Small molecules derived carbon dots: synthesis and applications in sensing, catalysis, imaging, and biomedicine

Carbon dots (CDs) are the new fellow of carbon family having a size less than 10 nm and attracted much attention of researchers since the last decade because of their unique characteristics, such as inexpensive and facile synthesis methods, easy surface modification, excellent photoluminescence, outstanding water solubility, and low toxicity. Due to these unique characteristics, CDs have been extensively applied in different kind of scientific disciplines. For example in the photocatalytic reactions, drug-gene delivery system, in vitro and in vivo bioimaging, chemical and biological sensing as well as photodynamic and photothermal therapies. Mainly two types of methods are available in the literature to synthesize CDs: the top-down approach, which refers to breaking down a more massive carbon structure into nanoscale particles; the bottom-up approach, which refers to the synthesis of CDs from smaller carbon units (small organic molecules). Many review articles are available in the literature regarding the synthesis and applications of CDs. However, there is no such review article describing the synthesis and complete application of CDs derived from small organic molecules together. In this review, we have summarized the progress of research on CDs regarding its synthesis from small organic molecules (bottom-up approach) via hydrothermal/solvothermal treatment, microwave irradiation, ultrasonic treatment, and thermal decomposition techniques as well as applications in the field of bioimaging, drug/gene delivery system, fluorescence-based sensing, photocatalytic reactions, photo-dynamic therapy (PDT) and photo-thermal (PTT) therapy based on the available literature. Finally, the challenges and future direction of CDs are discussed.


Introduction
Nano-crystals of carbon materials having dimensions smaller than 10 nm is known as carbon quantum dots (CDs) [1]. They exhibit different size reliant optical properties such as photoluminescence, chemiluminescence, electrochemical luminescence and photoinduced electron transfer [1,2]. Besides, the high aqueous dispersibility, biocompatibility, good elasticity in modification, high resistance to photobleaching and chemical inertness make it well applicable in bio-imaging [3,4], bio-sensing [5,6], chemical-sensing [7], and biomedical applications [8]. Being a new kind of fluorescent nanomaterial and having excellent biocompatibility, CDs are widely used in the area of bio-imaging both in vitro and in vivo and in diagnosis purposes [9], Photothermal as well as photodynamic therapy and drug/gene delivery carriers [10,11]. CDs could also been applied for the determination of cellular levels of biomolecules and ions (bio-sensor), such as Cu 2+ [12], Hg 2+ [13], NO 3 − [14], C 6 H 12 O 6 [15], pH [16], H 2 O 2 [17], etc. CDs could also act as a promising photocatalyst after co-doping with heteroatoms, such In 2004, during electrophoretic purification of singlewalled carbon nanotubes (SWCNTs) fluorescent carbon nanoparticles were accidentally discovered by Xu et al. [21], and 2 years later Sun et al. synthesized CDs from graphite powder and cement using a laser ablation technique [22]. Mainly two types of methods are available in the literature for the synthesis of CDs; top-down approach and bottom-up approach. Top-down approach refers to breaking down larger carbon structures, such as carbon nanotubes and graphite into smaller carbon structures having dimensions less than 10 nm using arc discharge, laser ablation and electrochemical methods [23]. One such electrochemical method was adopted by Wang et al. for the synthesis of CDs from MWCNTs [24]. Bottom-up approach refers to the synthesis of CDs from smaller carbon units (small organic molecules) by electrochemical/chemical oxidation, laser ablation, hydrothermal/solvothermal treatment, microwave irradiation, ultrasonic treatment, and thermal decomposition techniques [25]. For example, Zhu et al. synthesized CDs via by heating a solution of poly (ethylene glycol) and saccharide in 500 W microwave oven for 2 to 10 min [26].
Various chemical precursors have been identified as the source of CDs, such as citric acid [27], glycerol [28], l-ascorbic acid [29], glucose [30], citric acid-urea [31] and thiourea [32]. To convert these precursors into fluorescent CDs various synthetic processes are used, such as ultrasonication [33], simple heating [34], arc discharge [35], solvothermal [36], hydrothermal [37][38][39], chemical oxidation [40], and laser ablation [41]. Plentiful efforts have been made to expand the usability of CDs to fulfill the growing demand for high-performance techniques, such as bio-imaging, drug-gene delivery, chemical sensing, as well as photocatalysis. However, it is also necessary to regulate the dimensions of CDs during its synthesis to attain uniform properties for a particular application. A large number of reports established the methods of purifying the as-prepared CDs via post-treatment, for example, centrifugation, filtration, gel-electrophoresis and column chromatography [42,43]. Besides, monitoring the dimensions of CDs during its formation is also preferred.
In the past few years, many review articles have been published on the synthesis, properties and applications CDs which are derived from either small organic molecules (chemical) or green sources [44]. However, the number of review articles on CDs, which describe its synthesis only from chemical precursors and applications, like fluorescence-based sensing, bio-imaging, photocatalysis, drug/gene delivery carriers, photothermal as well as photodynamic therapy is still limited, and this article made an effort to fill the gap. In this current review article, we have described the recent progress of small molecule-derived CDs in the field of biomedical as well as chemical applications to date and their future perspective.

