Advances in the design and use of carbon dots for analytical and biomedical applications

Carbon dots (CDs) have garnered significant interest for their potential use in multiple applications due to their size, fluorescent properties, high photostability, low toxicity and biocompatibility. CDs can be tailored for specific needs, as they can be synthesized with diverse precursors and techniques for functionalization. Since the applications of CDs are rapidly expanding, this review highlights recent developments in this burgeoning field. Specifically, we describe advances in CD synthesis tailored for applications that include pH and temperature sensing, biochemical analysis, and bioimaging. We also discuss various challenges and practical solutions that will drive CD-based research forward. Challenges include the lack of standardized synthesis and purification methods for CDs, the lack of clarity regarding their mechanism of action, and procedural flaws in their applications. In conclusion, we provide recommendations for collaboration among disciplines to bridge existing knowledge gaps and address the key challenges required for CDs to be fully commercialized.


Introduction
The distinctive characteristics of carbon nanomaterials, which include carbon dots (CDs), have captured global attention.
Carbon's ability to form several allotropes is often attributed to its valency [1].CDs are fluorescent carbon nanomaterials with polymeric surface moieties covering an amorphous or crystalline nano-graphitized core [2,3].CDs are quite small (<10 nm) and have distinct, structure-dependent physicochemical properties that include high fluorescence quantum yields, blue to near-infrared emission, exceptional photostability, and ideal dispersion characteristics with low toxicity [4].CDs can be categorized as carbon nanodots (CNDs), graphene quantum dots (GQDs) or polymer dots (PDs) depending on their composition and structure [5].PDs  Original content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence.Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI.carbon core that is surrounded or composed of aggregated linear polymers or polymer chains, CNDs have a graphite-or amorphous-like structure, and GQDs are made up of one to two graphene layers [5,6].The physicochemical properties of CDs make them suitable for applications, such as sensors, electronic devices, and energy-modulating instruments, as they overcome challenges, such as photobleaching, phototoxicity, and interference from background autofluorescence [7][8][9].
CDs with photoluminescence properties that change with the environment can be useful for analytical applications [10,11].For example, emission intensities at different fluorescence spectra can change in response to variations in pH or temperature, making CDs attractive for use in industrial, agricultural, environmental and biomedical sectors where pH and temperature sensing are important [12].CDs are also suited for biomedical applications including diagnostics [13].
For example, recent studies demonstrated that sulphur and nitrogen-doped CDs could be used to image cancer cells, supporting their potential use as a diagnostic tool [14][15][16].Due to their small size and low toxicity, it is speculated that CDs can readily enter cells within tissues and can cross both the glomerular filtration and blood-brain barriers [17][18][19][20][21].The entry mechanism of CDs will vary with size, surface charge, and surface functionalization, as well as the cell type and their microenvironment (figure 1).Their distinct optical features and simple functionalization make them ideal for deep-tissue fluorescence imaging as they can be tuned for excitation and emission in far-red wavelengths [22].
In the last decade, CD-related research has evolved rapidly with their development for analytical use and biological applications.Several interesting reviews emphasize the synthesis, physicochemical, and/or optical characteristics of CDs, as well as their biological applications [1,4,10,23,24].This review will uniquely describe some of the more recent advances in these areas, as well as discuss the challenges that need to be overcome to move CDs from the bench and into the commercial domain.We believe that by highlighting the many benefits of CDs and their wide range of applications, this review will contribute to the growth of this burgeoning area of research.

Synthesis of carbon dots from organic precursors
The physicochemical properties of CDs are determined by their precursors and methods of synthesis.Conventionally, several methods have been reported for the synthesis of CDs, which include 'top-down' and 'bottom-up' approaches [25].The 'bottom-up' method describes the synthesis of CDs from selected chemical precursors under specific conditions, while the 'top-down' method generates CDs through the fragmentation of large carbonaceous materials into nanoparticles [26][27][28].Both approaches include methods for in situ doping and functionalization, among others, to create CD cores and surfaces with the desired physicochemical properties [29,30], which is essential for their functionality and potential use.Importantly, CDs are a green alternative to many other nanomaterials and organic dyes.Natural resources, biowaste, and other agricultural waste can be used as green carbon sources for the synthesis of CDs.This approach makes CDs attractive due to their global availability/accessibility, eco-friendliness, lack of toxic by-products and biodegradability.Furthermore, from ecological safety and sustainability perspectives, the synthesis of CDs from natural resources could mitigate environmental pollution and assist with waste management by repurposing agricultural waste into valueadded products.Other natural precursor materials include black fungus [31], bagasse, coffee grounds, banana juice, potatoes, leaves, palm shell powder, orange juice, vegetable waste and soybean juice, among others [32][33][34][35][36].
Different precursors can impact the photoluminescent properties of CDs, such as fluorescence emission wavelength (s), quantum yield, and photostability.For example, Manioudakis et al [37] reported that CDs made with citric acid and amine passivated agents as precursors resulted in CDs with blue fluorescence (excitation of ∼350 nm and emission of ∼450 nm) and excellent photoluminescent properties, including a high quantum yield of 33%, and prolonged chemical and photostability.In another study, Pan et al [38] demonstrated that CDs synthesized with citric acid and formamide exhibited strong fluorescence across the entire visible spectrum making them suitable for multicolor bioimaging assays.
The methods used for CD synthesis could also affect their physicochemical properties.Table 1 reveals that the hydrothermal method remains a widely utilized technique for the preparation of CDs.The benefit of hydrothermal/ solvothermal processes is that they do not require additional passivation steps to ensure water dispersibility.This process produces CDs that have predominantly hydrophilic moieties on their surfaces, such as carboxyl and amino groups, depending on the precursors.Another approach is the microwave method, which is quick and effective.The quality and yield of the CDs can be considerably increased by in situ transient heating using this method.While some CDs have low quantum yields, rendering them unsuitable for bioimaging, recent studies have shown that doping CDs with heteroatoms enhances their fluorescence characteristics.In comparison to other atoms, nitrogen-doped CDs have greater quantum yields by stabilizing surface defects and enhancing fluorescence emission [17].Given the improvement in optical properties by passivating or doping CDs with nitrogen and sulphur-containing groups, there are advantages to using natural precursors, such as the innate presence of sulphur and nitrogen groups in biomass, cost effectiveness and ecofriendliness [39][40][41].

