**2. Synthesis methods of carbon dots**

In 2004, during the purification process of single-walled carbon nanotubes (SWCNTs) using gel electrophoresis, the CDs were discovered [10]. Later, various procedures established for the synthesis of CDs. Broadly, the CDs synthesis methods can be classified into as "top-down" and "bottom-up" approaches (**Figure 1**).

### **2.1 Top-down method**

In the "top-down" approach, the breakdown of bulk carbon materials into nano size carbon occurs under relatively harsh conditions such as oxidative acid treatment, electrochemical exfoliation, laser ablation, and arc discharge [14]. The first described CDs were produced by the top-down process via laser ablation of graphite in the gaseous phase, subsequently acid oxidative treatment [15]. Later, numerous methods such as arc discharge, etching, electrochemical oxidation, ultrasonication, chemical exfoliation, and nitric acid/sulfuric acid oxidation developed to obtained CDs by reducing the size of bulk carbon materials [14, 16]. Mostly, graphite, graphene or graphene oxide (GO) sheets, carbon nanotubes (CNTs), carbon fibers

*Naturally Derived Carbon Dots as Bioimaging Agents DOI: http://dx.doi.org/10.5772/intechopen.96912*

and carbon soot etc. used as a precursor material in these methods. Even though these methods were successfully used to prepare CDs, they are limited with harsh conditions, complicated synthesis strategies, low quantum yield, expensive, eco-unfriendly, and unsuitable for the production of CDs in industrial-scale. These methods rarely used for the preparation of CDs from natural sources.

#### *2.1.1 Bottom-up method*

In the "bottom-up" approach, the CDs are synthesized from carbon-containing small molecules in a "polymerization– carbonization" process. Several methods include combustion, hydrothermal, solvothermal, and microwave-assisted pyrolysis developed in bottom-up synthesis [16]. Typically, in these methods carbon precursor such as small organic molecules taken in a liquid or gas stage are ionized, dissociated, sublimated or evaporated and then condensed via condensation, carbonization, polymerization and passivation to form nanosize CDs. Compared with the "top-down" strategy, the "bottom-up" approach is extensively used for the green synthesis of CDs using natural renewable sources. Here, we discussed some important synthesis methods of CDs.

**Acid oxidation:** In this method, CDs were synthesized by exfoliation and cleaving of activated carbon, graphene oxide, carbon nanotubes, carbon fibers and soot etc. by using concentrated acids such as sulfuric acid and nitric acid [17, 18]. Typically, this method involves the decomposing of the bulk carbon into nanoparticles and simultaneously introducing hydrophilic groups on the carbon core. Generally, these raw materials are low in cost, readily available and feasible for simple operation. This method can be extended for the synthesis of hetero atom doped carbon dots. For example, the heteroatom N doped CDs prepared using activated carbon as precursor and Nitric acid as oxidizing agent [19]. However, this method limited with some disadvantages such as harsh conditions and timeconsuming process to eliminate excessive acid.

**Electrochemical exfoliation:** This is a facile green and large-scale approach in which, CDs were prepared by avoiding excess concentrated acid, complex separation and purification process [20, 21]. In this method, high purity graphite used as anode and Pt wire used as a counter electrode. Distilled water can be used as electrolyte but the rate of reaction is very slow. In order to increase the rate of reaction, ionic liquids like 1- butyl-3 methylimidazolium tetrafluoroborate and 1-butyl 3-methylimidazolium hexafluorophosphate can be mixed with distilled water and can be used as electrolyte [22]. The electrochemical exfoliation carried by applying static potential through direct power which leads to corrosion of graphite anode and hence formation of CDs. The mechanism involves releasing carbon dots because of the electrochemical scissors OH− and O− ions from the water's anodic oxidation. Depending on the type of electrolyte nitrogen, phosphorus or boron can be doped in carbon dots.

**Laser ablation method:** The term ablation refers to the removal of surface atoms. Laser ablation method involves the absorption of highly energetic laser pulse by the carbon precursors and stripping of electrons from the atoms through a process like photoelectric effect generating a high electric field. Production of CDs takes place due to the repulsive force generated between positive ions and solid material [23, 24]. The size of the CDs can be controlled by a laser furnace. The precursors for laser ablation method are toluene, bulk graphite, graphene oxide and graphite powder etc. Laser ablation method provide high quality product with great velocity depending on the purity of the target and ambient media (gas or liquid). The size and other properties of carbon dots were controlled by irradiation time and laser fluence. The limitations of the method are requirement of high input energy and sophisticated equipment.

