Advances in the application of biosynthesized carbon dots as fluorescent probes for bioimaging

CDs can be synthesized via two broad approaches: top-down methods that break down carbon structures into nanosized dots and bottom-up methods that build them up from molecular precursors. Early synthesis of CDs was dominated by top-down techniques, including arc discharge, laser ablation, and electrochemical oxidation. For instance, Raniszewski et al. [29] prepared fluorescent CDs of around 3–5 nm diameter by arc discharging graphite. The coarse carbon soot formed was then oxidized with nitric acid to give watersoluble luminescent nanoparticles. Laser ablation has also been implemented by irradiating graphite or carbon nanopowder suspended in liquids with pulsed laser beams, fracturing them into CDs of 3–10 nm size [30]. Electrochemical approaches (Fig. 1A) rely on anodic oxidation of carbon electrodes in electrolyte solutions, which generate carbonaceous species that nucleate into CDs [31, 32]. However, such physical techniques offer little control over properties or yield.

Fig. 1.

In contrast, bottom-up wet chemical methods via molecular assembly of small precursors have become more popular as they allow better regulation of CD size and optical properties. Hydrothermal treatment (Fig. 1B) is one of the most facile and economical methods—direct heating of precursors like citric acid or plant extracts in water generates emissive CDs within a few hours [33, 34]. Microwave-assisted heating (Fig. 1C) can produce CDs even faster by rapid decomposition of precursors [35]. Ultrasonication has also been implemented to break down carbon-rich starting materials. Such bottom-up assembly-based growth allows tuning particle size and surface chemistry to desired specifications. It also enables the use of biomass-derived precursors for simple and sustainable biosynthesis compared to top-down bulk disintegration techniques.

Therefore, while physical top-down techniques marked the first reports on CD synthesis, more recent work has focused extensively on bottom-up assembly of molecular precursors as it offers unparalleled flexibility in materials design. Environmentally benign synthesis routes utilizing plant extracts or waste biomass as starting materials add further value for scalability. Careful optimization of these bottom-up techniques also facilitates excellent control of properties like size, surface chemistry, and fluorescence emissions, which can be tailored to the needs of bioimaging and other applications.

The choice of precursors is crucial as it defines components and surface chemistry of as-obtained CDs to a large extent. Many carbon-rich small molecules have been utilized, with citric acid being the most common due to its carbon scaffold and abundance of carboxyl groups for surface passivation [36]. Other organic acids like tartaric acid [37] and amino acids [38] have also yielded fluorescent CDs. Plant extracts provide a direct one-step biosynthetic route utilizing the rich bioactive components (Table 1) – leaves, fruits, peels and stem extracts from sources like spinach [39], onion [40], apple [41], orange [42], and neem [43] have been implemented. Waste biomass like silk cocoons [44], coffee grounds [45], and waste frying oils [46] offer sustainable precursors while milk [47], egg white [48], and honey [49] exemplify bioderived materials for simple synthesis.

More recently, algal extracts from Chlorella [50], Sargassum [51], and other genera [52, 53] have been explored for facile biosynthesis of CDs. Algae cultivation does not compete for arable land and utilizes unwanted CO₂, providing environmental and ethical advantages. Many algae are also rich in antioxidants, vitamins, and other healthpromoting components, which translate into beneficial surface functionalization. Advanced material extraction without sacrifice makes algal biomass an exciting renewable precursor. Agnol et al. [54] prepared bright green fluorescent CDs in a single step by hydrothermal treatment of commercially procured Spirulina powder. The protein-rich microalgal extract likely provided both scaffolding and capping agents. In another report, nitrogen and sulfur-doped CDs were synthesized using Dunaliella salina macroalgal biomass as a precursor [55]. Advantageously, compared to synthesis with other biomass sources, the yield of CDs from this seaweed source was found to be exceptionally high.

While a wide range of carbon-rich precursors have been utilized, building CDs from waste or naturally derived biomaterials confers them biocompatibility, sustainability, and potential biological functionality. Abundant surface groups also aid water dispersibility and defer agglomeration compared to graphene or carbon nanotubes. Environmentally sound biomass precursors coupled with facile synthesis provide a convenient biosynthetic toolbox for generating CDs [11]. Algal sources, in particular, offer renewed biomaterials advantage. Bottom-up assembly harnessed with such bioderived, value-added components allows the design of bespoke bright fluorescent CDs in an economical and eco-friendly manner.

