Towards Red Emissive Systems Based on Carbon Dots

Carbon dots (C-dots) represent an emerging class of nontoxic nanoemitters that show excitation wavelength-dependent photoluminescence (PL) with high quantum yield (QY) and minimal photobleaching. The vast majority of studies focus on C-dots that exhibit the strongest PL emissions in the blue/green region of the spectrum, while longer wavelength emissions are ideal for applications such as bioimaging, photothermal and photodynamic therapy and light-emitting diodes. Effective strategies to modulate the PL emission of C-dot-based systems towards the red end of the spectrum rely on extensive conjugation of sp2 domains, heteroatom doping, solvatochromism, surface functionalization and passivation. Those approaches are systematically presented in this review, while emphasis is given on important applications of red-emissive suspensions, nanopowders and polymer nanocomposites.


Introduction
The dynamic presence of carbon dots (C-dots) in the field of nanoemitters over the last 15 years is directly related to their unique combination of three desired characteristics: inexpensive preparation, nontoxic nature and superior photophysical properties in terms of light absorption, chemiluminescence, electroluminescence, phosphorescence and upconversion [1][2][3][4][5]. Typically, C-dots are spherical nanoparticles with size 2-20 nm that are composed of C, O and H along with heteroatoms such as N, P and S.
Their carbogenic cores can be predominantly amorphous or highly graphitic in nature [6], while the term graphene quantum dots (GQDs) is typically reserved for nanodisks of single-or few-layered graphene sheets [7]. In that sense, GQDS are considered as a distinct subgroup of C-dots and are synthesized by various strategies including chemical vapor deposition on hexagonal boron nitride substrates [8], all-organic synthesis starting from polyphenylene dendrimers [9] and ring opening of fullerenes adsorbed to Ru metal terraces [10]. Alternatively, GQDs can be prepared by oxidation or electrooxidation of carbon-rich sources such as coal [11], carbon black [12], graphite [13], carbon fibres [14] and carbon nanotubes (CNT) [15].
The PL properties of aqueous solutions of C-dots have been systematically investigated with emphasis on theragnostics [22], gene delivery [22], drug delivery [23], antimicrobial applications [24], bioimaging [25] and biosensing [26]. At the same time the PL properties of polymer-based nanocomposites [27,28] and powder formulations based on

Strategies to Generate Red-Shifted C-Dots
The main strategies that give rise to red-shifted C-dots rely on: (i) heteroatom doping, (ii) extensive conjugation length, (iii) surface functionalization and passivation and (iv) solvatochromism.