Methods of synthesis of CDs
CDs can be synthesized mainly via two routes: (i) topdown approach and (ii) bottom-up approach (Fig. 1). Top-down approach refers to breaking down larger carbon structures via chemical oxidation, discharge, electrochemical oxidation, and ultrasonic methods [25]. However, drawbacks of this approach includes the requirement of expensive materials, harsh reaction conditions, and long reaction time [45]. On the other hand, the bottom-up approach refers to the conversion of smaller carbon structure into CDs of the desired size. This bottom-up approach is consisting of hydrothermal treatment, ultrasonic treatment, thermal decomposition, pyrolysis, carbonization, microwave synthesis and solvothermal method to synthesize CDs. Here we will discuss both the top-down and bottom-up approach for the synthesis of CDs obtained from small organic molecules. Table 1 summarizes the several methods which are used for CD synthesis via the Top-down approach.

Electrochemical/chemical oxidation
Electrochemical/chemical oxidation is the most common top-down synthetic route for the synthesis of CDs, because of several remarkable advantages, such as high yield, high purity, low cost, and easy control over size. However, only a few articles are reported, so far regarding the electrochemical oxidation of small molecules to synthesize CDs. Zhou et al. demonstrated the first synthesis of CDs from carbon nanotube via electrochemical oxidation method [26]. Later, Ray et al. used carbon soots as the carbon source for the synthesis of CDs, and this approach can be used for the mg scale synthesis of CDs [40]. Peng [46]. Fabrication of CDs from low molecular-weight alcohols was reported by Deng et al. [47]. They observed that the diameter of CDs was highly dependent upon applied potentials [47]. Hou et al. synthesized CDs from sodium citrate, and urea and the observed diameter of the CDs were in the range of 1.0 to 3.5 nm with 11.4% quantum yield (QY) [48]. Liu et al. used graphite electrode as the carbon source to synthesize CDs with an average diameter of 4 ± 0.2 nm [49].

Laser ablation
Laser ablation is another standard method used by the researchers for the synthesis of CDs. Yu et al. prepared CDs using toluene as the carbon source via laser irradiation technique. They controlled the size of CDs using laser furnace [41]. Nguyen  of CDs from graphite powders via femtosecond laser ablation. They observed that the size of CDs and photoluminescence properties could easily be controlled by changing the parameters including spot size, irradiation time and laser fluence. Smaller size CDs can be synthesized by increasing the irradiation time [50].

Ultrasonic treatment
Ultrasonic treatment is also a very convenient method as the large carbon materials can be broken down by the action of very high energy of ultrasonic sound wave. Wang et al. synthesized N-doped CDs from ascorbic acid and ammonia via ultrasonic treatment [51]. Dang et al. fabricated CDs using oligomer-polyamide resin as the carbon source by ultrasonic treatment. The as-prepared CDs were well dispersed, had low crystallinity, and functional groups at the surface [33].

Microwave-assisted synthesis
Microwave assisted synthesis is a fast and low-cost method to synthesize CDs via the irradiation of electromagnetic radiations having a wavelength ranging from 1 mm to 1 m through the reaction mixture containing the precursor molecules (  [76]. Several other researchers have also reported the microwave-assisted the synthesis of CDs [27,31,[77][78][79][80][81].

Thermal decomposition
Researchers have also used the thermal decomposition technique as another standard bottom-up method to synthesize CDs (Table 4). In ordinary thermal decomposition, a substance or compound decomposes chemically by the action of heat. Thermal decomposition reactions are generally endothermic. This type of decomposition reactions are either irreversible (decomposition of starch, proteins) or reversible (decomposition of ammonium chloride, limestone). This method offers various 12.
Arginine and glucose Microwave synthesis -Blue 1-7 [76] advantages, such as easy to operate, less time consuming, low cost, and large scale production [45]. Wang et al. synthesized highly luminescent CDs by the thermal decomposition of citric acid as the carbon source and organosilane, N-(β-aminoethyl)-γ-aminopropyl methyl dimethoxy silane (AEAPMS) as the passivation agent. They heated the reaction mixture at 240 °C for only 1 min, and the observed diameter of CDs was ~ 0.9 nm [82]. Later, Wang et al. synthesized CDs by this method from citric acid. They heated citric acid on a hot plate at 200 °C for 30 min; neutralized with sodium hydroxide solution, and finally dialyzed for purification. The size of CDs was observed within the range from 0.7 to 1 nm [34]. These CDs showed both excitation-dependent as well as independent photoluminescent (PL) properties, and different QY depending on different synthesis conditions. Wan et al. used the thermal decomposition of 1-butyl 3-methyl imidazolium bromide and l-cysteine for the synthesis of CDs at 240 °C. AFM study showed that the height of the CDs was ranging from 1.0 to 3.5 nm [83]. Some other researchers also reported the synthesis of CDs from small organic molecules via this method [84][85][86].