While a diversity of precursors have been utilized, the synthesis technique plays an equally crucial role in directing the formation of CDs with desired optical properties. Hydrothermal treatment is one of the most common and versatile methods for CD biosynthesis. Direct heating of precursor solutions in sealed autoclave vessels at high temperatures (100–220°C) and autogenous pressures creates favorable conditions for dehydration, polymerization, and carbonization reactions to occur [56]. The harsh solvothermal environment provides energy for bond breakage and reformation – small molecules assemble into larger carbon scaffolds, which crystallize into CDs a few nanometers wide [57]. Zhang et al. [58] synthesized six types of nitrogen-doped CDs (NCDs) at different hydrothermal temperatures from 120°C to 220°C using citric acid and ammonia solution. Results showed that as temperature increased, carbonization degree and carbon/oxygen ratio increased while functional groups, nitrogen doping level, and fluorescence lifetime remained similar (Fig. 2A). The quantum yield, UV-vis absorbance, and photoluminescence intensity of the NCDs initially increased from 120°C to 180°C and then decreased at higher temperatures with a maximum of 35.4% at 180°C.

Fig. 2.

Microwave-assisted synthesis works on similar principles but offers a faster response as polar precursor molecules rapidly heat up under microwave irradiation. Dielectric heating induced by an alternating electric field causes molecular friction and decomposition [59]. As temperature rises exponentially, it creates high pressures in sealed reaction vials, leading to CD nucleation. The whole process finishes within minutes. For example, palm kernel shell biomass waste was used as a precursor to synthesize CDs via a microwave irradiation method [60]. CDs were successfully produced with sizes of 6-7 nm in diameter. All CDs contained hydroxyl and alkene functional groups from the transformation of the palm kernel shell. Simsek et al. [61] synthesized fluorescent CDs from Nerium oleander leaf extracts using two microwave-based methods—a domestic microwave oven (MWO) and a microwave-assisted hydrothermal (MAH) synthesizer (Fig. 2B). They investigated how factors like extract type, reaction time, temperature, and adding a surface passivation agent (polyethylene glycol) affected CD properties like particle size, surface charge, and photoluminescence intensity. The results showed that MWO synthesis for 40 minutes with an added passivation agent gave CDs a ten times higher fluorescence compared to shorter reaction times. MAH synthesis at 250°C gave the highest photoluminescence intensity. Adding a passivation agent dramatically decreased particle size from 288–825 nm to 2.6– 5.7 nm for MAH synthesis. For MAH synthesis, the increasing temperature made the CD surface charge less negative. The extract type affected particle size and surface charge depending on the solvent used. Overall, MWO gave better photoluminescence enhancement compared to MAH, which allowed synthesis in shorter times. Faster kinetics favor the formation of smaller particles with better quantum confinement. Microwave heating also consumes much less energy, making it attractive for scalable manufacturing.

Sonication techniques use powerful ultrasonic waves to create acoustic cavitation, which provides the energy for precursor decomposition and CD assembly. By avoiding harsh heating conditions, sonochemical methods provide more control over the growth process [62]. However, extended reaction durations of two hours to even 96 hours are usually required to produce brightly emissive CDs. The technique has shown promise for scaling up due to its easier processing. Autoclave-free sonication-enabled synthesis also ensures safety.

In summary, hydrothermal and microwavebased methods have been the most widely adopted as facile, energy-efficient tools for rapid biosynthesis of fluorescent CDs. Sonication approaches offer moderate alternatives avoiding harsh temperatures or pressures. The technique employed plays a definitive role in all stages of precursor transformation - breakage, nucleus formation, growth, and termination of CDs. Fine-tuning of synthesis parameters hence allows customization of optical properties for intended bioimaging and sensing applications.

The bright photoluminescence of CDs spanning the visible and near-infrared (NIR) spectrum is their defining signature that has greatly enabled bioimaging applications. Understanding and controlling the optical properties is, hence, key to further unlocking their analytical and biomedical potential. CD emissions tend to be broadly tunable and excitation wavelength dependent, which offers opportunities but also poses challenges regarding mechanistic interpretations.