Heteroatom Doping
Heteroatom doping is a well-explored strategy to modify the electronic and optical properties of C-dots, ultimately leading to red-shifted C-dots [38]. It has been supported that N doping can reduce the bandgap and improve bandgap uniformity of C-dots, thus enhancing red emissions with a narrow full width at half maximum. Moreover, B and S doping leads to reduced nonradiative recombinations, in essence intensifying red signals. Incorporation of La results in local structural rearrangements, increased charge transfer and ultimately stronger red emissions [35].
To that end, Hola et al. [39] synthesized N-doped graphitic C-dots following the solvothermal treatment of urea and CA in formamide (Figure 1a) that were subsequently subjected to column chromatography yielding blue-(b-C-dots), green-(g-C-dots), yellow-(y-C-dots) and red-(r-C-dots) emissive fractions as shown in Figure 1b,c,d,e, respectively. In water, at λ ex = 440 nm, the QY of b-C-dots, g-C-dots, y-C-dots and r-C-dots were 13.3%, 10.0%, 11.6% and 4.0%, respectively. Transmission electron microscopy (TEM) imaging suggested that the average diameter (d av ) of these C-dots was 2-3 nm (Figure 2a-d). X-ray photoelectron spectroscopy (XPS) indicated a lower concentration of oxygen, hydroxyl and carboxyl groups and an increased content of graphitic nitrogen for y-C-dots and r-C-dots (Figure 2e-l), while nitrogen stemming from surface amide groups was dominant in b-Cdots and g-C-dots. Fourier transform infrared spectroscopy (FT-IR) confirmed the presence of carboxylic C=O bonds and surface C−N bonds for b-C-dots and g-C-dots, whereas the most pronounced FT-IR peaks were ascribed to C−N= and C=N bonds for y-C-dots and r-C-dots, verifying the dominant role of graphitic nitrogen for the red-shifted fractions. Nanomaterials 2021, 11, x FOR PEER REVIEW 3 of whereas the most pronounced FT-IR peaks were ascribed to C−N= and C=N bonds for C-dots and r-C-dots, verifying the dominant role of graphitic nitrogen for the red-shift fractions.  Moreover, Ding et al. [40] fabricated red graphitic C-dots (QY = 28% at λ ex = 533 nm) with d av = 4.6 nm by means of solvothermal treatment of lemon juice in ethanol (λ ex stands for excitation wavelength). The PL spectra of these C-dots suggested λ ex -dependent behaviour, with the maximum of their emission peak at λ max = 615 nm. FT-IR suggested the presence of O-H, N-H, -COOH and C-O on their surface, while C=C/C=N and C-N vibrations were attributed to the polyaromatic structures of the carbogenic cores. XPS confirmed the presence of C-C/C=C, C-N, C-O, C=O/C=N and COOH groups along with the presence of pyridinic C-N-C, pyrrolic C 2 -N-H and graphitic N-C 3 groups.
Capitalizing on similar concepts, Guo et al. [41] used various ratios of two types of functionalized polythiophene derivatives to prepare a series of graphitic C-dots with tuneable PL emissions ranging from blue to near infrared (NIR). All C-dots consisted of C, O and S, while the N content was higher for the red-shifted fractions. Pan et al. [42] synthesized graphitic C-dots with d av = 6.8 nm, through microwave heating of CA and formamide solution in an autoclave with QY close to 11.9%, 16.7% and 26.2% at λ ex = 360 nm, 450 nm and 540 nm, respectively. Likewise, Pan et al. [43] followed microwave-assisted heating of glutathione formamide to synthesize graphitic NIR-emitting C-dots with d av =2.9 nm and QY = 16.8% at λ ex = 420 nm. High-resolution C 1s and N 1s XPS spectra for (e,i) b-C-dots; (f,j) g-C-dots; (g,k) y-C-dots and (h,l) r-C-dots. Reprinted (adapted) with permission from [39]. Copyright 2021 American Chemical Society.
Moreover, Ding et al. [40] fabricated red graphitic C-dots (QY = 28% at λex = 533 nm) with dav = 4.6 nm by means of solvothermal treatment of lemon juice in ethanol (λex stands for excitation wavelength). The PL spectra of these C-dots suggested λex-dependent behaviour, with the maximum of their emission peak at λmax = 615 nm. FT-IR suggested the presence of O-H, N-H, -COOH and C-O on their surface, while C=C/C=N and C-N vibrations were attributed to the polyaromatic structures of the carbogenic cores. XPS confirmed the presence of C-C/C=C, C-N, C-O, C=O/C=N and COOH groups along with the presence of pyridinic C-N-C, pyrrolic C2-N-H and graphitic N-C3 groups.
Capitalizing on similar concepts, Guo et al. [41] used various ratios of two types of functionalized polythiophene derivatives to prepare a series of graphitic C-dots with tuneable PL emissions ranging from blue to near infrared (NIR). All C-dots consisted of C, O and S, while the N content was higher for the red-shifted fractions. Pan et al. [42] synthesized graphitic C-dots with dav = 6.8 nm, through microwave heating of CA and formamide solution in an autoclave with QY close to 11.9%, 16.7% and 26.2% at λex = 360 nm, 450 nm and 540 nm, respectively. Likewise, Pan et al. [43] followed microwave-assisted heating of glutathione formamide to synthesize graphitic NIR-emitting C-dots with dav =2.9 nm and QY = 16.8% at λex = 420 nm.
In terms of multiheteroatom doping, Huang et al. [44] followed a hydrothermal method using 3-aminobenzeneboronic acid and 2,5-diaminobenzenesulfonic acid as precursors to synthesize N, B and S codoped C-dots (NBS-C-dots) with d av = 2.8 nm and QY = 11.6% at λ ex = 520 nm. NBS-C-dots showed λ ex -independent behaviour with λ max = 605 nm for 410 nm ≤ λ ex ≤ 530 nm ( Xu et al. [45] followed a hydrothermal treatment of L-methionine and urea using water and dimethylformamide (DMF) as the solvent to produce red-(N,S-doped) and blue-emitting (N-doped) graphitic C-dots, respectively. Miao et al. [46] followed a hydrothermal treatment of CA and thiourea in acetone to synthesize graphitic red-emitting N,S-doped C-dots with QY = 22% in water. Ge et al. [47] followed a hydrothermal treatment of polythiophene phenylpropionic acid (PPA) to synthesize graphitic, red-emissive S-doped C-dots with d av = 10 nm exhibiting λ ex -independent properties with QY = 2.3%. Yang et al. [48] followed a hydrothermal treatment of 2,5-diaminobenzenesulfonic acid to In situ chromophore doping can lead to red-shifted C-dots by introducing new electron transition pathways that alter their highest occupied molecular orbital and lowest unoccupied molecular orbital (HOMO-LUMO) levels. Karami et al. [49] developed a synthetic procedure to synthesize chromophore-doped graphitic C-dots with d av = 5 nm via a hydrothermal treatment of glucose and 3-nitroaniline dissolved in sulfuric acid. The C-dots exhibited three λ ex -independent emissions (blue, red and yellow) under acidic, neutral and alkaline environments, respectively. At pH = 2 the C-dots showed λ max = 385 nm (QY = 43.0%), but at pH = 3 a weak red peak appeared along with the dominant blue peak. Upon increasing the pH, the intensity of the blue emission peak declined, whereas the red emission peak was enhanced, and at pH = 7 there was a single emission peak at λ max = 610 nm (QY = 14.4%), which at pH =12 shifted to λ max = 565 nm (QY = 24.3%). FT-IR spectra showed peaks related to O-H and N-H groups, C-H stretching vibrations, the C=O band of amide and carbonyl groups, C=C stretching/N-H bending bands and C-S/C-N/C-O stretching vibration bands. At the same time, a pH-dependent FT-IR peak centred at 1545 cm −1 was attributed to the presence of azo groups (-N=N), showing maximum intensity at neutral pH and minimum intensity at acidic pH.