**Ultrasonic treatment:** In this green synthetic method, carbon materials can be broken down by the action of very high energy of ultrasonic waves [25, 26]. Ultrasonic waves create high pressure and low-pressure waves in liquid medium resulting in the formation, growth and violent collapse of small vacuum bubbles. The collapse of the bubbles lead to local high temperature and pressure up to 5000 K and 1000 atm respectively, producing the CDs. The precursors used for making CDs in this method are crab shell powder, glucose, active carbon, polyethylene glycol, citric acid, tri-ammonium citrate, and arginine. N, S, and P elements doped CDs can also be prepared by this method.

**Microwave synthesis:** This method involves the irradiation of electromagnetic radiations within a range of 1 mm to 1 m through the carbon precursor containing reaction mixture, which results from rapid and uniform heating. The microwaves absorbed by the solvent and precursor leading to the activation of molecules directly and its leads to formation of CDs [27, 28]. So, reaction volumes as such as 200 μl to >100 ml can be used without difficulty. The advantages of microwave irradiation are fast, higher efficiency and require less purification. The microwave irradiation can be controlled instantaneously so the risk of overheating is also minimized. However, the main drawback of this method is that solvents with lower boiling point cannot be used. This method widely used to convert bio-waste and natural sources such as plant materials, sea food waste and kitchen waste into CDs [29–31].

**Thermal decomposition:** In ordinary thermal decomposition, a carbon containing compound or substance decomposes chemically by action of heat and converted into CDs [32]. In general, CDs were synthesized from the variety of precursors like citric acid and L-cysteine etc. by simple heating under pyrolytic condition and controlled pressure using ionic liquid like 1-butyl 3-methyl imazonium bromide [33, 34]. The advantages of ionic liquid are high thermal stability, chemical stability; low melting point and low vapor pressure. At very high temperature, an irreversible thermal decomposition of organic matter takes place in inert mixture. Low cost, easy to operate, less time consuming and large-scale production are the advantages of the thermal decomposition method.

**Pyrolysis:** Pyrolysis is an irreversible thermal decomposition reaction in which decomposition of organic materials take place in inert atmosphere and at high pressure. Pyrolysis of the carbonaceous material is a simple, clean and inexpensive route for synthesizing CDs because no need of additives, acids or bases [35, 36].

*Naturally Derived Carbon Dots as Bioimaging Agents DOI: http://dx.doi.org/10.5772/intechopen.96912*

In this method, solid residues with high carbon content were formed from organic materials by prolonged pyrolysis in an inert mixture. During pyrolysis, dehydration and fragmentation occurs. The natural precursors used for producing CDs in this method are cheap biowaste materials like rice husk, coffee grounds, watermelon peel, sago waste, peanut shells and wool etc.

**Hydrothermal or solvothermal synthesis:** In hydrothermal synthesis, carbon precursors undergoes "polymerization–carbonization" and leads to formation of CDs in water media taken in a sealed container under high temperature and pressure [37]. In solvothermal synthesis, organic solvents like methanol, ethanol, n-butanol and N, N- dimethylformamide etc. can be used as the solvent instead of water [38, 39]. This method was proved to be a cheap and eco-friendly route to the synthesis of carbon dots. However, solvothermal reactions can lead to an explosion in a few cases because the temperature rises rapidly in limited space. This can be avoided by taking a small quantity of solvent and reactant.

#### *2.1.2 Natural materials as a green precursor for preparation of CDs*

The green synthesis of CDs relies on natural precursors such as plant materials, protein products and waste materials [40]. Compared with bulk carbon materials (Graphene, Graphene Oxide and carbon tubes etc.) and toxic organic compounds including aromatic molecules, natural materials are renewable, economical, eco-friendly, safer and easier to get industrial-scale production. Mostly, "bottomup" methods adopted for the synthesis of CDs due to the existing small organic molecules in natural sources can be carbonized into CDs at a specific temperature. A few top-down methods developed for the waste bulk carbon materials or biowaste are broken down/cut into small-sized CDs. Among the various techniques, hydrothermal and microwave approaches are extensively used to prepare CDs from multiple natural materials. Most natural sources or biomass materials are made with small organic molecules, converted into CDs by carbonization and pyrolysis.

In recent years, various kind of plant material such as coriander leaves, ginger, garlic, grass, coffee beans, lemon, orange juice etc. due to the existence of various carbon containing organic molecules including carbohydrates, cellulose and phenolic compounds [41]. Compared with commercial precursors, the plant material derived CDs showed enhanced fluorescence emission with high quantum yield due to heteroatoms such as nitrogen, sulfur and phosphorus. Hence, the optical and structural properties of CDs are mainly relying on the selection of natural precursors. Besides this, with the growing concern about environmental pollution and sustainability, variety of waste materials including different kind of agriculture, kitchen, fruit peel and seafood waste etc. used as a starting material for the preparation of CDs [30, 42, 43]. These waste resources, also containing organic molecules, can be polymerized and followed by carbonized to form CDs.