By rationally choosing precursors and judiciously controlling synthesis parameters, emissions can be tuned spanning blue, green, yellow, orange, and red spectral windows [63]. Extending photoluminescence (PL) into the red and NIR regions has been important to minimize background interference from autofluorescence of biological samples for more sensitive bioimaging [64]. The use of sulfur and nitrogen-rich precursors generally allows the obtaining of long wavelength emissive CDs. For instance, Li et al. [39] developed a method to synthesize NIR fluorescent CDs (R-CDs) from spinach via a one-step solvothermal treatment. The R-CDs exhibited maximum fluorescence emission at 680 nm, high quantum yield of 15.34%, excellent water solubility, remarkable photostability, negligible toxicity, and superior bioimaging capability. Jiang et al. [65] synthesized a new type of CD (N-CDs-F) by doping nitrogen and fluorine using ammonium fluoride during a solvothermal process. The resulting N-CDs-F had a donor-π-acceptor structure, which gave them unique optical properties. Compared to undoped carbon dots, the N-CDs-F exhibited broad UV-vis-NIR absorption from 250–1050 nm and efficient excitation upcon-version fluorescence under two-, three-, and four-photon laser excitation up to 2000 nm. The QY of the N-CDs-F and the undoped CDs at λex/λem of 710/772 nm are calculated to be 9.8% and 8.1%, respectively. The N-CDs-F also showed larger multiphoton absorption cross sections, with the three-photon cross section over two times greater than previous organic compounds. Analysis suggested the special chemical structure of N-CDs-F, facilitated by extensive hydrogen bonding networks, enabled delocalized conjugated systems that lowered the HOMO-LUMO gap and enhanced nonlinear optical behavior. Such careful modulation of synthesis conditions hence allows the development of application-specific fluorescent CDs. Similar performance has also been observed by many biosynthesized CDs (Fig. 3) [66, 67].

Fig. 3.

In addition to tunable emissions under a single excitation wavelength, CDs also demonstrate prominent excitation-dependent behavior where the emitted PL peak progressively red shifts with increasing excitation wavelength. This optic signature has been observed across CDs derived from sources as diverse as lemon juice [68], Aegle Marmelos [69], spices [70], and Azadirachta indica [71]. While excitation-dependent emissions can be a practical way to obtain multicolor readouts from biocompatible nanoprobes on a single testing platform, the mechanism behind this phenomenon still remains ambiguous and under debate. Explanations invoke varying emissive sites, a distribution of particle sizes, as well as diverse surface defects and functional groups, resulting in a manifold of energy levels that lead to versatile recombinations under assorted excitation energies [72]. Further research using advanced spectroscopic techniques and theoretical simulations is still needed to completely resolve the structure-property-function relationships.

In summary, facile tunability of emissions across the visible spectrum, access to NIR region to avoid background interference, and signature excitation-dependent behavior leading to myriad color readouts offer both opportunities as well as riddles regarding biosynthesized CDs as fluorescent bioprobes. Rational optimization of all these dimensions can vastly expand their applicability for sensitive quantification, multiplexed assays, and multimodal imaging of complex biological phenomena.

While CDs can demonstrate excitation-tunable emissions spanning the visible spectrum, careful optimization of synthesis parameters allows the direction of desired optical outputs tailored to imaging needs. Furthermore, the as-obtained, crude CDs have inferior quantum yields that can be enhanced via surface engineering with ligands and polymers. Such surface passivation impacts not only brightness but also spectral distribution of emitted photons.

Harsh hydrothermal syntheses generally result in greater carbonization and more surface defects, which impact optical signatures. For instance, Han et al. [73] systematically analyzed the effects of hydrothermal reaction temperature on the photoluminescence properties of CDs. They found that a reaction temperature of 200°C was optimal for synthesizing CDs with strong fluorescence. The CDs prepared at 200°C exhibited an emission peak at 414 nm when excited at 340 nm and a quantum yield of about 22% (Fig. 4A). This finding is consistent with the results reported by Li et al., [74] who also investigated the influence of hydrothermal reaction temperature on CD properties. Their work further confirmed that 200°C is a critical threshold for optimizing CD fluorescence. They observed that increasing the reaction temperature from 170°C to 200°C enhanced the fluorescence intensity of the CDs (Fig. 4B). However, temperatures exceeding 200°C led to a significant decrease in fluorescence. Li et al. provided additional insights into the underlying mechanisms. They proposed that temperatures below 200°C are insufficient for effective CD core formation and surface defect reduction. In contrast, temperatures above 200°C cause excessive carbonization, destroying the surface fluorophores and quenching the emission. Furthermore, their work revealed that the reaction temperature also impacts the nitrogen doping level in CDs, with higher temperatures promoting more effective decomposition of nitrogen precursors.