Extensive Conjugation Length
Early studies indicated the crucial role of quantum confinement in π-bonded hexagonal carbon clusters on the broadband emission of carbon-based films [50]. More recently, it has been shown that the HOMO-LUMO gap energy decreases as the size of GQDs increases [51]. Moreover, it has been demonstrated that the emission wavelength shows a linear size dependence in defect-free, functionality-free, zigzag-edged GQDs and that the fluorescence emission covers a broad spectrum from deep UV (λ em = 235.2 nm) to NIR (λ em = 999.5 nm) as the size varies from 0.46 nm to 2.31 nm [52]. At the same time, the presence of armchair edge widens the band gap in and blue shifts the emissive signals of GQDs [53].
Early studies indicated the crucial role of quantum confinement in π-bonded hexagonal carbon clusters on the broadband emission of carbon-based films [50]. More recently, it has been shown that the HOMO-LUMO gap energy decreases as the size of GQDs increases [51]. Moreover, it has been demonstrated that the emission wavelength shows a linear size dependence in defect-free, functionality-free, zigzag-edged GQDs and that the fluorescence emission covers a broad spectrum from deep UV (λem = 235.2 nm) to NIR (λem = 999.5 nm) as the size varies from 0.46 nm to 2.31 nm [52]. At the same time, the presence of armchair edge widens the band gap in and blue shifts the emissive signals of GQDs [53].
Yeh et al. [54] relied on the oxidation of GO sheets to generate GQDs that were separated (via polyethersulfone membranes with different cutoff molecular weights) to fractions with dav = 1.0 nm (QD10), 1.6 nm (QD16), 2.6 nm (QD26), 5.4 nm (QD54), 6.1 nm (QD61) and 7.9 nm (QD 79), all of which exhibited excitation-wavelength independent PL with λmax at 460 nm, 490 nm, 520 nm, 540 nm, 560 nm and 580 nm, respectively ( Figure 4). Li et al. [51] explored alkali-assisted electrooxidation of a graphite rod by applying different current densities to fabricate a series of C-dots and demonstrated that small-(1.2 nm), medium-(1.5-3 nm) and large-sized (3.8 nm) C-dots emit within the UV (350 nm), visible (400-700 nm) and NIR (800 nm) regions, respectively (Figure 5a,b). FT-IR spectroscopy indicated the presence of carbonyl (C=O) groups on the surface of these C-dots that can be eliminated via hydrogen plasma treatment without altering the PL properties of Cdots, an effect that points to the dominant role of quantum confinement for this class of materials. Figure 5c suggests that as the size of the C-dots increases, the HOMO-LUMO energy gap decreases, and therefore the PL emission is shifted towards the NIR region of the spectra. Li et al. [51] explored alkali-assisted electrooxidation of a graphite rod by applying different current densities to fabricate a series of C-dots and demonstrated that small-(1.2 nm), medium-(1.5-3 nm) and large-sized (3.8 nm) C-dots emit within the UV (350 nm), visible (400-700 nm) and NIR (800 nm) regions, respectively (Figure 5a,b). FT-IR spectroscopy indicated the presence of carbonyl (C=O) groups on the surface of these C-dots that can be eliminated via hydrogen plasma treatment without altering the PL properties of C-dots, an effect that points to the dominant role of quantum confinement for this class of materials. Figure 5c suggests that as the size of the C-dots increases, the HOMO-LUMO energy gap decreases, and therefore the PL emission is shifted towards the NIR region of the spectra. Yuan et al. [55] followed solvothermal treatment of CA and diaminonaphthalene in ethanol and allowed different reaction times in the presence of concentrated sulfuric acid, yielding GQDs with dav = 1.9 nm, 2.4 nm. 3.8 nm, 4.9 nm and 6.7 nm emitting respectively within the blue, green, yellow, orange and red regions, in agreement with the expected behaviour owing to quantum confinement effects. Similar trends were confirmed by Tian Yuan et al. [55] followed solvothermal treatment of CA and diaminonaphthalene in ethanol and allowed different reaction times in the presence of concentrated sulfuric acid, yielding GQDs with d av = 1.9 nm, 2.4 nm. 3.8 nm, 4.9 nm and 6.7 nm emitting respectively within the blue, green, yellow, orange and red regions, in agreement with the expected behaviour owing to quantum confinement effects. Similar trends were confirmed by Tian et al. [56], who explored the solvothermal treatment of CA and urea in water, glycerol, DMF and their mixtures to generate C-dots with tuneable size and PL emissions.

Surface Functionalization and Passivation
Surface functionalization strategies have been shown to dramatically modify the PL properties of C-dots via defect and intrinsic state emission mechanisms [57] and charge transfer effects [58]. In principle, the presence of functional groups on C-dots generates structural defects and midgap states facilitating π*→midgap states→π transitions, thus enhancing the red components on their PL spectra [59]. An interesting study showed that within a series of C-dots with similar size distribution and graphitization degree, the bandgap was gradually reduced upon increasing the degree of surface oxidation, leading to a pronounced red shift from 440 to 625 nm [60]. In electrochemically oxidized C-dots it was shown that the PL mode stemming from the organic fluorophores was blue-shifted, while the PL component originated by the carbogenic cores was red-shifted [61].
Xiong's group [60] synthesized a series of graphitic C-dots via hydrothermal treatment of urea and p-phenylenediamine (p-PD) yielding a mixture of four fractions (A, B, C and D in Figure 6a) that could be separated via silica column chromatography because of their different polarities. TEM imaging indicated that all fractions consisted of C-dots with d av = 2.6 nm, while FT-IR spectra revealed the presence of O-H, N-H, C-O and -COOH groups on their surfaces. XPS indicated a systematic enhancement of the oxygen content when moving from fraction A to D, a trend that is accompanied by a systematic shift from blue to red PL emission at λ ex = 365 nm. It has been suggested that as the population of surface oxygen atoms increases, the HOMO-LUMO bandgap gradually narrows, ultimately facilitating red PL emission (Figure 6b). Liu et al. [62] followed a microwave-assisted pyrolysis of m-PD, o-PD and p-PD in formamide to generate three types of C-dots that exhibited λex-independent emission with λmax at 360 nm, 450 nm and 530 nm, respectively. On the basis of FT-IR and XPS data, the authors demonstrated that the red-shifted C-dots exhibited a higher level of surface oxidation and possessed a larger population of amino groups on their surface. Zhang et al. [63] reported that blue-emitting C-dots (derived via pyrolysis of polyethylene glycol (PEG)) can be turned into green-and orange-emissive systems when treated thermally with PEG and 2,20-(ethylenedioxy)-bis(ethylamine), respectively.
Pang et al. [64] suggested that the PL of C-dots is directly related to surface-state Liu et al. [62] followed a microwave-assisted pyrolysis of m-PD, o-PD and p-PD in formamide to generate three types of C-dots that exhibited λ ex -independent emission with λ max at 360 nm, 450 nm and 530 nm, respectively. On the basis of FT-IR and XPS data, the authors demonstrated that the red-shifted C-dots exhibited a higher level of surface oxidation and possessed a larger population of amino groups on their surface. Zhang et al. [63] reported that blue-emitting C-dots (derived via pyrolysis of polyethylene glycol (PEG)) can be turned into green-and orange-emissive systems when treated thermally with PEG and 2,20-(ethylenedioxy)-bis(ethylamine), respectively.
Pang et al. [64] suggested that the PL of C-dots is directly related to surface-state emission, but the energy gap is controlled not only by the nature of the surface, but also by the size of the carbogenic core. In particular, they reported the synthesis of Cdots via HNO 3 -assisted oxidation of carbon fibres (CFs). By means of ultrafiltration and using membranes with molecular cutoffs < 3 kDa, 3-10 kDa and 10-30 kDa, three different fractions were isolated with d av = 2.7, 3.3 and 4.1 nm, respectively. Evidently, low concentrations of HNO 3 facilitate the commencement of exfoliation from the defect sites of CFs, leading to small and blue-shifted C-dots. In contrast, higher concentrations of HNO 3 and larger oxidation times give rise to larger, red-shifted C-dots with a higher degree of surface oxidation. By accurately controlling the oxidation conditions (reaction time, size of cutoff membranes, concentration of nitric acid), a series of well-defined C-dots were prepared that exhibited λ ex -independent emission, but their λ max varied from 430 nm to 610 nm (λ ex = 360 nm).