Carbonization synthesis
Carbonization of the precursor molecules is one of the best, inexpensive, simple, and ultrafast one-step methods to fabricate CDs (Table 4). Carbonization is a chemical process in which solid residues with higher content of carbon are formed from organic materials by prolonged pyrolysis in an inert atmosphere. Wei et al. synthesized N-doped CDs using this ultrafast carbonization method within 2 min from glucose as a carbon source, and ethylenediamine as the nitrogen source. The observed size of the CDs was in the range of 1 to 7 nm with 48% of QY [87]. Wang et al. fabricated blue luminescent thermally-reduced CDs (t-CDs) with size ranging from 4.8 to 9 nm using citric acid carbonization. They used thermogravimetric analyzer for thermal reduction of CDs, which resulted in the five times increment of QY compared with non-reduced-CDs

Pyrolysis synthesis method
Pyrolysis method for the synthesis of CDs from precursor molecules is also preferred by some researchers (Table 4). Pyrolysis is an irreversible thermal decomposition reaction in which decomposition of organic materials take place in an inert atmosphere. It involves physical as well as chemical changes in organic materials resulting in solid residue containing carbon. Generally pyrolysis takes place at very high temperature and under controlled pressure. Bourlinos

Solvothermal method
There are some reports where researchers used solvothermal method for the synthesis of CDs from small organic molecules as the carbon source (Table 4) The average size of the as-prepared B-doped CDs was ~ 16 nm [15].

Ultrasonic treatment
There are limited numbers of studies, where researchers used ultrasonic treatment method to synthesize CDs (Table 4). For example, Li et al. [30] synthesized watersoluble fluorescent CDs having a size in the range of 5-10 nm through the acid assisted ultrasonic treatment of glucose. In the same year, Li et al. synthesized watersoluble fluorescent CDs from activated carbon using one-step H 2 O 2 assisted ultrasonic treatment method. According to the TEM results, the average size of CDs was ranging from 5 to 10 nm, and the surface of CDs was rich in hydroxyl groups [30,92].

Bioimaging
CDs are considered as a potential candidate for bioimaging application due to its unique fluorescent nature, high photobleaching resistivity, less cytotoxicity, and better aqueous solubility ( Fig. 2 and Table 5 In case of in vivo imaging, they injected CDs in the nude mice bearing xenograft tumor and checked the whole body fluorescence at an excitation wavelength of 530 nm and an emission wavelength of 600 nm. It was observed that the CDs were mostly accumulated within the tumor region due to the EPR effect [112].

Drug/gene delivery
CDs can be used as a vehicle for drug/gene delivery due to their synthesis from cheap sources, facile surface functionalization, tiny size, and higher biocompatibility (Table 6). Besides, the drug/gene delivery pathway is also tracked due to the intrinsic fluorescence nature of CD; therefore considered as an excellent alternative  [114]. After that Yang et al. fabricated β-cyclodextrin (βCD), oligoethylenimine (OEI) and Phosphoric acid based green luminescent CDs for a drug delivery system. They further functionalize CDs with HA (hyaluronic acid) for active targeting onto tumor cells. The nanoparticles were noncytotoxic and exhibited green fluorescence in H1299 cells. They also demonstrated that DOX-loaded CD nanocomplexes exhibited higher cytotoxicity towards H1299 cells than that of free DOX [115]. Wang et al.
used CDs/DOX nanocomplexes as the image-guided drug delivery system. They synthesized the CDs form citric acid and o-phenylenediamine, and the positively charged DOX was loaded on the surface of negatively charged CDs through electrostatic interactions. However, the fluorescence of CDs decreased after loading with DOX, while after the release of DOX from the surface of CDs, fluorescence was regained and thereby acted as an image-guided drug delivery system [116]. Further, the CDs/DOX nanocomplexes showed higher cytotoxicity towards cancerous cells (HeLa) compared with normal cells (L929, mouse fibroblast cells). Recently Kong et al. fabricated a citric acid and ethylenediamine based CDs for drug delivery systems. According to their results they observed that DOX could be quickly loaded on CDs through electrostatic interaction and the CDs/DOX complexes showed better cellular uptake and antitumor efficiency on the breast cancer MCF-7 cells compared with free DOX [117]. Similarly, some other researchers have also used CDs loaded with DOX for simultaneous cell imaging and drug delivery systems [27,114,[118][119][120]. Zheng 26. Citric acid, urea and sodium fluoride Red Glioma C6 cells [112] et al. fabricated fluorescent CDs with an anticancer drug, oxaliplatin (CD-Oxa) via condensation reaction between the carboxyl groups of oxaliplatin derivative and the amino groups on the CD surface to increase its anticancer efficacy. They demonstrated that the assynthesized CD-Oxa could enter into hepatic cancer cells and release the active drug under reducing environment. They also showed that the CDs were biocompatible towards normal fibroblast cells, whereas the CD-Oxa was cytotoxic towards hepatic cancer cells. The charge convertible CDs were obtained via further functionalizing the CDs with PEG-poly(allylamine) and polydimethylmaleic acid (PEG-(PAH/DMMA). From the in vitro experiments, they demonstrated that the charge convertible nano-carriers offer higher therapeutic capability in the tumor extracellular environment compared with normal physiological conditions. Besides, they also used zenograft tumor bearing mice model and found that intravenous injection of the nanoformulations exhibited better tumor cells inhibition capability with less systemic toxicity [11].
Wang et al. fabricated CDs based upon citric acid and tryptophan (as passivation agent) for imaging-guided survivin siRNA delivery in the gastric cancer cells. The as-prepared CDs were further coated with PEI which binds the negatively charged siRNA. The western blot and qRT-PCR experiments assured the survivin gene silencing via nanoparticle-mediated siRNA delivery into the MGC-803 cells. They also showed the secondary effects of survivin gene silencing in the MGC-803 16. Arginine and glucose -pSOX-9 Chondrogenic differentiation of mouse embryonic Fibroblasts [76] cells, such as cell cycle arrest at the G1 phase and enhanced apoptosis [60].
Wu et al. used folate-conjugated reduction-sensitive polyethyleneimine passivated CDs for targeted EGFR and cyclinB1 siRNA delivery and targeting in H460 lung cancer cells. The combined siRNAs were released in reducing intracellular conditions and increased the anti-cancer activity in H460 cells [123]. Recently Cao et al. fabricated dual functional cationic CDs from glucose and arginine for imaging and SOX9 plasmid delivery in mouse embryonic fibroblasts (MEFs). They observed that CDs/pSOX9 nano-particles possessed low cytotoxicity towards MEFs, helped in the intracellular tracking, and SOX9-dependent chondrogenic differentiation [76]. Several other researchers have also worked with CDs, as non-viral gene delivery vectors [124,125].