Fig. 4.

Apart from synthesis parameters, surface functionalization of as-prepared crude CDs has proven pivotal for amplifying fluorescence brightness. Diverse surface passivating agents have been implemented, spanning small ligands, crosslinkers, macromolecular coatings, and thin-layered shells [75]. Amino acids like cysteine and organic species with multiple amine or thiol moieties interact via electrostatic attractions or covalent linkages with abundant carboxyl and hydroxyl groups on biosynthesized CDs to null surface dangling bonds. For instance, Watcharamongkol et al. [76] used ethylenediamine (EDA) as a nitrogen doping agent to synthesize NCDs via a hydrothermal method from cassava pulps. EDA was used together with cassava pulp to prepare NCDs and introduce nitrogen functional groups into the carbon dots. With this nitrogen doping it was possible to improve the optical and chemical properties of the CDs. NCDs synthesized with EDA (NCDs-EDA) exhibited five times higher fluorescence intensity compared to undoped carbon dots under 360 nm excitation. NCDs-EDA also showed higher fluorescence quantum yield (36.9%) than undoped CDs (6.6%) synthesized under the same conditions. NCDs-EDA demonstrated higher photostability under UV illumination and visible light compared to undoped CDs (Fig. 4C). Polymer coatings like PEG and oligochitosans also similarly curtail non-radiative relaxations. An interesting review by Peng et al. [22] concluded that PEG has been widely used to passivate CDs, enhancing properties like fluorescence intensity. For example, one study showed that amine-terminated PEG1500 greatly increased the brightness of C-dots made from laser-ablated graphite (Fig. 4D) (77). PEG has also been explored as a solvent, matrix, or carbon source for making CDs. The authors discussed how the molecular weight and structure of PEG impact properties like C-dot size and emission wavelength. Smaller PEG chains tend to produce smaller, blue-emitting C-dots. The proposed mechanism involves PEG chains breaking down and aggregating into aromatic, electron-rich cores surrounded by oxygen-rich hydrophilic surfaces. Therefore, engineering the interfacial layer around the lightemitting CD core directly amplifies brightness to levels comparable with toxic semiconductor quantum dots.

Many biomass precursors used for CD synthesis, such as plant extracts, amino acids, and proteins, are inherently chiral in nature. The asymmetric arrangement of surface functional groups or the incorporation of chiral biomolecules during hydrothermal carbonization can result in CDs with chiral surfaces [78]. This chirality can be probed through techniques like circular dichroism spectroscopy and circularly polarized luminescence (CPL) [79].

Zhang et al. [80] developed CDs-composited G-quartet hydrogels capable of emitting CPL. Utilizing a novel one-step microwave-assisted synthesis, this research not only simplified the fabrication process but also introduced a method to control the chirality and CPL properties of the hydrogels through the addition of metal ions, particularly potassium ions. The composite hydrogels demonstrated exceptional CPL performance, with a dissymmetry factor (g_lum) reaching up to 10^-1, markedly surpassing most existing CPL materials in terms of efficiency. This remarkable achievement indicates a significant leap forward in the development of CPL materials, offering potential applications in anti-counterfeiting and information encryption technologies. The study further explored the influence of K+ ions on the helical structure of the hydrogels, achieving reversible control between left-handed and right-handed configurations. These examples demonstrate that the synthetic methods described in our work, particularly hydrothermal treatment and microwave-assisted synthesis, can preserve the chiral nature of biomass precursors and translate it to the resulting CDs. However, the extent of chirality transfer and its impact on CD properties likely depends on factors such as precursor type, reaction conditions, and post-synthesis modifications.

While the carbon core imparts characteristic photoluminescence, the surface chemistry and availability of functional groups on CDs play a pivotal role in governing their interactions and compatibility in biological systems. The surface state also determines lifetime via colloidal stability in aqueous dispersions. Further bioconjugation for sensitive bioimaging hinges on tailoring the interfacial layer appropriately.