Solvatochromism
Solvatochromism effects are common in C-dot dispersions and have been attributed to extensive nanoparticle-solvent dipole-dipole interactions and H-bonding [65]. By virtue of their highly responsive nature, solvatochromic C-dots have been explored as sensitive optical sensors to detect volatile organic compounds [66]. Interestingly, emissive contributions arising from edge and surface bands might induce solvatochromic shifts in the opposite directions [67]. At the same time, pyrrolic nitrogen-and amino nitrogenenriched C-dots show wider solvatochromic shifts and impart higher QY [68].
Wang et al. [69] used p-PD and diphenyl ether as precursors to synthesize spherical C-dots with d av = 2. In other words, the PL of C-dots systematically red-shifts as the polarity of the aprotic solvent increases. In protic solvents (such as methanol, ethanol, benzyl alcohol, 1-hexanol and 1-octanol), the C-dots emitted identical red PL regardless of the nature of the solvent, an effect that has been attributed to the formation of hydrogen bonds between the N and O atoms on the surface of C-dots and the -OH groups of the solvent molecules, which stabilize the excited states.
In addition, Li et al. [70] followed thermal treatment of CA, DMF and urea to synthesize C-dots that showed enhanced red (λ max =640 nm) and NIR (λ max =760 nm) emission in aprotic solvents such as DMSO, DMF and N-methyl-2-pyrrolidone. However, in water, these C-dots exhibited weaker signals in the red region and showed no NIR emission. This solvatochromic effect was ascribed to electron-acceptor groups (S=O/C=O) of the aprotic solvents attached to the outer layers and the edges of the C-dots. Yang et al. [71] synthesized triphenylphosphine (P(Ph) 3 ) functionalized C-dots (p-C-dots) with d av = 4 nm and QY up to 58%. It was observed that λ ex of p-C-dots systematically red-shifted from 431 to 641 nm upon increasing the dielectric constant (ε) of the dispersion medium (Figure 7), an effect that has been attributed to the reduced electron-donating capacity of the P(Ph) 3 groups in environments with increased polarity.

Bioimaging
Red/NIR-emissive C-dots are considered to be ideal for bioimaging applications because they allow larger penetration depth without damaging the surrounding tissue. To that end, Liu et al. [72] suggested a rapid synthesis of red-emitting C-dots (λ max = 630 nm) with QY = 10.8% in water and 31.5% in ethanol (λ ex = 540 nm) using o-PD as a precursor in diluted HNO 3 via a one-step hydrothermal procedure. The C-dots showed low level of toxicity against mice osteoblasts MC3T3-E1 and bone marrow stromal cells (BMSCs) (Figure 7a,d). The bright field and FL images (λ ex = 530 nm) of MC3T3-E1 and BMSCs incubated with C-dots at a concentration of 200 µg/mL for 24 h at 37 • C are displayed in Figure 7b,c,e,f, suggesting that C-dots are predominantly distributed into the cytoplasm and not the nucleus, thus facilitating high resolution cell imaging without affecting the replication or the transcription of DNA.
Tan et al. [73] followed electrochemical exfoliation of graphite in 0.01 M K 2 S 2 O 8 solution to synthesize red C-dots with d av = 3 nm and λ max = 610 nm at λ ex = 500 nm. Those C-dots, when used without any further surface treatment, showed minimal toxicity against HeLa cells and were able to stain both their cell membranes and their cytoplasm, offering clear images under an FL microscope while exhibiting excellent photostability over a prolonged period.
Nanomaterials 2021, 11, x FOR PEER REVIEW 9 of 21 the solvent, an effect that has been attributed to the formation of hydrogen bonds between the N and O atoms on the surface of C-dots and the -OH groups of the solvent molecules, which stabilize the excited states. In addition, Li et al. [70] followed thermal treatment of CA, DMF and urea to synthesize C-dots that showed enhanced red (λmax =640 nm) and NIR (λmax =760 nm) emission in aprotic solvents such as DMSO, DMF and N-methyl-2-pyrrolidone. However, in water, these C-dots exhibited weaker signals in the red region and showed no NIR emission. This solvatochromic effect was ascribed to electron-acceptor groups (S=O/C=O) of the aprotic solvents attached to the outer layers and the edges of the C-dots. Yang et al. [71] synthesized triphenylphosphine (P(Ph)3) functionalized C-dots (p-C-dots) with dav = 4 nm and QY up to 58%. It was observed that λex of p-C-dots systematically red-shifted from 431 to 641 nm upon increasing the dielectric constant (ε) of the dispersion medium (Figure 7), an effect that has been attributed to the reduced electron-donating capacity of the P(Ph)3 groups in environments with increased polarity.