Bio-sensing
CDs have also been employed by researchers as a biosensing and chemical-sensing nano-materials due to their unique properties like excitation-dependent emission, higher photostability, low cytotoxicity and better aqueous solubility (Table 7) [45]. The changes in their fluorescence property take place via different mechanisms, such as resonance energy transfer, inner filter effect, photo-induced electron and charge transfer [126]. These nanomaterials can be used for sensing of several biological molecules and intracellular metal ions, such of H 2 O 2 , Fe 3+ , C 6 H 12 O 6 , Vitamin B 12 , l-cysteine, galactose, etc. via changing their fluorescence intensity (Fig. 4). Wu et al. used l-glutamic acid derived CDs for effective detection of H 2 O 2 . The asprepared CDs possessed peroxidase-like activity which was utilized for H 2 O 2 detection in the presence of 2, 2 1 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) with the detection limit of 20 μM [17].
Qian et al. used hydroquinone, and SiCl 4 derived CDs as the sensor for Fe 3+ , H 2 O 2 , and melamine. The detection of H 2 O 2 was attained by an electron transfer mechanism between Si-doped CDs and H 2 O 2 , while both electron and energy transfer mechanisms were operating for detecting Fe 3+ . Formation of a stable adduct between H 2 O 2 and melamine following melamine addition recovered the fluorescence and thereby acted as a sensitive detector for melamine as well [29]. Similarly, Shan et al.  CDs from lactose and NaOH by simple heating. They observed that folic acid could quench the fluorescence of the as-prepared CDs upon binding and employed those CDs as the selective probe for folic acid detection in the human urine [129]. The detection of Fe 3+ using fullerene (C60) derived CDs was demonstrated by Lan et al. [130]. They passivated the CD surface with hydroxyl and carboxyl groups which interact with Fe 3+ , thereby quenching the fluorescence [130]. Yang et al. introduced boronic  10. Citric acid and melamine Blue Glutathione [134] acid functionalized CDs as the biosensor nanomaterials for detecting galactose. The boronic acid moiety present on the CD surface reacted with cis-diol units of galactose to give rise cyclic boronate esters, leading to quenching and selective detection of galactose in human urine [131]. Bhattacharya et al. also used aminoguanidine based CDs for nitric oxide (NO) detection via formation of azo dye [132]. Recently Iqbal et al. fabricated N-doped CDs from citric acid and melamine (passivation agent) and applied them for sensing glutathione and Hg 2+ in the biological system via fluorescence quenching [133].