Photoluminescent properties of CDs
The unique photoluminescent properties of CDs make them ideal for applications in sensing and bioimaging.CDs typically fluoresce at different wavelengths with a range of quantum yields and photostability depending on their physicochemical properties.As shown in figure 2, the photoluminescence of CDs can be influenced by their size, shape, surface charge and functionalization, which is largely determined by the precursors and methods used for synthesis.For example, as shown by Behi et al [67], CDs <5 nm have higher photostability and quantum yields compared to larger CDs.While it is not clear why this occurs, one hypothesis is that the larger surface area could trap electrons causing a decrease in surface energy [68].Furthermore, the surface state caused by passivation, such as nitrogen-sulfur interactions, as well as interactions with the carbon backbone play key roles in conferring distinct fluorescent properties [69].In particular, solvents, such as water, dimethylformamide (DMF), ethanol, and acetic acid (AA) produce CDs that emit fluorescence of different colours (i.e.blue at 470 nm, green at 500 nm, yellow at 539 nm, and orange at 595 nm, respectively), demonstrating that different functional groups cause different effects on the absorption spectra in the visible light band [70].Huo et al [70] showed that CDs prepared solvothermally from citric acid in solvents, such as DMF, or DMF mixed with ethanol or acetic acid rather than water, displayed red fluorescence with greater intensity.Furthermore, FTIR and XPS results revealed that the graphitic-N in the carbon core and COOH functional groups on the surface of the CDs likely caused the red shift of emission peaks.
The surface functionalization of CDs can also influence their photoluminescent properties.Surface modifications can introduce functional groups that enhance the stability and fluorescence properties of CDs.For example, Fu et al [71] reported that surface passivation of CDs with polyethylene glycol (PEG) improved their quantum yield and photostability compared to commercially available dyes.The PEG 6000 /CD nanocomposite had better resistance to photobleaching and exhibited two emission wavelengths at 560 nm (green) and 613 nm (red), making them ideal for long-term sensing applications and ratiometric measurements [71].The presence of functional groups containing nitrogen and oxygen, as well as the sp 2 hybridized carbon on PEG 6000 /CD confer dual photoluminescence properties.In a study by Zhang et al, protonation with 2,3-diaminophenazine resulted in CDs with NIR-induced two-photon fluorescence emission at 630 nm and 680 nm, and long wavelength one-photon fluorescence emission at 620 nm (full width at half maximum [FWHM] ∼24 nm) [72].Edge-amino protonation alters the chemical state of CDs by reducing the photon transition band gap, causing red fluorescence to emit with a narrow peak.The effects of 2,3-diaminophenazine on emission demonstrate how precursors can be chosen to achieve the desired photoluminescence properties.However, whether the optical properties of CDs are affected by their interaction with analytes and/or arise due to differences in the environmental conditions (variation in pH, temperature) is not clear.
As described above, CDs can have excitation-dependent emission that is tunable, however the mechanism remains debatable [14].This phenomenon has been attributed to the quantum confinement effect, electronegativity of heteroatoms, surface traps, giant red-edge effect, edge states, among other models [73,74].Furthermore, differences in size, or surface groups (i.e.polydispersity) could cause variable band gaps and/or selective excitation of the differently-sized layered structures [75][76][77][78].Hence, extensive purification to obtain a monodisperse population is recommended for more consistent fluorescence characteristics [79,80].Fluorescence can emanate from both the carbon core and bound fluorophores, where the core is speculated to produce weaker excitationdependent fluorescence compared to the surface [81][82][83].
Furthermore, changes in temperature and pH, and the presence of analytes, such as inorganic and organic substances, can quench the fluorescence of CDs [84][85][86].Hence, CDs can be utilized as a sensor to detect analytes and changes in CD's environment/medium based on fluorescence quenching.Förster resonance energy transfer (FRET), surface energy transfer (SET), photoinduced electron transfer (PET), inner filter effect (IFE), Dexter energy transfer (DET), static quenching and dynamic quenching were some of the quenching mechanisms proposed and used to elucidate the detection of analytes and other analytical applications [87].While some studies suggest that quenching is influenced by the crystallinity of the core and solution chemistry [88][89][90], other studies support that quenching can be attributed to interactions between the core and molecular states of CDs and various analytes [85,90,91].These hypotheses and mechanistic theories point to the necessity for in-depth research to clarify the optical characteristics of CDs in relation to their physicochemical properties and environment.

CDs as bioimaging tools
CDs can be used for biological imaging applications, such as monitoring cellular oxidative stress, changes in intracellular acidity or temperature, sensing metallic species, in addition to other applications.However, it is crucial to understand the impact of CDs on biological systems, their mechanism of uptake by cells, and factors that may influence how they interact with the subcellular and extracellular environment.