Bioimaging
Red/NIR-emissive C-dots are considered to be ideal for bioimaging applications because they allow larger penetration depth without damaging the surrounding tissue. To that end, Liu et al. [72] suggested a rapid synthesis of red-emitting C-dots (λmax = 630 nm) with QY = 10.8% in water and 31.5% in ethanol (λex = 540 nm) using o-PD as a precursor in diluted HNO3 via a one-step hydrothermal procedure. The C-dots showed low level of toxicity against mice osteoblasts MC3T3-E1 and bone marrow stromal cells (BMSCs) (Figure 7a,d). The bright field and FL images (λex = 530 nm) of MC3T3-E1 and BMSCs incubated with C-dots at a concentration of 200 μg/mL for 24 h at 37 °C are displayed in Figure  7b,c,e,f, suggesting that C-dots are predominantly distributed into the cytoplasm and not the nucleus, thus facilitating high resolution cell imaging without affecting the replication or the transcription of DNA.
Tan et al. [73] followed electrochemical exfoliation of graphite in 0.01 M K2S2O8 solution to synthesize red C-dots with dav = 3 nm and λmax = 610 nm at λex = 500 nm. Those Cdots, when used without any further surface treatment, showed minimal toxicity against HeLa cells and were able to stain both their cell membranes and their cytoplasm, offering

Sensing/Biosensing
Oftentimes, heteroatom doping is coupled with surface patterning strategies and sp 2 domain engineering in order to optimize red-shifted emissions. Along those lines, Li et al. [74] followed hydrothermal treatment of CA and thiourea to synthesize redemitting S,N codoped C-dots that were functionalized with phenylboronic acid tags yielding S,N-C-dots-PBA with QY = 23% and λ max = 593 nm under λ ex = 550 nm. The S,N-C-dots-PBA showed excellent biocompatibility and were able to illuminate PC12 cells under the FL microscope 20 min after their injection (Figure 8a). Interestingly, the PL intensity decreased upon the addition of 1.0 and 5.0 µM Fe 3+ (Figure 8b,c), and the corresponding intracellular PL was compared to the S,N-C-dot-free sample (control) (Figure 8d). The quenching efficiency was 73% and 96% for 1.0 µM and 5.0 µM Fe 3+ ions, respectively. This behaviour indicates that S,N-C-dots-PBA can be explored for the highly sensitive intracellular detection of Fe 3+ . microscope 20 min after their injection (Figure 8a). Interestingly, the PL intensity d creased upon the addition of 1.0 and 5.0 μM Fe 3+ (Figure 8b,c), and the correspondi intracellular PL was compared to the S,N-C-dot-free sample (control) (Figure 8d). T quenching efficiency was 73% and 96% for 1.0 μM and 5.0 μM Fe 3+ ions, respectively. T behaviour indicates that S,N-C-dots-PBA can be explored for the highly sensitive intrac lular detection of Fe 3+ .  In addition, Gao et al. [75] followed hydrothermal treatment of CA and neutral red dye (NR) (ratio of 1000:1) to prepare red-emissive C-dots, the PL of which quenched in the presence of Pt 2+ (ethanol as solvent) due to a rapid electron-transfer process between the metal ions and the C-dots' surface. Moreover, zebrafish (ZF) that were loaded with Pt 2+ and C-dots exhibited PL properties in a manner that critically depended on the concentration of the internalized Pt 2+ (Figure 9), indicating that C-dots can be used as sensors to quantify the Pt 2+ levels present in living organisms.
In addition, Gao et al. [75] followed hydrothermal treatment of CA and neutral red dye (NR) (ratio of 1000:1) to prepare red-emissive C-dots, the PL of which quenched in the presence of Pt 2+ (ethanol as solvent) due to a rapid electron-transfer process between the metal ions and the C-dots' surface. Moreover, zebrafish (ZF) that were loaded with Pt 2+ and C-dots exhibited PL properties in a manner that critically depended on the concentration of the internalized Pt 2+ (Figure 9), indicating that C-dots can be used as sensors to quantify the Pt 2+ levels present in living organisms. Figure 9. FL imaging of Pt 2+ in ZF: (a1−e1) Optical images and (a2−e2) FL images stemming from Cdots in ZF loaded with several concentrations of Pt 2+ : 0 (a1,a2); 30 (b1,b2); 60 (c1,c2); 100 (d1,d2) and 150 μM (e1,e2). Reprinted (adapted) with permission from [75]. Copyright 2021 American Chemical Society.