Chemical-sensing
The interaction of metal ions and surface functional groups of CDs lead to the formation of new electronhole recombination, with the help of energy transfer route and results in the change of the fluorescence nature of CDs (Fig. 4). This property of CDs is utilized for sensing different types of metal ions [7], such as Hg 2+ [134], Ag + [146], Cu 2+ [12], Fe 3+ [142] etc. (Table 8).
The heavy metal ion, Hg 2+ is highly toxic, and CDs are employed for Hg 2+ detection on several occasions. Zhou et al. demonstrated the use of unmodified CDs for the detection of Hg 2+ and biothiols (glutathione, cysteine, and homocysteine) with higher selectivity and sensitivity. They observed that the addition of Hg 2+ to CDs caused fluorescence quenching. However, subsequent addition of biothiols to the Hg 2+ /CDs recovered the fluorescence via the removal of Hg 2+ ions which has a high affinity towards thiol (-SH) groups [134]. After that Guo et al. synthesized CDs from sodium citrate/citric acid via hydrothermal treatment for the selective and sensitive detection of Hg 2+ ions [13].  [6,54]. Later, Xu et al. demonstrated the use of blue fluorescent Mn-doped CDs for the highly selective and sensitive detection of Hg 2+ ions via fluorescence quenching with a nM detection limit due to its higher chemical affinity towards Hg 2+ ions compared with various other metal ions. The fluorescence quenching by Hg 2+ might be due to the electron transfer or energy transfer from CDs [135]. Pan at el. fabricated green fluorescent CDs from phenolphthalein and ethylenediamine for multimode sensing. They observed that the fluorescence emission of CDs was selectively quenched by Hg 2+ ions (with detection limit 5.8 μM) due to the photo induced electron transfer mechanism between them. Furthermore, CDs exhibited significant fluorescent response to lemon yellow dye, H 2 O 2 and Fe 2+ with detection limits of 0.5, 1.1 and 1.2 μM respectively [136]. Wang et al. synthesized N-doped CDs from citric acid and triethylamine via hydrothermal treatment for rapid and selective detection of Hg 2+ ions. They observed that the N-doped CDs could act as highly sensitive sensor to Hg 2+ ions in tap water samples, with a detection limit of 2.8 nM [137]. Recently Li et al. fabricated blue/yellow emissive CDs from o-phenylenediamine as the multi-mode sensor materials and applied for the selective detection of Hg 2+ , Cl − and Cr 4+ ions. They observed that blue emission of CDs get quenched by Hg 2+ (turn off ) and recovered on addition of Cl − ions (turn on). Furthermore, blue/yellow fluorescence of CDs get quenched significantly on addition of Cr 4+ ions [138]. Zhang et al. synthesized graphitic CDs as fluorescent material for selective and sensitive detection of the Fe 3+ ions. They observed that the hydroxyl and carboxyl groups present on the surfaces of CDs help in interaction with Fe 3+ ions and thereby enabling CDs to act as the fluorescence sensor for detecting Fe 3+ ions with a detection limit of 2 nM [139]. Recently the detection of Fe 2+ with the help of CDs based upon cetyl pyridinium bromide as the source molecule was reported by Kaur et al. [140]. They observed that the fluorescence intensity of CDs was decreased gradually with the addition of Fe 2+ ions with increasing concentration. This technique is also used in determining the Fe 2+ ions in syrup and iron tablets [140]. Wu et al. synthesized folic acid/3-aminopropyl trimethoxy silane based N, Si-doped CDs via one-step hydrothermal method and applied for sensing Fe 3+ ions. They observed that Fe 3+ ions could be detected using the as-synthesized CDs with a detection limit of 3.8 nM [141]. Sun et al. fabricated red-emitting CDs via microwave irradiation of phenylenediamine and applied for detection of Fe 3+ ions in aqueous solutions with a detection limit of 15 nM. They demonstrated that fluorescence of CDs get quenched significantly in presence of Fe 3+ due to electron transfer mechanism [142]. Similar articles relating to the detection of Fe 3+ were also published in the past few years [105, 143-145, 155, 156]. Qian, et al. synthesized blue fluorescent N-doped CDs from CCl 4 and diamines for the detection of Ag + ion. Their results suggested that fluorescence intensity of the as-prepared CDs was inversely proportional to the pH values ranging from 5.0 and 13.5, indicating the possibility of using those CDs as a pH indicator. Besides, they also used the CDs for detection of Ag + , which increased the fluorescence intensity of CDs upon binding with the N-atoms on their surface [146]. Binding of Ag + ions with the amino groups on the CDs surface leads to positive charges on CDs which decreased the collision among them and increased the fluorescence. Borse et al. also used N-doped CDs for the 'turn-off ' dynamic fluorescence-based sensing of Ag + ions with detection within the concentration range of 1-1000 μM [111]. Ren et al. (2017) synthesized blue fluorescent N-doped CDs from uric acid for the selective detection of Ag + and Hg 2+ ions with detection limit of 1 and 4.8 nM respectively. Besides, the quenching of fluorescence of N-doped CDs could be recovered on the addition of EDTA [147]. Li et al. fabricated 1,2-diaminobenzene based N-doped CDs as a sensitive detection probe for Ag + with a detection limit of 0.5 nM [148]. Wang et al. employed citric acid and amino acid derived carbon dots (CDs)-gold nanoparticles (AuNPs) hybrid for sensing Ag + ions in presence of glutathione. They demonstrated that on the addition of Ag + ions to a solution containing CDs-AuNPs and GSH, solution color changes from red to blue due to the aggregation of AuNP. This method could be used to detect Ag + ions with a detection limit of 50 nM [149]. Recently, Qin et al. synthesized blue fluorescent CDs via pyrolysis of uric acids and N, S co-doped CDs from citric acid and guanidine thiocyanate via hydrothermal method for the selective detection of Ag + ions [150,151]. Lin et al. used citric acid and poly(ethylenimine) based CDs in a zeolitic imidazolate framework and act as a selective detecting agent for Cu 2+ ions with a detection limit of 80 pM. The intense blue emission of CD-nanocomposites was quenched following the addition of Cu 2+ ions [12]. Zhang et al. fabricated citric acid based N doped CDs (NCDs) as the chemical sensing materials and applied for "turn off " detection of Hg 2+ ions. That NCDs-Hg 2+ system was further used for the selective and sensitive detection of l-cysteine via "turn on" mechanism with a detection limit of 79 nM [6]. Fong et al. synthesized blue fluorescent CDs from sodium alginate for the detection of Fe 3+ and ascorbic acid. They showed that Fe 3+ ions decreased the fluorescence intensity (turn off ) of the CDs, which was recovered by ascorbic acid addition (turn on) [91]. Sun et al. employed ascorbic acid, and glycol derived green fluorescent CDs for sensitive and selective detection of Al 3+ ions with the detection limit of 0.39 µM via fluorescence enhancement response. They demonstrated that the combination of Al 3+ ions with the hydroxyl groups on CDs at nearly neutral pH resulted in the creation of much more surface state of the CDs, thereby increasing the fluorescence intensity. However, they also used the formed CDs-Al 3+ ion system for selective and sensitive detection of F − anions with a detection limit of 0.14 µM via fluorescence "on-off " mechanism due to the strong affinity of F − ions towards Al 3+ ions [152].
Devi et al. used nitrogen-rich ligands functionalized CDs for selective detection of selenite (SeO 3 2− ) via fluorescence quenching mechanism (with the detection limit of 0.1 ppb) due to the formation of strong Se-N bonds [153]. N and S co-doped CDs were prepared by Chen et al. form citric acid and 1,6-diaminohexane dihydrochloride and employed those for Cr 6+ detection with the detection limit of 0.86 μM. They proposed that the fluorescence quenching of CDs by Cr 6+ ions were due to the inner filter effect [154].