Cytotoxicity and biocompatibility of CDs
Cytotoxicity and biocompatibility are important to test when considering the use of CDs in vivo.CDs could affect cell viability (cytotoxicity), but also have different accessibility and functionality in tissues due to factors, such as retention and immune response (biocompatibility).Cytotoxicity is evaluated in vitro using cultured cells, where CDs are directly added to the cells and their effect on cell viability is assessed [92].Since assays for metabolic activity (i.e.MTT, WST8) measure the proportion of 'live' cells, performing these assays after 1-3 population doubling times will not distinguish between an increase in death versus a decrease in proliferation, and additional studies are needed to determine how CDs affect cells more precisely.Cytotoxicity has implications for the safety and suitability of CDs for biomedical applications [93].On the other hand, biocompatibility is important to evaluate in tissues and whole organisms to determine how the accessibility and efficacy of CDs are altered, to ensure they do not trigger an immune response, as well as how well they are retained after systemic delivery [94][95][96].Importantly, CDs have been reported to have low cytotoxicity and good biocompatibility in various models in vitro and in vivo, making them attractive for biomedical use.
The low cytotoxicity and good biocompatibility of CDs can be attributed to their organic precursors, as well as their physicochemical properties.As shown in table 2, CDs made from carbohydrates, amino acids and other natural sources, such as citric acid, glucose, or chitosan all exhibit excellent biocompatibility and low cytotoxicity [97][98][99][100][101].In addition, the small size of CDs (<10 nm) could facilitate cellular uptake, and their surface properties could be functionalized to reduce toxicity and/or improve biocompatibility [102][103][104].
For instance, CDs coated with polyethylene glycol (PEG) showed reduced cytotoxicity compared to those without [105][106][107].It is not clear why PEG modifications reduced toxicity in this study, but their surface charge may have been neutralized, or PEG may have provided a coating more compatible with the cell surface [105,108].Notably, PEG is commonly used to improve delivery and reduce the toxicity of therapeutic molecules and nanomaterials.The properties imparted by PEG could make surfaces more inert, improve retention and reduce immunogenicity, all of which could improve the biocompatibility of CDs [109].Even without PEG, prior studies demonstrated that CDs have good biocompatibility in biological contexts.For example, CDs can be efficiently cleared from the body through renal excretion [110].CDs are filtered by the kidneys and released into the urine, which reduces their accumulation in organs and tissues, minimizing toxicity that could arise after multiple doses [111,112].Importantly, CDs are stable and retain their optical properties in blood and serum without aggregating or precipitating [113][114][115].This potentially makes CDs suitable for systemic administration for in vivo applications.Despite these promising studies, there is a need to study CDs in human tissues and more complex environments, as the biocompatibility and bioimaging capability of CDs may vary in comparison to mice or other animal models where previous studies have been done.

Mechanisms controlling the cellular uptake of CDs
The cellular uptake of CDs has not been well-studied but likely involves endocytic pathways including clathrin-coated pit endocytosis, fast endophilin-mediated (FEME; dynamindependent) endocytosis, clathrin-independent carriers, macropinocytosis, phagocytosis (specific to immune cells) or caveolin-dependent endocytosis depending on their physicochemical properties [124].For example, clathrin-or FEMEmediated endocytosis requires specific ligand-receptor interactions and are the least likely paths for CD entry (or any nanomaterial) unless they are functionalized with targeting ligands or other functional groups [125].CDs functionalized with transferrin had increased uptake in HeLa, HT29 and MCF-7 cells with overexpressed transferrin receptors compared to non-functionalized CDs [126,127].In a separate study, Zhang et al [128] functionalized CDs with folic acid (FA) and demonstrated their selective uptake by HepG2 cells over-expressing folate receptors.Presumably, the ligands on the surface of CDs remain accessible to receptor binding, but biochemical studies are needed to show that their binding properties (e.g.kinetics) remain intact.Due to their small size, CDs could enter cells via less selective mechanisms, such as micropinocytosis, which describes the constant uptake of small molecules that are nearby via minute vesicles [124,125].
The surface charge of CDs could play a role in cellular entry by impacting selectivity and/or efficacy.A recent study found that CDs synthesized by citric acid and PEI 25000 used multiple routes to enter human adenoid cystic carcinoma cells [129].In general, positively charged CDs seem to have higher cellular uptake compared to negatively charged CDs [106].The negatively charged polar head groups on membrane phospholipids could electrostatically interact with positively charged particles and cause them to accumulate near the cell surface where they can more easily enter cells via endocytic mechanisms.CDs that are very small (i.e.1-2 nm) with amphipathic properties could enter cells via passive entry or less-selective porins or channels although this needs to be properly tested.Finally, the microenvironment surrounding cells could also influence the uptake of CDs.Feng et al observed an increase in uptake for dual-responsive CDs made of citric acid and diethylenetriamine in MCF-7 cells maintained at lower pH [130].In this study, the lower pH could have changed the chemical properties of membrane phospholipids to increase permeability without necessarily increasing the selectivity for CDs.
Importantly, it is difficult to speculate how studies using cultured cells grown as a monolayer in vitro can compare to tissues or tumors in vivo, which are more complex and contain multiple cell types, connective tissue and vasculature.For example, the uptake of CDs made with various oligo (ethyleneimine)-based passivating reagents was reduced in macrophage cells, possibly due to the formation of a protein corona on the CD surface by serum proteins.This corona could have increased its size and altered the net surface charge of the CDs, hindering their interaction with the membrane and/or receptors [131,132].While these studies highlight some of the key features of CDs that impact their uptake, more studies are needed to properly explore the mechanisms by which they enter cells, especially different cell types and in more relevant contexts.