Photothermal Therapy (PTT)
Photothermal therapy (PTT) relies on radiation-absorbing particles to cause thermal ablation of tumour cells and subsequent cell death. Red-shifted C-dots are ideal candidates as PTT agents due to their low phototoxicity, great photothermal conversion effectiveness and deep tissue penetration.
As mentioned in the Heteroatom doping section, Ge et al. [47] developed a hydrothermal approach of PPA to fabricate red-emissive C-dots (u-C-dots) that showed a wide absorption range from 400 to 750 nm and 38.5% photothermal conversion. Those properties make u-C-dots suitable for PL and photoacoustic (PA) imaging as well as PPT. In Figure  10a IR images of a mouse that was subjected to intertumoral injection of u-C-dots indicate that the temperature of the tumour cells rose up to 58.4 °C within 10 min of laser radiation, Figure 9. FL imaging of Pt 2+ in ZF: (a 1 -e 1 ) Optical images and (a 2 -e 2 ) FL images stemming from C-dots in ZF loaded with several concentrations of Pt 2+ : 0 (a 1 ,a 2 ); 30 (b 1 ,b 2 ); 60 (c 1 ,c 2 ); 100 (d 1 ,d 2 ) and 150 µM (e 1 ,e 2 ). Reprinted (adapted) with permission from [75]. Copyright 2021 American Chemical Society.

Photothermal Therapy (PTT)
Photothermal therapy (PTT) relies on radiation-absorbing particles to cause thermal ablation of tumour cells and subsequent cell death. Red-shifted C-dots are ideal candidates as PTT agents due to their low phototoxicity, great photothermal conversion effectiveness and deep tissue penetration.
As mentioned in the Heteroatom doping section, Ge et al. [47] developed a hydrothermal approach of PPA to fabricate red-emissive C-dots (u-C-dots) that showed a wide absorption range from 400 to 750 nm and 38.5% photothermal conversion. Those properties make u-C-dots suitable for PL and photoacoustic (PA) imaging as well as PPT. In Figure 10a IR images of a mouse that was subjected to intertumoral injection of u-C-dots indicate that the temperature of the tumour cells rose up to 58.4 • C within 10 min of laser radiation, thus causing permanent damage to cancer cells. Following a 16-day course, the tumour disappeared and the mouse fully recovered, while the mouse receiving saline treatment failed to do so (Figure 10b,c). At the same time, no distinct inflammation, cell necrosis or apoptosis in the heart, liver, spleen, lung or kidney were observed, confirming the absence of undesired side effects induced by u-C-dots (Figure 10d).

Photodynamic Therapy (PDT)
Photodynamic therapy (PDT) has distinct advantages over traditional cancer treatments (surgery, chemotherapy and radiotherapy) such as low toxicity, minimal damage to healthy tissue and mild side effects. In PDT, a laser beam excites the photosensitizer to generate reactive oxygen that eventually destroys the tumour cells.
Jia's group [76] followed solvothermal treatment of Hypocrella Bambusae to synthesize red-emissive HBC-dots with d av = 4.8 nm and λ max = 610 nm at λ ex = 540 nm. After 10 min of laser irradiation (635 nm at 0.8 W/cm 2 ), the temperature of the HBC-dot solution (200 µg/mL) rose by 26.9 • C, while it increased by only 3.7 • C upon irradiation at 0.1 W/cm 2 . The results confirmed that the HBC-dots, at 0.1 W/cm 2 , could produce 1 O 2 , facilitating PDT, but at 8 W/cm 2 they could generate both 1 O 2 and thermal energy, facilitating both PDT and PTT treatments. Upon a 6-hour incubation of HeLa cells with HBC-dots, it was observed that the cytoplasm emitted red PL (Figure 11a). The coincubation of HeLa cells with HBC-dots and 2 ,7 -Dichlorofluorescin diacetate (DCF-DA), a green dye that is commonly used to probe intracellular 1 O 2 formation, followed by 635 nm irradiation gave rise to strong green PL emission (Figure 11b), thus confirming the presence of 1 O 2 . Moreover, standard MTT assay showed that HBC-dots exhibited low toxicity against HeLa cell under dark conditions (black bars in Figure 11c). The cell viability was compromised at power density at 0.1 W/cm 2 (PDT system, red bars in Figure 11c), and the effect was more pronounced at power density at 0.8 W/cm 2 (PDT+PTT system, blue bars in Figure 11c), given that 99% of HeLa cells incubated with 200 mg/mL HBC-dots did not survive. After intravenous injection of HBC-dots in mice, the PL intensity in cancer sites rose with time, reaching its maximum value after 8 h (Figure 11d). standard MTT assay showed that HBC-dots exhibited low toxicity against HeLa cell under dark conditions (black bars in Figure 11c). The cell viability was compromised at power density at 0.1 W/cm 2 (PDT system, red bars in Figure 11c), and the effect was more pronounced at power density at 0.8 W/cm 2 (PDT+PTT system, blue bars in Figure 11c), given that 99% of HeLa cells incubated with 200 mg/mL HBC-dots did not survive. After intravenous injection of HBC-dots in mice, the PL intensity in cancer sites rose with time, reaching its maximum value after 8 h (Figure 11d). Ge et al. [77] synthesized C-dots with excellent water solubility using polythiophene derivatives as the precursor via a hydrothermal method. The C-dots showed interesting properties such as a broad absorption region from the visible to the NIR, superior biocompatibility and photostability, high 1 O2 yield and strong emission with λmax = 680 nm at λex = 488 nm.

Solid-State Red C-Dots and Their Applications
Significant progress has been made in solid-state red C-dot-based systems such as hybrid powders and polymer nanocomposites typically derived via in situ polymerization, mechanical and melt mixing and solution blending. Ge et al. [77] synthesized C-dots with excellent water solubility using polythiophene derivatives as the precursor via a hydrothermal method. The C-dots showed interesting properties such as a broad absorption region from the visible to the NIR, superior biocompatibility and photostability, high 1 O 2 yield and strong emission with λ max = 680 nm at λ ex = 488 nm.