Photocatalytic application
CDs exhibit potential applications in the field of photocatalytic reactions due to their unique properties like better water solubility, very less toxicity, and high chemical stability compared with another common photocatalysts, such as ZnO, CdS, TiO 2 , etc. (Table 9). In addition, CDs can exhibit up-converted photoluminescence (UCPL) and photoinduced electron transfer properties which attract much interest in designing photocatalysts utilizing CDs [44]. Qu et al. fabricated S, N co-doped CDs/ TiO 2 composites for the degradation of Rhodamine B (RhB) as a visible light photocatalyst. They proposed that S, N: GQDs absorbed the visible light, and electronic excitation occurred from valence band (VB) to conduction band (CB). The electrons were then injected from the CB of CDs to CB of TiO 2 (CDs acts as a sensitizer) and promoted the charge separation process. This charge separation process further generates superoxide and hydroxyl radicals which subsequently degraded the dye [157]. Yu et al. prepared CDs modified TiO 2 (CDs/ P25) nanocomposites for efficient photocatalytic hydrogen evolution under both UV and visible light irradiation. When CDs/P25 was exposed to the UV light, CDs acted as electrons reservoir and improved the efficient separation between photo-induced electron-hole pairs. However, under visible light irradiation, CDs acted as a sensitizer and promote electron excitation in P25 [158]. Wang et al. used vitamin-C derived CDs/TiO2 nanocomposites for hydrogen production from water. They demonstrated that the efficient electron transfer ability and up-conversion properties of CDs contributed to the enhanced photocatalytic behavior of TiO2 [159]. Martins et al. synthesized N-doped CDs/TiO2 composite with improved photocatalytic activity [160]. Cai et al. prepared S, N co-doped CDs/g-C 3 N 4 nanocomposites for the high catalytic performance in degrading RhB under visible light irradiation. They observed that S, N doped GDs/g-C 3 N 4 nanocomposites exhibit better photocatalytic activity than pure g-C 3 N 4 due to electron transfer from the CB of g-C 3 N 4 to CB of S, N doped GDs/g-C 3 N 4 (CDs act as an electron reservoir), results in the efficient separation of photogenerated electrons and holes [161]. Recently Zhang et al. fabricated CDs/La 2 Ti 2 O 7 nanocomposites for the degradation of RhB. The as-synthesized CDs absorbed visible light and converted it into UV light (due to its up-conversion properties) which was utilized by La 2 Ti 2 O 7 for charge separation. Besides, the CDs were also capable of accepting electrons from La 2 Ti 2 O 7 , thereby promoting efficient separation of photogenerated electrons and holes [162]. Similar kind of photodegradation of RhB was also reported by several other researchers using CDs nanocomposites [163][164][165][166]. The zinc oxide reduced graphene oxide (ZnO-RGO) nanocomposites were synthesized by Li et al. for the RhB photodegradation and CO 2 photoreduction. They observed that ZnO-RGO nanocomposites showed enhanced photocatalytic activity and the main reasons were: (1) RGO acted as electron acceptor, i.e. photoexcited electrons of ZnO transferred from its CB to the CB of RGO; (2) RGO absorbed a large number of dye molecules on their surface due to pi-stacking interaction [167]. They found that under the visible light irradiation g-C 3 N 4 could be utilized for the photoreduction of CO 2 to organic fuels. The urea derived g-C 3 N 4 showed better CO 2 photoreduction due to their porous like structure and larger surface area than that of melamine derived g-C 3 N 4 [168]. Sahu et al. fabricated Au-doped CDs from small acetic acid for the photoreduction of CO 2 [169]. Cao et al. used Au or Ptcoated PEG-functionalized CDs as an active photocatalyst for the reduction of CO 2 . Surface functionalization with PEG increases the solubility of the catalysts allowing homogeneous reaction in the aqueous phase. The coated Au or Pt over the CD surface soaks up the photogenerated electrons, thereby disrupting the electron/hole recombination process [170].
Martindale et al. fabricated citric acid derived CDs via straightforward thermolysis route and combined it with molecular Ni catalyst [Ni-bis(diphosphine)] for solar hydrogen production [85]. Yan et al. used N doped CDs-ZnNb 2 O 6 /g-C 3 N 4 nanocomposite for photocatalytic hydrogen production. Under visible light irradiation, NGQDs absorbed light with wavelength ≥ 550 nm and converted them to shorter wavelength < 460 nm that was further used to excite g-C 3 N 4 and to promote the generation of more electron-hole pairs. The electrons of g-C 3 N 4 in CB were further injected into the CB of the ZnNb 2 O 6 and trapped by the Pt nanoparticles [171]. Ecofriendly CDs/N-doped ZnO nanocomposites were used by Muthulingam et al. for the degradation of methylene blue, fluorescein and malachite green dyes under the sunlight irradiation. It was noticed that CDs helped in electron/hole separation in ZnO via accepting the electrons from its CB and also exerted anti-photocorrosion property due to its coating over the ZnO surface [172]. Chen et al. synthesized CDs/Ag/Ag 2 O nanocomposites as an excellent photocatalyst for pollutant degradation via incorporating the photoinduced electron transfer and up-converted properties of CDs [163]. Yang et al. prepared ultrafine iron oxyhydroxide/g-C3N4 nanocomposites heterojunctions as an efficient photocatalyst in the presence of visible light. They observed that the ultrafine nanocomposites helped to increase the visible light 28. Ag-CDs/g-C 3 N 4 Citric acid, ethylenediamine Naproxcen [179] 29. CDs/g-C 3 N 4 /MoO 3 Citric acid, urea and dicyandiamide Degradation of tetracycline (TC) [180] absorbance, shortened the band gap of photogenerated carriers, and also helped in the formation of a large number of heterojunctions with g-C 3 N 4 [ Several other researchers also used CDs nanocomposites for the photocatalytic degradation of ampicillin antibiotic [174], organic/industrial dyes [175][176][177], NO [178], naproxcen [179], and tetracycline [180]. The overall mechanisms of action behind the photocatalytic applications CDs have been depicted in Fig. 5.