Pristine CDs synthesized from biomolecules and waste sources using green techniques may offer sustainability but lack long-term stability due to irreversible aggregation arising out of interparticle hydrophobic interactions and π-π stacking. Surface oxidation techniques, as well as response reactions with amines, thiols, and carboxylic acids, help imprint hydrophilic groups like hydroxyls [81] and carboxyl [82] as well as amides and amines [83] to promote water dispersibility. Amphiphilic polymers like oligochitosans [84] and PEGylated lipids [85] can also be grafted to produce stable glycoprotein-like nanoparticulates. The introduction of zwitterionic moieties or the construction of hydration layers prevents opsonization and unwanted biofouling for prolonged shelf life.

In addition, surface passivation is also mandated to mitigate the potential cytotoxicity of nascent CD formulations. While toxicity is generally lower compared to graphene and CNTs due to lack of sharp edges, factors like dose, dimensions, and surface groups do play a role. Conversion of reactive groups and enrichment with biocompatible ligands hence enhances bioresponse. For instance, Havrdova et al. [86] found the surface functionalization and resulting surface charge of CDs greatly affect their toxicity profile (Fig. 5). Pristine CDs with a negative charge from carboxylic groups (CDs-Pri) showed a stimulatory effect on cell proliferation at low doses of 5–50 μg/mL. However, at higher doses, they induced G2/M cell cycle arrest, caused higher oxidative stress and reactive oxygen species (ROS) production compared to other CDs, and altered cell morphology. Despite this, they did not enter the cell nucleus. CDs-Pri had an IC50 value of 300 μg/mL. In contrast, PEG-modified CDs with a neutral charge (CDs-PEG) did not affect cell morphology, cell cycle progression, or ROS levels even at high doses. They were the most biocompatible CDs and also did not enter the cell nucleus. Their toxicity (IC50) was similar to CDs-Pri at 300 μg/mL. Positively charged PEI-modified CDs (CDs-PEI) exhibited the highest toxicity of the three CDs, with the lowest IC50 of around 50 μg/mL. CDs-PEI entered the cell nucleus, caused significant morphological changes, induced G0/G1 cell cycle arrest, and stimulated considerable oxidative stress. Likewise, oligochitosan coating of peanut-derived dots was important for biocompatible imaging applications, as reported by Ma et al. [87] Therefore, rational surface passivation through oligomer grafting and polymer entrapment integrates colloidal stability and biological stealth for wider imaging adoption.

Fig. 5.

While pristine CDs offer tunable fluorescence, their bioimaging sensitivity and selectivity are enhanced manifold through surface functionalization with recognition elements for the molecular targets. Common functionalization strategies rely on coating CDs with polymers, biomolecular ligand attachment, and integration of stimuli-responsive small molecule groups that render them “smart.”

Among polymeric coatings, oligo-peptides [88], proteins like BSA [89], as well as charged species like chitosan [90] and polyethyleneimine [91] have been extensively implemented to boost water solubility. Hydrophilic polymers like PEG provide extensive steric stability and modify pharmacokinetics by reducing renal clearance and increasing circulation half-life. Gonçalves and Silva [92] used PEG200 to functionalize CDs is multifold. In terms of performance, functionalization with PEG200 and mercaptosuccinic acid yielded fluorescent CDs with an average size of 267 nm, maximum excitation at 320 nm, and peak emission at 430 nm. The fluorescence intensity of these dots is affected by the solvent, pH (showing an apparent pKa of 7.4), and iodide, which causes dynamic quenching but is not impacted by other metal ions such as Hg, Cu, Cd, Ni, and Zn. The fluorescence lifetimes have three complex decay components at around 2.7 ns, 7.4 ns, and 0.4 ns, and these lifetimes are not influenced by the PEG200 chain length nor the addition of mercaptosuccinic acid. In addition, PEGylation allows the carbon dots to be further functionalized with other molecules like mercaptosuccinic acid to develop novel nanosensors.

Small bioactive ligands like folic acid [93], hyaluronic acid [94], glucose [95], arginineglycine-aspartic acid (RGD) peptide [96] and many more have also been integrated to instill CDs with sensitivity for overexpressed cancer biomarkers and related metabolic dysregulation. Amino acids like L-aspartic acid impart intrinsic brain cancertargeting ability as elaborated by Zulfajri et al. [97] The incorporation of such targeting ligands activates uptake mechanisms, like receptor-mediated endocytosis, for selective tracing of diseased cells overexpressing them.