#### **2.2 General properties**

The general properties of CDs illustrated in **Figure 2**. Structurally, CDs belongs to the quasi-spherical zero-dimension carbon nanomaterials class with a size of less than 10 nm [44, 45]. They are amorphous or nanocrystalline cores with a typical sp2 carbon hybridization. The absorption band of CDs exhibits UV–Visible region to the NIR region and contains various functional groups. The electrifying properties of CDs are their excitation wavelength-dependent emission spectrum, high photostability and resisting to photobleaching, which permits CDs for multicolour and long-term imaging applications, respectively [45]. The cytotoxicity and preclinical biocompatibility of CDs on various models such as cell lines, zebrafish, mice

**Figure 2.** *Properties of carbon dots.*

displayed CDs have no apparent toxic effects. Extensive studies are to be done on the toxicological and biocompatibility properties to translate from preclinical to clinical application. For targeted bio-imaging, the surface functional groups such as hydroxyl, amine and carboxylic groups allow conjugation with targeting agents.

### *2.2.1 Optical properties*

Among all the CDs' properties, optical properties such as absorbance and fluorescence are vital for bio-imaging applications [45, 46]. Usually, CDs exhibited a strong absorption band at UV region with a falling intensity absorption tail increased to the visible light region. Usually, absorption peak around 230–340 nm was typically ascribed due to π-π\* transition of the C=C bonds of the carbon core. Similarly, the absorption band of 350–550 nm is ascribed to the surface functional groups on the carbon core. Besides this, one exciting feature of CDs is their excitation wavelength-dependent emission spectrum by varying excitation light wavelength, which is commonly observed in most CDs, which allows for multicolour imaging applications. CDs' exact PL mechanism is currently debatable due to the various methods available for the preparation of CDs and the lack of consistency in CDs' PL behavior. Nevertheless, three main mechanisms have been proposed to explain the PL of CDs: (1) The intrinsic band gap arising from the quantum confinement effect or the conjugated π domains, determined by CDs carbon core. (2) The creation of trap states (such as surface defects) in the band gap due to the surface functionalization and CDs doping. (3) The presence of individual fluorescent molecules (fluorophores) on or within the CDs. According to these theories, the wide tunable emissions of CDs have been attributed to their broad size distribution, variable surface chemistry, and the uncontrolled preparation conditions. Another fascinating PL property about CDs is that they generally exhibit high photostability, resisting photobleaching, which is very important for long-term imaging applications.

## *2.2.2 Elemental doping and surface functionalization*

Usually, most of the bare CDs showed comparatively weak FL ability than traditional semiconductor quantum dots or organic dyes. In this line, CDs' structure altered by incorporating elements or surface functionalization strategies to improve fluorescence properties which are essential for fluorescence-based bio-imaging applications. So far, a variety of element doping is adopted to obtain CDs with charming FL properties. At present, heteroatoms such as N, S, Si, P, B, Ga, halogen (Cl, Br, I), Se, Ge, Mg, Cu, Zn, Tb, Ru and Mn incorporated into CDs during the synthesis process [47, 48]. Besides this, large functional groups such as carboxylic acid, amine, hydroxyl and amide groups presence on the surface of CDs facilitate the opportunity to conjugate with various passivate agents [38, 49]. Therefore, several groups focused on improving the fluorescence efficiency through conjugation with variety of passivating agents such as Polyethylene glycol, polyethyleneimine, poly (propionyl ethyleneimine co-ethyleneimine), 4,7,10-trioxa-1,13-tridecanediamine, 1-hexadecylamine, poly(ethylenimide)-b-poly(ethyleneglycol)-b-poly (ethylene imide) and amino acids etc. Generally, these passivating conjugate with CDs via electrostatic interactions, covalent bonds, and hydrogen bonds. More interestingly hetero element doped or surface modified CDs also improved water solubility, photostability, biocompatibility, NIR absorbance and multicolour fluorescence emission, which are the vital parameters for bioimaging. Moreover, unique surface functional CDs have been prepared based on individual cell membrane lipids, proteins, targeting ligands and biomarkers of different cells to develop impactful imaging applications. Furthermore, variety of targeting moieties including peptides (transferrin), aptamers, antibodies and small molecules (folic acid) which have been selected to integrate on the surface of CD through N-hydroxy succinimide (NHS) or 1- ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) chemistry [50]. These targeting moieties offer the internalization of CDs into cells or tissues via a ligand-receptor interaction. Remarkable specific cell targeting bioimaging besides adequate circulation of CDs avoid the side effects originating from the nonspecific interactions. The targeting moiety linked to CDs improves the specificity of bioimaging. Moreover, various cancer therapeutic drugs, anti-microbial agents and photosensitizers were conjugated with the CDs' surface for image guided therapeutic applications.