The use of CDs in bioimaging
One of the potential applications of CDs is for bioimaging due to their unique fluorescence properties.As an example, CDs synthesized with asparagine (Asp) showed high contrast biodistribution 15 min after their injection into mice.A significantly stronger fluorescent signal was measured in glioma (cancerous glial cells) compared to normal brain tissue, indicating their ability to penetrate the blood-brain barrier and become preferentially enriched in the tumor [133,134].This preferential localization of CDs is consistent with other nanomaterials that become enriched in tumors due to their disorganized vasculature, and is not specific per se.However, these findings suggest that CDs could be developed as theragnostic tools, such as an imaging agent to diagnose cancers.CDs can also be used for imaging at the subcellular level.Several studies showed that CDs localize to organelles, such as endosomes and lysosomes, ER, golgi, nucleus and mitochondria (e.g.figure 3) [135,136].It is important to note that unless CDs are specifically functionalized, they are most likely going to be in endosomes or lysosomes based on their predicted mechanisms of uptake.For example, Liu et al [137] reported that pH-dependent CDs synthesized from ophenylenediamine precursors with emission at 556 nm colocalized with the commercial lysosome probe (Lyso-Tracker™ Deep Red) in HepG2 cells.Similarly, Guo et al [138] showed that CDs synthesized with citric acid and N,Ndimethylaniline with emission at 520 nm colocalized with LysoTracker™ in HeLa cells.Their CDs showed superior resistance to photobleaching in comparison to the commercial probe with little effect on cell viability.Some CDs have also been speculated to localize to other endomembrane components, such as the endoplasmic reticulum (ER), golgi, and nucleus.For instance, Li et al developed an amphiphilic CD using o-phenylenediamine (o-PD) and phenylalanine with emission at 584 nm that localized to the ER in HeLa cells based on overlap with a commercial dye called ER-Tracker.Importantly, the authors used STEDsuper resolution imaging to detect the localization of their CDs with higher spatial resolution than conventional confocal imaging [139].Huang et al [140] synthesized CDs with L-ascorbic and L-cysteine, which localized to the golgi in HeLa cells based on co-localization with NBD C6-Ceramide.Wang et al [141] synthesized CDs from citric acid and claimed that they accumulated in the nucleus in A549, HEK293 and MDA-MB-231 cells, and speculated that this occurred due to the association of CDs with chromatin via electrostatic interactions.However, more experiments are needed to properly assess the subcellular location of CDs with higher spatial resolution.
Functionalized CDs have also been designed to localize to the mitochondria.Wu et al [142] synthesized CDs coupled to triphenylphosphonium (TPP), a mitochondria-targeting moiety.These CDs localized to the mitochondria in MCF-7 cells as revealed by Mito-tracker and had excellent photostability that permitted long-term imaging of mitochondria dynamics [142].Kaminari et al [143] also designed CDs that selectively localized to the mitochondria.These CDs generated with o-phenylenediamine precursors and conjugated with TPP were capable of tracking mitochondria in MCF-7 cells as shown by MitoTracker.In a related study, Gao et al [144] developed CDs from glycerol and APTMS for targeting the mitochondria, and reported an increase in fluorescence from APTMS-CDs in HepG2 and MCF-7 cells compared to macrophage RAW 264.7 cells, which could be due to differential uptake or quenching (figure 4).Cellspecific differences in the uptake and/or quenching of APTMS-CDs could reveal interesting dynamic changes in mitochondrial function among different cell types in vitro.
While studies of CD subcellular localization are promising, it is important to note that the lack of cell biology expertise may have caused misinterpretation of data.For example, without using a specific marker that is known to localize to a specific organelle, claims of localization may not be accurate.Many images lack spatial resolution or are saturated and have not been quantified using methods that would accurately support statements of colocalization.Further, adding specific signals (e.g.peptides) that could target CDs to precise locations may not be accessible for binding to the factors required for their entry into specific organelles, and/or could be affected by proteins in the culture media.Further, comparing cell types from vastly different tissues and making claims of 'cancer' versus 'non-cancer' could be misleading.In many cases, differences could arise due to the nature of that cell type and tissue of origin versus whether it is cancerous or not.Thus, incorporating more cell biological methodologies will enable CDs to transition to clinical use.