Solid-State Red C-Dots and Their Applications
Significant progress has been made in solid-state red C-dot-based systems such as hybrid powders and polymer nanocomposites typically derived via in situ polymerization, mechanical and melt mixing and solution blending.
FT-IR proved the presence of -NO 2 and -NH 2 for the GnC-dots and the C=O in the quinone structure for the RdC-dots. The dispersion of GnC-dots into methyltriethoxysilane (MTES) and RdC-dots into 3-triethoxysilylpropylamine (APTES) gave rise to transparent nanocomposite gel glasses that under UV radiation appeared green and red, respectively (Figure 12c). To form WLEDs, the GnC-dots/MTES gel was deposited (and allowed to dry) on a blue LED chip (27 lm/W at 20 mA, λ ex = 460 nm), followed by the deposition of RdC-dots/APTES gel (Figure 12d). The electroluminescence (EL) spectra of both cold and warm WLED lamps revealed three emission peaks located at 460, 508 and 615 nm, corresponding to the emissions of the LED chip, GnC-dots and RdC-dots (Figure 12e), respectively. Moreover, the warm WLED lamp showed a high colour rendering index (CRI) of 92.9, compared to CRI = 81.1 for the cold lamp.
As aforementioned, Wang et al. [69] followed a solvothermal treatment of p-PD into diphenyl ether to synthesize graphitic C-dots that exhibited λ max at 511, 525, 545, 554, 568, 602 and 615 nm in CCl 4 , toluene, CHCl 3 , acetone, DMF, CH 3 OH and H 2 O, respectively. To prepare liquid LEDs, C-dot dispersions in toluene, DMF and methanol were encapsulated in silica glass boxes and packed above a UV-LED chip (λ em = 370 nm) in order to generate green, yellow and red PL contributions respectively. Moreover, the green-emissive C-dots in methyl methacrylate nanocomposite and the red-emissive C-dots in PVA nanocomposite were deposited on a UV-LED chip to assemble solid LEDs.
Zhang et al. [80] synthesized a colour-tuneable solid-state luminescent material via a hydrothermal procedure using CA, urea and Eu (DPA) 3 and a 2D-layered-structure nanoclay. The hybrid material obtained showed tuneable emission colours from red to blue under different λ ex , where the Eu 3+ and C-dots mainly contributed to the red and blue emission, respectively, making them potential candidates for WLED applications. Yuan et al. [81] synthesized amorphous, red-emissive C-dots using 1,2,4-triaminobenzen as carbon source and PEG200 as passivation agent via a solvothermal method. The C-dots showed λ exindependent behaviour, with a QY up to 25% in ethyl acetate (λ ex = 460 nm), and were combined with silica to fabricate red PL powder in order to construct warm WLEDs.

Pollutant Sensing
Liang's group [82] followed the carbonization of sugarcane bagasse in a mixture of concentrated sulfuric and phosphoric acid to synthesize red-emitting C-dots that were subsequently coated on polyvinylidene fluoride (PVDF) membranes. The PVDF/C-dot membranes exhibited red PL (λ ex = 365 nm) that was quenched in the presence of ammonia, but not in the presence of acetonitrile, o-nitrotoluene, toluene, cyclohexylamine, hydrazine, ethanediamine, dimethyl sulfoxide, acetic acid, acetone, chloroform or hydrochloric acid (Figure 13a,b). The PVDF/C-dot system was shown to be a highly selective and highly sensitive sensor for ammonia with a detection limit of 1.7 ppm and a response time of 30 s (Figure 13c). Lu et al. [83] followed hydrothermal treatment of citric acid and ethylenediamine in formamide−water binary systems to generate a mixture of blue and red-emissive C-dots. Subsequently, ion imprinted mesoporous polymers were prepared using Cr 3+ and Pb 2+ as templates and the mixture of blue and red C-dots as fluorescent probes to generate a dual channel detection system, given that Cr 3+ only quenches the emission of blue Cdots and Pb 2+ only quenches red C-dots. The detection limits for Cr 3+ and Pb 2+ were 27 nm and 34 nm, respectively. Hu et al. [84] followed a solvothermal treatment of ophenylenediamine and selenourea in HCl to synthesize red-emissive Se,N,Cl codoped C-dots with QY = 23.6%, the emission of which can be selectively quenched in the presence of malachite green (MG), thus functioning as nanosensors for MG with a limit of detection close to 20 nM.

Nanoforensics
Li et al. [85] synthesized red-emissive, λ ex -independent, graphitic C-dots (pC-dots) with d av = 12.5 nm following solvothermal treatment of p-PD. FT-IR of pC-dots showed the stretching vibrations of N-H, C=N, C=C and C=C, and XPS spectra revealed they consisted of C (79.56%), N (15.61%) and O (4.83%). As shown in Figure 14a, incorporation of pC-dots into starch gave rise to highly PL powders (the PL spectra of pC-dots in ethanol is also displayed for comparison). The pC-dot/starch powder was used to develop latent fingerprints deposited on a glass substrate and was able to reveal well-defined ridges with minimal background interference (Figure 14b). Lu et al. [83] followed hydrothermal treatment of citric acid and ethylenediamine in formamide−water binary systems to generate a mixture of blue and red-emissive C-dots. Subsequently, ion imprinted mesoporous polymers were prepared using Cr 3+ and Pb 2+ as templates and the mixture of blue and red C-dots as fluorescent probes to generate a dual channel detection system, given that Cr 3+ only quenches the emission of blue C-dots and Pb 2+ only quenches red C-dots. The detection limits for Cr 3+ and Pb 2+ were 27 nm and 34 nm, respectively. Hu et al. [84] followed a solvothermal treatment of o-phenylenediamine and selenourea in HCl to synthesize red-emissive Se,N,Cl codoped C-dots with QY = 23.6%, the emission of which can be selectively quenched in the presence of malachite green (MG), thus functioning as nanosensors for MG with a limit of detection close to 20 nM.