Surface functionalized CDs can be further bioconjugated with marker-specific recognition elements like antibodies, aptamers, and affinity molecules to selectively tag desired cells and biomolecules for sensitive optical detection. Immobilization of such biorecognition probes transforms CDs into targeted imaging contrast agents.

Antibodies that selectively bind corresponding cell surface protein antigens have been conjugated on CDs for specific cell labeling applications. For example, CDs chemically grafted with anti-EpCAM and anti-CD44 antibodies allowed differentiating epithelial vs mesenchymal type cancer cells overexpressed with the respective biomarkers [98, 99]. For example, Li et al. [100] developed a multifunctional nanoparticle called anti-EpCAM@PDA-CDs@Pt(IV) that combines imaging, targeted drug delivery, and chemophotothermal therapy for treating liver cancer cells. The nanoparticles were made by attaching an antibody called anti-EpCAM and a chemotherapy drug called Pt(IV) to polydopamine CDs (PDA-CDs). Anti-EpCAM enabled targeted delivery to liver cancer cells that overexpress the EpCAM protein, while the PDA-CDs allowed fluorescence imaging to track the nanoparticles (Fig. 6A). The researchers showed that anti-EpCAM@PDA-CDs@Pt(IV) nanoparticles exhibited strong fluorescence for bioimaging, with high stability across varying pH and ionic concentrations. In vitro tests demonstrated targeted uptake in liver cancer cells and significant cytotoxicity triggered by near-infrared light irradiation attributed to combined chemo and photothermal therapy. In vivo results in a mouse model revealed targeted tumor accumulation and an 87.6% tumor growth inhibition rate through the synergistic treatment, higher than either chemotherapy or photothermal therapy alone. Histological analyses confirmed the destruction of tumor tissue without noticeable toxicity in other organs.

Fig. 6.

In addition to protein-based agents, oligonucleotide affinity molecules like aptamers also offer attractive probes for functionalizing CDs to generate targeting effects. Aptamers undergo conformational changes in the presence of the target, modulating the quenching efficiency of adjacent fluorophores [101]. Liu et al. [102] developed a new nanomedicine called CD-IR820-Aptamer (Fig. 6B) made of carbon dots, IR820 dye, and an anti-vascular endothelial growth factor (VEGF) aptamer. They showed that this nanomedicine could selectively target tumor cells overexpressing VEGF receptors. Once taken up by cancer cells, the nanomedicine is localized to the mitochondria. When irradiated with an 808 nm near-infrared laser, the nanomedicine generated reactive oxygen species through type I photodynamic therapy, singlet oxygen through type II photodynamic therapy, and heat through photothermal therapy. Unlike traditional photodynamic therapies relying on singlet oxygen, this multifunctional nanomedicine retained efficacy even under hypoxic conditions in vitro and in vivo. In mouse models of breast cancer, the combination treatment of laser irradiation plus intravenous injection of CD-IR820-Aptamer resulted in significant tumor growth inhibition and damage compared to laser plus carbon dots or laser plus IR820 dye alone.

In vitro cell labeling, tracking intracellular fate, and organelle imaging applications constitute the most widely investigated implementations of fluorescent CDs before potential in vivo adoption. Due to innate size-dependent uptake and lack of specificity, most pristine CD preparations exhibit panspecific staining of standard cell lines. Hence, strategies rely on conjugation with targeting vectors for selective imaging.