Photo-dynamic therapy (PDT) and Photo-thermal (PTT) therapy
Photo-dynamic therapy (PDT) and Photo-thermal therapy (PTT) are applied for the treatment of cancer using laser light (most often by near IR radiations). PTT refers to the use of NIR which is absorbed by a photoabsorber to generate local heat, and destroy diseased tissue. PTT offers several advantages over the conventional chemotherapy, radiotherapy, and surgery; therefore attracting much interests of the researchers in the field of cancer treatment. However, the effectiveness of nanomaterials as photothermal agent (gold nanostructures, graphene and graphene oxide) in PTT is still under consideration due to the difficulties in their synthesis, and high production cost. Unlike the PTT, PDT requires O 2 to produce ROS and destroy targeted cells. In PDT, a photosensitizer is irradiated with laser light that produces ROS which ultimately destroys the cancer cells/tissue. Upon excitation with a suitable wavelength, photosensitizer gets excited from its ground singlet state to higher energy singlet state and suffers intersystem crossing to form long lived triplet state. The triplet state reacts with oxygen molecule, resulting in ROS that can effectively destroy cancer cells [181]. Recently CDs are intensively used by researchers in PTT and PDT due to its smooth and low-cost synthesis, facile surface functionalization and superior caring capacity, excellent biocompatibility and ability to convert absorbed light into heat due to a large number of pi electrons (Table 10). Both PDT and PTT are involving the use of light having less energy and hence is less injurious to healthy cells/tissue [113]. Ge et al. fabricated red-fluorescent CDs from polythiophene phenylpropionic acid for Furthermore, the π-π stacking between CDs and organic dye also enhances its degradation upon light irradiation photoacoustic/FL (NIR) imaging and in vitro as well as in vivo photo-thermal therapy. They demonstrated that the as-synthesized CDs showed extensive cytotoxicity towards HeLa cells upon NIR laser irradiation. The CDs were also found to show in vivo PTT efficacy in HeLa tumor bearing mice without any sign of systemic toxicity [182]. Zheng et al. fabricated CDs from hydrophobic cyanine dye and poly(ethylene glycol) for NIR imaging and in vitro as well as in vivo photothermal therapy. The as-prepared CDs exhibit superior photothermal effect in HepG2 and CT26 cells upon NIR laser irradiation. The CDs were also effective in inhibiting tumor growth in CT26 xenograft Babl-c mice upon NIR laser irradiation [183]. Li et al. synthesized supra-CDs from citric acid, and urea via electrostatic interactions and hydrogen bonding. The as-prepared supra-CDs showed strong visible-NIR absorption with high photothermal conversion efficiency [184]. Beack et al. synthesized CDs-Ce6-HA, hyaluronic acid modified CDs conjugated with chlorine-Ce6 (Ce6, a photosensitizer) for targeted therapy of melanoma skin cancer. They observed that CDs conjugation to Ce6 increased the photodynamic reaction of Ce6, and produce more singlet oxygen than free Ce6. Transdermal administration of CDs-Ce6-HA resulted in suppression of B16F10 melanoma cells in tumor mice upon laser irradiation [185]. Yang et al. fabricated Mg/N doped CDs and used them as a carrier of Ce6. This CDs/Ce6 composite showed high fluorescence resonance energy transfer (FRET) efficiency and subsequently enhanced the PDT effect. They also demonstrated that CDs conjugation to Ce6 increased the production of singlet oxygen almost two times than the free Ce6, and a significantly enhanced PDT effect was observed in HepG2 cancer cells compared with free Ce6 [10]. Zheng et al. synthesized carbon nitride (C 3 N 4 ) doped CDs and functionalized it with a targeting agent and a photosensitizer to increase the targeted PDT efficiency in solid tumor under hypoxic condition. They demonstrated that the nanocomposite produces oxygen inside the hypoxic tumor region via splitting of water upon light exposure, thereby producing ROS and showed enhanced PDT efficiency under both in vitro and in vivo conditions [186].
Wu et al. synthesized porphyrin implanted CDs from citric acid and 5,10,15,20-tetrakis(4-aminophenyl)porphyrin via selective pyrolysis treatment. They demonstrated that the as-prepared CDs could act as an active agent for photoacoustic molecular imaging under NIR irradiation. Furthermore, cetuximab-conjugated CDs exhibit superior photodynamic effect in HCC827 and MDA-MB-231 cancer cells as well as MDA-MB-231 tumor bearing mice [187].
Recently Hua et al. synthesized CDs from l-cysteine and m-phenylenediamine. The as-prepared CDs showed nucleus targeting property, thereby could be used as a nucleus staining agent. They further conjugated the CDs with protoporphyrin IX (a photosensitizer), which showed enhanced PDT efficacy in vivo without affecting healthy cells [188]. Guo et al. synthesized Cu, N codoped CDs via one-step hydrothermal treatment using EDTA·2Na and CuCl 2 . They demonstrated that the assynthesized Cu, N co-doped CDs significantly inhibited cancer (B16 melanoma) cell growth via photodynamic and photo-thermal therapy. In addition it could also act as an active agent in FL cell-imaging and IR thermal imaging to visualize in vivo and in vitro treatment process [189]. The Role of CDs in photo thermal (PTT) and photo dynamic (PDT) therapy has been depicted in Fig. 6.

