the phenomenon [20]. A closely related issue, which has drawn researchers' attention, is the quantum yield (QY), the classical signature of the PL properties of C-dots. To increase the QY, several approaches such as surface passivation, inorganic salt or element doping, and purification procedures have been surfaced from time to time and are significantly influencing the current trends in the biological applications of C-dots.