Intrinsic endocytic internalization allows the majority of sub-10 nm sized CDs to spontaneously permeate plasma membranes and localize inside cells. For example, Rai et al. [103] reported a green method to synthesize reduced fluorescent CDs (r-FCDs) from lignosulfonate lignin using microwave irradiation. The resulting r-FCDs had an average diameter of 4.9 nm and maximum photoluminescence intensity at 475 nm when excited at 440 nm. The quantum yield was 47.3%. The r-FCDs showed high stability over a wide pH range and in saline solutions. The r-FCDs were incubated with A549 and SW480 cancer cell lines, and imaging showed cytoplasmic and nuclear localization, indicating cellular uptake (Fig. 7). The hemolysis assay demonstrated low toxicity, with only 2.89% hemolysis at 0.5 mg/mL r-FCDs concentration. Vasimalai et al. [70] synthesized CDs from four common spices - cinnamon, red chili, turmeric, and black pepper—using a green one-pot hydrothermal method. The CDs showed high fluorescence quantum yields of up to 43.6%, with particle sizes around 3–4 nm measured by TEM. The authors systematically evaluated the cytotoxicity and bioimaging potential of these spice-derived CDs in vitro using human glioblastoma cancer cells (LN-229) and non-cancerous human kidney cells (HK-2). The CDs exhibited differential toxicity in the cancer and non-cancer cells. After 24 hours of exposure, the spice CDs, especially those from black pepper, inhibited LN-229 cancer cell viability in a concentration-dependent manner, with 2 mg/mL yielding 35-75% growth inhibition. However, the CDs showed negligible toxicity in HK-2 cells. Confocal imaging demonstrated higher cellular uptake and cytoplasmic accumulation of spice C-dots in LN-229 versus HK-2 cells. Control citric acid CDs did not affect the viability of either cell type. The results suggested that spice CDs, particularly from turmeric and black pepper, have potential therapeutic applicability as cancertargeting bioimaging agents with minimal toxicity to normal cells.

Fig. 7.

In terms of the subcellular distribution, the majority of naked anionic CDs localize in the cytoplasm and peri-nuclear spaces. Cationic CDs with amine enrichment demonstrate higher nuclear affinity due to electrostatic attractions for negatively charged DNA and better permeation through nuclear pores. For example, Zhang et al. [104] synthesized NCDs via a one-step hydrothermal method. Characterization showed the NCDs had small size (4.35 nm), bright green fluorescence, abundant surface functional groups, and excellent fluorescence stability. The NCDs also demonstrated good biocompatibility with low cytotoxicity. When incubated with HeLa and PC12 cells, the NCDs could rapidly and specifically stain the nucleoli without needing additional washing steps. Further tests revealed the NCDs had a strong binding affinity to ribonucleic acid (RNA) abundant inside nucleoli, significantly enhancing NCD fluorescence. Digesting cellular RNA using ribonuclease inhibited NCD nucleoli staining, confirming the fluorescence enhancement mechanism. Similarly, Sachdev et al. [105] developed a dualfunctional nanocomposite made of fluorescent CDs decorated on silver-zinc oxide nanoparticles (CDs-Ag@ZnO NCs) and evaluated its performance for bioimaging in cancer cells. The intrinsic fluorescence of the CDs allowed real-time monitoring of cellular uptake and localization of the NCs in MCF-7 breast cancer and A549 lung cancer cells using fluorescence microscopy, with higher uptake and nuclear localization observed compared to normal cells. The CDs served as an effective alternative to conventionally used organic dyes for tracking intracellular nanoparticle trafficking.

While pristine CDs permit bulk staining of cells, surface engineering with targeting ligands imparts selectivity for discriminatory recognition of cancer cells, stem cells, and neurons. Taking clues from in vitro assays, the approach can be eventually translated to identify diseased tissues in more complex in vivo environments.

Cancer cell imaging applications leverage the binding of overexpressed surface biomarkers like folate receptors, hyaluronic acid, EpCAM, and mucins, as well as aberrant pH and protease microenvironments. Glycosylated liposomes conjugated CDs showed preferential labeling of HepG2 correctly reporting on metabolic dysregulation [106]. Mohammadi et al. [107] developed a highly sensitive ratiometric fluorescence assay for microRNA-21 detection using a nanohybrid of blue-emitting CDs (B-CDs) and yellowemitting CDs (Y-CDs). The B-CDs were functionalized with a DNA probe to specifically recognize the microRNA-21 target. In the presence of microRNA-21, the fluorescence intensity of B-CDs at 410 nm decreased due to the inner filter effect, while the intensity at 540 nm increased. Using the ratio of fluorescence intensity change (ΔF540/ΔF410) as readout, the assay demonstrated a wide linear detection range of 0.15 fM to 2.46 pM and an ultralow detection limit down to 50 aM for microRNA-21. The assay could also quantify intracellular microRNA-21 in MCF-7 cancer cells over a range of 3,000 - 45,000 cells/mL (Fig. 8). Ratiometric CD probes targeted to cathepsin protease biomarkers also helped differentiate cancerous over normal cells by selective fluorescence light-up [108]. Through growth factor analog decoration, Shu et al. [109] traced HER2 receptors among the SK-BR-3 family, enabling the differentiation of breast cancer lines. Such surface re-engineering allows moving beyond panstaining towards molecular discrimination vital for cytological applications.