Conclusion and future perspective
The first report of CDs synthesis was registered in 2004 as the new fellow of carbon family and attained tremendous attention in the area of chemistry and biology due to their economical and facile synthesis methods, easy surface modification, excellent photoluminescence, and superior water solubility.
Diaminohexane and carboxylic group of Ce6 Ce6-HA (hyaluronate) B16F10 melanoma [185] 3. Acrylic acid, 1, 2-ethylenediamine (EDA) and Mg(OH) 2 Mg/N HepG2 [10] 4. Hydrophobic cyanine dye and poly(ethylene glycol) -HepG2, CT26 [183] 5. Citric acid and urea -HeLa [184] 6. Dopamine -Hela cells [181] 7. Urea Carbon nitride (C 3 N 4 ) 4T1 [186] 8. Citric acid and 5,10,15,20-tetrakis(4-aminophenyl)porphyrin Cetuximab (C225) HCC827 and MDA-MB-231 cells [187] 9. m-Phenylenediamine and l-cysteine Protoporphyrin IX (PpIX) HeLa [188] 10. EDTA·2Na and CuCl 2 -Murine melanoma (B16) cells [189] CDs can be extensively applied in photocatalytic reactions, in vitro, and in vivo bioimaging, drug-gene delivery system, chemical and biological sensing as well as in photodynamic and photothermal therapies. This review summarized the progress of research on CDs regarding its synthesis from small organic molecules and applications in biological as well as chemical field till date. Although significant development has been done in those areas over the past decade, still some difficulties need to be addressed related to the synthesis and applications of CDs as follows: • The synthesis of CDs with uniform size distribution is somehow tricky. The size distribution plays a crucial role to decide its toxicity and fluorescence properties which may obstruct further biological applications, particularly in vivo. CDs reproducibility is also a big issue for clinical applications. Besides, different synthesis methods of CDs result in different QY, size, fluorescence color, etc. which obstruct CDs commercialization. • Despite enormous research on CDs synthesis and its modifications, the real mechanism of CDs formation is not understood fully till date. Furthermore, simple and controllable surface modifications are still crucial problems, which may help for designing CDs with excellent photoluminescence property and other applications with high efficiency.
• Although CDs based chemical and, bio-sensing technology is well applicable in real life samples, but studies on several other toxic metal ions, like Cd 2+ , Mn 2+ , As 3+ , Po 3+ , etc. are still missing. Therefore, further research with those metal ions should be done to understand the full potential of CDs as a sensing agent. • More efforts are needed to apply the CDs in the field of in vivo imaging, drug and gene delivery systems as well as cancer therapies to broaden the area of its applications. Furthermore, dual drug-gene delivery systems are not fully explored to date. • Finally, the use of CDs in the area of energy storage is needed to be explored. Therefore, researchers should concentrate on those above-said issues and CDs will gain significant interest in the future undoubtedly after proper addressing such problems.