Temperature sensing using CDs
CDs can also be functionalized and/or modified to sense changes in temperature.Temperature is a thermodynamic factor that significantly affects the functionality of chemical and biological systems [145].Nanothermometry refers to the use of nanomaterials to sense changes in temperature in a defined environment, and a variety of nanomaterials synthesized with metals, polymers, rare-Earth-doped nanocomposites and organic materials have thermosensitive luminous characteristics [146][147][148][149][150]. With these nanomaterials, thermal measurements are derived from changes in signal intensity, fluorescence lifetimes, and spectral shifts, among other parameters [151].For example, quantum dots (QDs) have good photostability, high quantum efficiency, and tunable fluorescence, but also have inherent blinking and should be used for short-versus long-term monitoring [152,153].Additionally, QDs often contain heavy metals (e.g.cadmium, tellurium, selenium), preventing their use in biological and environmental applications.On the other hand, fluorescent nanodiamonds have a surface that is chemically resistant and inert, and are more biologically compatible [154].Recently, nanodiamonds were shown to have subdegree accurate intracellular temperature sensing for changes as low as 1.8 mK (sensitivity of 9 mK Hz −1 ) [155][156][157].An intracellular local heat generator and nanothermometer (nanodiamond-nanogel-indocyanine green; ND-NG-ICG) was shown to function as a temperature sensor in HeLa cells [158].Their temperature-sensing ability is due to fluorescence associated with nitrogen-vacancy centers, which arise when a nitrogen atom substitutes for a lattice carbon atom with an adjacent unoccupied lattice site [159,160].However, the use of fluorescent nanodiamonds is severely limited due to their irreproducible synthesis and low quantum yield [161], and local ND-NG-ICG heating sources >100 μg mL −1 produce too much heat causing cell death [158].Therefore, to overcome these limitations, CDs have emerged as an ideal alternative for temperature sensing in biological systems.
Several studies have reported the doping of CDs to modify their physicochemical properties and improve their use in nanothermometry [12,[162][163][164].For example, doping CDs with an epoxy resin caused an improved temperature response due to the enhanced dielectric constant caused by the epoxy resin [165].Aggregation or cooperative interaction between oxygen-containing functional groups and hydrogen bonds in response to temperature may cause quenching of fluorescence intensity that can be used as a read-out of temperature changes [166].As shown in figure 5, the fluorescence intensity of CD/epoxy composite declined linearly from 25 °C to 95 °C and displayed exceptional sensitivity and amazing reversibility/recoverability as a temperature sensor [165].In a recent study performed by Xu et al, [167] CDs synthesized from citric acid and thionine with excitation at 500 nm and emission at 650 nm, could sense temperature changes in MCF-7, Hep G2 and A549 cells without causing toxicity.These CDs were shown to have a strong response to temperature changes with a linear relationship, reversibility and reproducibility under heating and cooling treatments.However, this single-emission approach has some disadvantages in biological systems.For example, autofluorescence that occurs naturally in the cells from components like flavins, NADH and collagen can interfere with single-emitting CDs.In addition, other analytes could also quench the fluorescence, causing misinterpretation of data.An ideal way to overcome this challenge is to use ratiometric sensing.
CDs that differentially produce multiple spectra (dual emissive) have been developed for ratiometric measurements [168].Figure 6 highlights an example of CDs with dual emission, with two fluorescence bands at 370-500 nm and 640-730 nm when excited at 405 nm.The ratiometric approach involves measuring differences in the ratio of the intensity of two emission wavelengths, where one wavelength changes in response to temperature and the other does not and is used as a reference.This ratio can provide precise quantitative measurements independent of concentration [169,170].In a study by Macairan et al [171], dual emissive CDs (dCDs) that emit at 450 nm and 680 nm (blue/red) when excited at 405 nm were synthesized from glutathione and formamide and used to measure temperature changes in HeLa cells (figure 7(a)).Cells treated with dCDs were heated for 2 h between 32 °C-42 °C and quenching of the 680 nm peak occurred as the temperature increased, while the peak at 450 nm remained stable (figure 7(a)).Importantly, these results were comparable to those obtained when tested in water, supporting that the fluorescent properties of these dual emissive CDs were retained in the subcellular environment (figure 7(b)).Thus, CDs designed for ratiometric temperature sensing is a favoured approach for fluorescent  nanothermometry in cells [172].Future CD systems could consider combining the advantages of ratiometric sensing with doping to increase the temperature range and sensitivity.

pH sensing using carbon dots
CDs have also been designed to sense changes in pH.Several environmental and biological processes are influenced by pH, and changes in pH could reveal pollutants or diseases.CDs derived from black fungus demonstrated changes in fluorescence in solution in response to pH due to protonation-deprotonation of the surface functional groups [31].As shown in figures 8(a) and (b), the fluorescence intensity increased as the pH increased from 1 to 4, and then gradually decreased in a linear fashion to pH 13.Similarly, extracts from shiitake mushrooms were used to prepare doped CDs with amine, hydroxyl and carboxyl groups attached to the surface of CDs, which had good cell permeability and pH-sensitive changes in fluorescence (figure 8(c)) [173].Wang et al synthesized green fluorescent CDs from m-diaminobenzoic acid as nanoprobes for pH sensing [174].The fluorescence intensity of the CDs increased as the pH rose from 4.5 to 12, and were used to measure the pH of eight different types of mineral water samples with pH ranging from 5.9 to 8.1.Importantly, the pH values measured by the CDs were consistent with those measured by a pH meter, supporting their reliability [174].In another study, CDs were developed with three-colour emissions using citric acid as a precursor [175].At a pH of 1, these CDs emitted fluorescence as a single, moderately strong peak at 510 nm, while increasing the pH to 2 caused the CDs to produce three distinct emission peaks at 440 nm, 510 nm, and 620 nm, all of which continued to increase with pH.The CDs were used on test paper to demonstrate their ability to sense pH across the scale from 1 to 14.A vivid green was visible at pH 1, which progressively turned gray as the pH value rose to 8, then shifted to purple as the pH increased from 8 to 13, and finally to yellow at pH 14.In another study, Huang et al [176] developed pH-sensitive CDs that exhibited fluorescence lifetime changes in response to pH, enabling real-time monitoring of pH in HeLa cells.These CDs were generated from urea, citric acid and dimethylformamide precursors, and their fluorescence intensity and lifetime differed with pH in a non-linear fashion from 3.5 to 8.5, confirming their potential use as pH sensors.
Other CDs can sense pH ratiometrically, similar to those described for sensing temperature.For example, CDs synthesized from citric acid, 1,4-butanediamine, pH-sensitive dyes and PEG 400 emit spectra at 464 and 580 wavelengths, and ratiometrically sense pH changes within the mycelia of Pholiota adipose fungus [173,177,178].Figure 9(a) illustrates how the CD emission intensity at 580 nm increased with pH (3.0-8.5), while the emission intensity at 464 nm remained constant.Further, the ratiometric sensing of these CDs is linear between pH 4 and 8 (figure 9(b)) [177].Other studies also described the synthesis of CDs with dual emission spectra for ratiometric sensing of intracellular pH changes [179].Shi et al [180] generated CDs conjugated with a pH-sensitive dye (fluoresceinisothiocyanate; FITC) and a pH-insensitive dye (rhodamine B isothiocyanate; RBITC) for ratiometric pH sensing in HeLa cells.An increase in pH caused a greater change in fluorescein (515 nm) compared to rhodamine (575 nm), permitting real-time monitoring of pH changes with different treatments [180].
More recently, Macairan et al [86] demonstrated the use of dual emissive CDs synthesized from glutathione and formamide to sense changes in lysosomal pH in glioblastoma cells in response to diclofenac and metformin, which are currently in clinical trials to treat cancer.Diclofenac does not become protonated or alter the pH of the lysosome, while metformin can cause acidification.The CDs accumulated in the lysosomes and showed linear changes in the ratio of red fluorescence at 650 nm and 680 nm as both peaks changed in response to pH changes.As expected, no change in lysosomal pH occurred with diclofenac, while a change was observed after treatment with metformin.Other CDs that can be used for pH sensing include those with fluorescence resonance energy transfer between the core and surface groups, and passivation with large organic moieties could improve photostability and intensity [178,181].Table 3 shows examples of ratiometric CD-based probes, mechanisms for pH sensing and the range of linear responses obtained for the different probes.
One of the main physicochemical properties of CDs that is affected by pH is the zeta potential.For example, the deprotonation of carboxyl groups in basic pH could cause an increase in negatively charged surface moieties on the surface, which would result in more negative zeta potential values [182].Sun et al [183] used this change in zeta potential to measure pH changes.The zeta potential of their CDs was approximately 17 mV between pH 4-5, but as pH increased to 11, the zeta potential changed to −12 mV.These changes in zeta potential could affect the optical properties of CDs including quenching, and TEM revealed that CDs aggregated due to noncovalent molecular interactions facilitated by pH changes [10].
Although CDs may address some of the drawbacks of conventional pH sensors, some challenges remain: (i) the pH sensitivity and response range may not support practical applications, requiring alternative synthetic approaches, and (ii) the surface structures predicted to be in prototropic equilibrium with H + /OH − are the main basis for the pHsensitive mechanism of CDs, yet the precise structural arrangement that mainly facilitates pH sensitivity has yet to be fully understood.More studies are needed to reveal how a certain structure or group affects the pH sensitivity of CDs.