Nanoforensics
Li et al. [85] synthesized red-emissive, λex-independent, graphitic C-dots (pC-dots) with dav = 12.5 nm following solvothermal treatment of p-PD. FT-IR of pC-dots showed the stretching vibrations of N-H, C=N, C=C and C=C, and XPS spectra revealed they consisted of C (79.56%), N (15.61%) and O (4.83%). As shown in Figure 14a, incorporation of pC-dots into starch gave rise to highly PL powders (the PL spectra of pC-dots in ethanol is also displayed for comparison). The pC-dot/starch powder was used to develop latent fingerprints deposited on a glass substrate and was able to reveal well-defined ridges with minimal background interference (Figure 14b). Yuan et al. [86] followed a solvothermal treatment of o-phenylenediamine and 1,8diamino naphthalene in DMF in the presence of boric acid to synthesize red-emissive Bdoped C-dots with QY = 12%. The B-C-dot dispersion in PVA was used as an ink for advanced anticounterfeit applications, given that it appears blue upon UV excitation but becomes pink after alkali treatment and returns to blue after acid treatment in a reversible manner.

Conclusions
Although the large-scale production of well-defined, red-emissive C-dots remains an open challenge, a number of promising approaches towards this direction have been demonstrated. These synthetic strategies rely on heteroatom doping, surface functionalization and the formation of extensively conjugated domains, while a combination of two or more of those engineering principles is usually more effective in terms of PL Yuan et al. [86] followed a solvothermal treatment of o-phenylenediamine and 1,8diamino naphthalene in DMF in the presence of boric acid to synthesize red-emissive B-doped C-dots with QY = 12%. The B-C-dot dispersion in PVA was used as an ink for advanced anticounterfeit applications, given that it appears blue upon UV excitation but becomes pink after alkali treatment and returns to blue after acid treatment in a reversible manner.

Conclusions
Although the large-scale production of well-defined, red-emissive C-dots remains an open challenge, a number of promising approaches towards this direction have been demonstrated. These synthetic strategies rely on heteroatom doping, surface functionalization and the formation of extensively conjugated domains, while a combination of two or more of those engineering principles is usually more effective in terms of PL performance. In contrast to their blue and green counterparts, the synthesis of red C-dot-based systems is rather tedious and energy intensive and largely depends on the use of aromatic precursors, organic solvents and toxic compounds. Overcoming those barriers will allow the development of a new generation of photoactive materials that can potentially advance crucial biomedical treatments, sensing applications and lighting technologies.

Outlook
There is no doubt that C-dots are a highly promising class of nanomaterials with distinct advantages with respect to conventional organic fluorescent dyes because of their multicolour nature and enhanced structural stability, while they are superior in terms of environmental friendliness and preparation ease compared to heavy metal-based quantum dots. These nanoemitters can be used in isolation for a variety of applications [38] or can be combined with other materials to generate advanced nanocomposites and devices, such as polymeric hybrids [27], powder compositions [87], ordered mesoporous frameworks [88] and C-dot-based Forster resonance energy transfer systems [89]. At the same time, their photoluminescence mechanism is not fully understood, and more studies are needed in order to gain further insights on the structure-properties relationships of those complex systems. This observation is particularly true for red-emissive C-dots, which are only recently gaining significant attention from the scientific community, although both challenges and benefits associated with their production are well-recognized.
First is the observation that the majority of C-dots typically display weak emissions on the red end of the spectrum, while certain approaches allow the synthesis of predominantly red nanoemitters. A recent theoretical study indicated that improved red emission is expected for C-dots bearing abundant weakly interacting surface emission centres [90]. Simple post-modification treatments (such as the reaction with acetaldehyde [91]) can modulate the PL emission, essentially converting blue-emitting into red-emitting C-dots. In addition, C-dots with reversibly switchable green-red emissions have been reported [78,86].
Second is the realization that a number of effective strategies for the large-scale production of blue and green C-dots that require a relatively low capital investment are emerging in the literature, but those efforts are less advanced for their red-emissive counterparts. Nevertheless, cost-effective approaches that are compatible with standard industrial processing are present in the literature. For example, far-red C-dots with QY = 18.5% were prepared via a 3 min microwave treatment of glutathione in formamide followed by centrifugation and dialysis [92]. Moreover, red C-dots with QY = 53% can be produced on a gram scale by heating a formamide solution of citric acid and ethylenediamine [93].
Third is the understanding that the nature of the precursor material plays a significant role on the PL properties of the red-emissive C-dots. For example, red C-dots with QY = 84% are derived from the highly conjugated molecular precursor tris(4-aminophenyl)amine [94]. In analogy to blue and green C-dots, organic fluorophores developed in situ during the synthesis of red C-dots are expected to have a pronounced impact on their PL properties. We note that it has been suggested that red emission might be associated exclusively to organic fluorophores entrapped within the carbogenic structure [37]. In terms of biomass utilization, we note that red-emitting N,Mg codoped C-dots from the leaves extract of Bougainvillea plant showed λ ex -independent emissions with λ max = 678 nm and QY = 40% [95], while NIR-C-dots with QY = 31% and 15.3% were synthesized by heating a formamide solution of lemon juice [40] and via solvothermal treatment of spinach [96].
Author Contributions: Conceptualization, S.G. and A.K.; writing, S.G. and A.K. Both authors have read and agreed to the published version of the manuscript.
Funding: S.G. was supported by the University Alliance Doctoral Training Alliance under the Marie Skłodowska-Curie COFUND scheme.