Biochemical analysis using CDs
CDs have also been developed to detect specific components in complex samples, such as metals and organic molecules, which have importance in environmental or biological contexts [190].Metal ions such as sodium (Na + ), potassium (K + ), magnesium (Mg 2+ ), calcium (Ca 2+ ), and iron (Fe 3+ ) are required for biological processes, such as signaling, neuronal function and muscle contraction, and tracking changes in their concentration or distribution could be important for the diagnosis of various diseases [191,192].Current detection methods include spectroscopy, chromatography, mass spectrometry (MS), immuno-and enzymatic assays, which can be costly and/or rely on technically advanced infrastructure, and CDs could offer a more reliable, cost-effective solution.
CDs have been synthesized for use as fluorescent probes to sense ferric ions (Fe 3+ ) in solution or in cells [193].Liu et al [194] synthesized CDs doped with sulfur, nitrogen and boron with emission at 520 nm that quenched linearly in response to an increase in Fe 3+ concentration coupled with an increase in emission at 645 nm.This sensing was shown by imaging the doped CDs in HeLa cells, human urine and in serum samples in cuvettes.In another study, Yue et al [195] developed CDs with ethylenebis (oxyethylenenitrilo) tetraacetic acid that emit fluorescence at 460 nm with linear fluorescence quenching in response to intracellular Ca 2+ over a range of 15-300 μM and a detection limit of 0.38 uM in solution.These CDs were also tested in HeLa cells, where treatment with 1 mM of Ca 2+ caused quenching of fluorescence, demonstrating their potential use as Ca 2+ sensors [195].However, it is not clear how such high concentrations of Ca 2+ affected the health of the cells, and more studies are needed to determine how CDs, which tend to be trapped in endosomes and lysosomes, could be developed to detect changes in intracellular Ca 2+ , which tends to be released to the cytosol during signaling.
CDs have also been developed to sense important organic molecules in biological contexts.Reactive oxygen species (ROS) has been associated with signaling as well as redox processes and homeostasis, where changes could signify disease [196].Hydrogen peroxide is a ROS that can be generated intracellularly, and Du et al [197] developed multifunctional CDs from citric acid, glycerin and 2,2′-(Ethylenedioxy) bis (ethylamine) that have dual emission at 457 and 525 nm when excited at 370 nm and can sense changes in hydrogen peroxide (figure 10).These CDs showed a decrease at 457 nm and increase at 525 nm in response to changes in hydrogen peroxide in cells with a limit of detection of 0.75 μM [197].Other CDs have been generated with ligands that target mitochondria, such as triphenylphosphonium, p-phenylenediamine, etc, and are covalently linked with CDs for ROS sensing [142,198,199].The functionalized/doped CDs undergoes physicochemical changes, facilitating the FRET-based or PET-based  Label-free CDs pH-responsive basic fuchsin/ pH-insensitive citric acid pH 5.2-8.8[189] ratiometric sensing of ROS [142,197,200].A unique way to use CDs is to generate ROS through Fe-doped CD nanozymes, which have enzymatic activity that alters redox homeostasis and increases intracellular ROS.These CDs were further functionalized with a ligand to enable them to pass the blood-brain barrier, where they could inhibit the growth of drug-resistant glioblastoma multiforme (GBM) in mice [201].
CDs have also been developed to sense changes in other important biological molecules.Coenzyme A (CoA) supports energy production, fatty acid synthesis, and drug efficacy in biological systems [202].
Although not yet tested in cells, Xu et al developed a sensitive Cu 2+ -assisted biosensor using nitrogen-doped CDs for the detection of CoA.These CDs are effectively quenched by Cu 2+ at 480 nm, and CoA forms a stable complex with Cu 2+ permitting fluorescence recovery [203].Another study by Cui et al [204] synthesized dual emissive CDs from chlorophyll that could ratiometrically measure the concentration of CoA in cells.The CDs had selectivity towards CoA when compared to biomolecules, such as cysteine and glucose [205].Cytochrome C is another crucial player in metabolism, and its release triggers apoptosis.Ghayyem et al [206] developed an aptamer-conjugated CD to detect and quantify Cytochrome C levels.The synthesized CDs emitted a linear change in fluorescence intensity at 450 nm in response to Cytochrome C sensing by the aptamers.Although these CDs have not yet been tested in cells, they detected changes in Cytochrome C in human blood plasma [21].CDs have also been developed to detect nucleic acids [207].While current imaging probes are specific for DNA (e.g.intercalating dyes) or RNA (e.g.hybridization probes), Han et al [21] developed cationic CDs that emit distinct emission spectra when bound to double-stranded DNA (500-560 nm) versus single-stranded RNA (570-650 nm), enabling dynamic monitoring of DNA and RNA in single cells [208,209].
Lastly, CDs have also been designed for use in enzyme activity assays [210,211].Li et al [212] developed nitrogendoped CDs to detect changes in alkaline phosphatase (AP) activity.This enzyme is typically used to measure changes in biomolecules (e.g. via coupling to antibodies for the detection of proteins, or with probes that report for RNA expression) involved in physiological processes.The emission intensity of the nitrogen-doped CDs at 510 nm decreased in the presence of AP in live RAW 264.7 cells [212].These CDs have been used successfully in human serum, HeLa cells, HEK293 cells, and mice [213,214].Other biomolecules, such as glucose and insulin have been detected in human serum using dual emissive fluorescent nano-aptasensor CDs [215].Emission peaks at 450 nm and 585 nm associated with the aptamerprobe CDs and QDs quenched linearly between insulin and glucose concentration ranges of 0.2-2 nM and 0.5-7 nM, respectively [215].Overall, CDs have great potential for use in a range of biological applications to detect various inorganic and organic analytes.

Challenges to consider for the practical use of CDs
CDs have broader spectral profiles and quantum yields that less desirable compared to conventional semiconductor quantum dots and organic dyes, however, they have superior aqueous dispersibility, functionalization, resistance to photobleaching, lower toxicity profiles and higher biocompatibility [101,216].CDs can be synthesized with desired properties using chemical approaches that minimize the use of costly precursors and high-energy inputs [32].However, to increase water dispersibility and fluorescence properties, multiple steps are required that include the use of powerful acids and posttreatment surface passivation.The lack of standardized synthesis and purification methods for CDs can cause high variability from batch-to-batch when made with the same precursors.This variability can cause difficulties when comparing and reproducing results, which may hinder their development for commercial use, and the field would benefit from adopting uniform standardized preparation and purification protocols.
Advances in CD preparation techniques have generated CDs with different passivation to confer distinct physicochemical properties.Self-passivating CDs can be generated using microwave-assisted hydrothermal carbonization or onestep high-temperature hydrothermal carbonization of various carbon precursors [217,218].However, these techniques often require a lengthy preparation process, high reaction temperatures, and/or corrosive synthetic conditions.These methods are less safe and can be more costly, restricting their widespread usage.Another challenge is the lack of knowledge regarding how CDs emit distinct spectral profiles and quantum yields.Despite extensive research, the mechanism by which CDs emit fluorescence has not been fully elucidated, and further studies are needed to reveal the physical and/or chemical moieties that confer unique optical properties.This knowledge will drive the rational design of CDs with enhanced or tailored fluorescence properties for specific applications.Furthermore, while CDs made from renewable or cheap organic precursors using less expensive and environmentally friendly approaches is desirable, the yield can be low [219].Thus, there is a need for additional research into synthetic protocols that could improve reaction yields using organic precursors.
Although CDs can address some of the drawbacks of conventional pH and temperature sensors, several challenges need to be overcome before they can be used commercially.As mentioned above, the lack of standardized approaches for their synthesis makes reproducibility an issue.Further, more knowledge is needed to know how their core and surface properties cause different spectral profiles to better understand how to modify them for improved dynamic ranges.The pH sensitivity range of most CDs does not satisfy the demands of real-world applications.The surface moieties of CDs, which may be in prototropic equilibrium with H + /OH − , are the primary mechanism for pH-sensing with CDs.Temperature sensing may be attributed to CD aggregation, hydrogen bonds and the many oxygen-containing functional groups working together synergistically to quench fluorescence.However, the precise structural and functional properties responsible for pH and temperature sensing are yet to be fully understood with frequent debates in the literature as to how certain structures or chemical groups affect the measured sensitivity [165,166,[220][221][222].
CDs have the potential for use in a wide range of biomedical applications, especially as a nanoimaging tool for diagnostics.However, despite predictions that they should have low cytotoxicity toward any biological cell type and have good biocompatibility in most organisms, few studies have done this correctly.Notably, synthetic techniques, surface coating, or passivation among other processes could impact their toxicity and biocompatibility, as well as alter their optical properties, which could restrict their use for specific applications.CDs can interact with external and internal environments in cells and tissues, which could cause unintended biological effects.Therefore, careful evaluation of the cytotoxicity and biocompatibility of CDs in different biological systems is necessary to ensure their safe use in bioimaging applications.Further studies are needed to assess the long-term effects of CDs on cells and complex organisms, including their potential accumulation and clearance in vivo.One notable drawback concerns the assays performed for assessing cytotoxicity.These tests ought to be performed after multiple consecutive cell cycles after treatment with CDs, the length of which can vary drastically between cell types.There is little to no information that can be properly interpreted within the first 24 h of treatment with CDs (or other nanomaterials).
Despite these challenges, the potential use of CDs as analytical and diagnostic tools is very promising.Figure 11 provides a concise summary of the merits, challenges, and areas of development for specific applications.With advancements in synthesis methods, surface functionalization strategies, and characterization techniques that result in enhanced fluorescence, specificity for cell types or intracellular locations, and prolonged stability, CDs can be tailored for multiple applications.Moreover, combining CDs with other imaging modalities, such as magnetic resonance imaging (MRI) and computed tomography (CT), could enable multi-modal imaging with enhanced sensitivity and specificity for various biological processes.Lastly, the multiplexing of CDs with other nanoparticles or nanocomposites could synergize the advantages of multiple systems.

Conclusions and recommendations: bridging the gaps in CD-based research and development
CDs are promising tools for various analytical and biological applications in biomedical or industrial sectors, more research is needed before they can be commercialized for use.This requires interdisciplinary collaborations between researchers from disciplines with the requisite expertise.While chemistry and physics have helped to advance the CD field by improving and diversifying their physicochemical properties, biochemists, biologists, and physiologists are required to perform cell-based and in vivo studies to properly assess their efficacy, specificity and toxicity among other properties.Just as fieldstandards should be developed for manufacturing to ensure the reproducibility of their physicochemical properties, standards should also be developed for their biological assessment.This is crucial because any CD will need to have extensive preclinical testing before they can be considered for use in clinical studies by medical professionals, or similarly for the environmental and industrial sectors.Specifically, while some scientific fields contribute to the synthesis, characterization, understanding of the physicochemical properties, and potential environmental applications of CDs, others provide insights into the biological interactions, mechanisms of action, and potential applications of CDs in living cells and organisms.
Collaborations between these fields can foster synergetic, cooperative and harmonious interactions, leading to a deeper understanding of CD properties and behavior in biological organisms and the environment, as well as the development of safer and more effective CD-based technologies.Without interdisciplinary collaborations, there is a risk of overlooking crucial, practical aspects that need to be considered or misinterpretation of results, which can hinder progress and prevent CDs from being moved into real-world applications.

Figure 1 .
Figure 1.The different physicochemical properties affecting CD cellular uptake.

Figure 2 .
Figure 2. Photoluminescence of different CDs in solution under 365 nm UV light.

Figure 3 .
Figure 3. Localization of CDs (red) in cells outlined in yellow dotted lines and co-labeled for lysosomes, mitochondria or tubulin (green), and DNA (blue) [86].

Figure 4 .
Figure 4.An organosilane molecule (3-aminopropyl) trimethoxysilane (APTMS) and glycerol were used in a one-step solvothermal synthesis of APTMS CDs.(a) APTMS CDs differentially accumulated and/or were quenched in the mitochondria of HepG2 and MCF7 cells (left) compared to macrophage cells (right).Adapted with permission from Gao et al [144].

Figure 5 .
Figure 5. (a) The impact of temperature on CD/epoxy composite fluorescence emission over several cycles.(b) CD emission relationship to temperature.(c) Composites of CD and epoxy.(d) The emission of CD/epoxy composites is temperature-dependent.Reprinted from [165].

Figure 6 .
Figure 6.Fluorescence and absorbance spectra of a 50 g mL −1 dispersion of CDs at room temperature.Three absorption bands with peaks at 295-350 nm, 370-450 nm, and 590-690 nm are visible in the UV-Vis absorption spectrum of dCDs (black curve).Two fluorescence bands are seen at 370-500 nm and 640-730 nm after excitation at 405 nm (blue curve), whereas red fluorescence is seen from 645-730 nm after excitation at 640 nm (red curve); (inset) CD dispersion under white light (left) and UV light (ex = 365 nm; right).Both blue and red fluorescence contribute to the violet colour shown in the inset.Adapted with minor modifications from [171].

Figure 7 .
Figure 7. (a) CD-treated HeLa cell images captured by fluorescence microscopy.The fluorescence signals for cells incubated at various temperatures are displayed (left: λ ex = 640 nm; right: λ ex = 405 nm; right).Untreated HeLa cells revealed no fluorescence in the control sample at 42 °C.(b) Temperature-dependent changes in the red/blue fluorescence ratio of CDs in both the dispersion model and the intracellular model (λ ex = 405 nm).Both exhibit a linear ratiometric response to the temperature change.While having comparable slopes, the ratios in the cell model are slightly lower, most likely due to differences in detection sensitivity.Adapted with minor modifications from [171].

Figure 8 .
Figure 8.(a) A graph shows the correlation between fluorescence intensity of the synthesized CDs (excited at 370 nm) and pH from 1-13.(b) The line of best fit is shown for pH from 4-13.(c) A schematic (left) shows surface passivation of CDs, and images on the right show their use for intracellular pH sensing.Adapted with minor modifications from [31, 173].

Figure 9 .
Figure 9. (a) FL emission spectra of functionalized CDs at various pH levels.(b) Correlation between pH values in the range of 4.0-8.0 and the ratiometric fluorescence intensity (ratio of emission at 580 versus 464 nm).Adapted with minor modifications from [177].

Figure 10 .
Figure 10.Scheme showing the FRET-based ratiometric sensing of mitochondrial H 2 O 2 in living cell using CD-based nanoprobe.Adapted with permission from Du et al [197].
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Table 1 .
Comparisons of CDs derived from plant-based materials.

Table 2 .
Different carbon dots and their respective IC 50 for viability in various cell lines as well as potential applications.

Table 3 .
An overview of recent CD-based ratiometric probes for pH sensing.