Fig. 8.

Beyond cell-level recognition, targeted delivery of CDs to intracellular organelles like the nucleus, mitochondria, and lysosomes allows probing their microenvironments and associated enzymatic activities [110]. Aberrations therein are linked with diseases, including cancer progression, making stimuli-responsive organelle imaging important.

Due to strong Coulombic affinity towards negatively charged macromolecules, amine-rich cationic CDs demonstrate natural nuclear localization abilities. For example, yellow emissive zwitterionic CDs prepared by citric acid permeated cell and nuclear membranes within 2 h [111]. Clear nucleolar binding permitted monitoring rRNA transcription activities via selective quenching interactions (Fig. 9A) [112]. Through conjugation with DNA intercalators like propidium iodide, CDs platforms also enable real-time monitoring of drug uptake for nuclear delivery applications (Fig. 9B) [113].

Fig. 9.

Mitochondria, being the powerhouse and metabolic signaling center, contributes to the initiation and progression of disorders, including neurodegeneration and tumorigenesis. Geng et al. [114] developed mitochondria-targeted CD (MitoTCD) with tunable long-wavelength fluorescence ranging from green to red. The MitoTCD were synthesized via a retrosynthesis approach using citric acid and m-aminophenol derivatives as precursors. This allowed the incorporation of rhodamine as a luminescent center into the CDs. Four types of MitoTCD were made emitting at 520, 540, 580, and 600 nm with high quantum yields of 0.39, 0.44, 0.25, and 0.40, respectively (Fig. 10A). Targeted cellular imaging experiments demonstrated the MitoTCD could selectively localize to the mitochondria of HeLa cells after 4 h of incubation. Taking the MitoTCD 580 as an example, it was found to be independent of membrane potential and could continuously track the mitochondria for as long as six cell passages, indicating suitability for long-term imaging. Guo et al. [115] synthesized fluorescent CDs that could shuttle between mitochondria and the nucleolus to visualize cell viability. In healthy cells, the positively charged CDs accumulated in the negatively charged mitochondria. However, when cells were damaged and the mitochondrial membrane potential decreased, the CDs migrated to the nucleolus, likely due to electrostatic interactions with nucleic acids concentrated there. Adding hydrogen peroxide damaged the cells and caused CD migration to the nucleolus, with the fluorescence intensity ratio of nucleus to cell increasing from 0.29 to 0.57. Subsequent addition of the antioxidant ascorbic acid recovered cell viability, causing the CDs to move back to the mitochondria, and the fluorescence ratio decreased to 0.32. Thus, the study demonstrated CDs that act as in situ visual reporters of cell viability based on their dynamic migration between cellular compartments.

Fig. 10.

In addition, abnormal lysosomal enzymes and upregulated catabolism also mark pathological transformations. Geng et al. [116] developed fluorescent CDs that can detect adenosine triphosphate (ATP), specifically within acidic lysosomes in cells. In acidic solutions (pH 4.5-5.5) resembling the lysosomal environment, the CDs exhibited a yellow fluorescence when they interacted with ATP. This on-off fluorescence switching enabled selective detection of ATP within lysosomes without signals from ATP in the neutral pH cytoplasm or other organelles. The CDs had a linear ATP detection range from 0.01 to 0.3 mM ATP. When applied to human cancer cell lines, the CDs demonstrated colocalization with a commercial lysosome dye, indicating lysosome-specific accumulation. Further, the CDs’ fluorescence intensities changed in response to drugs that modulated intracellular ATP levels and lysosomal pH/integrity over time (Fig. 10B).

Translation of CDs utility from in vitro assays to in vivo administration mandates studies into pharmacokinetic fate and biodistribution trends for evaluating safety. Key considerations encompass circulation half-life, preferential accumulation, metabolism, and clearance pathways, which impact toxicity concerns-especially for therapeutic modalities like drug delivery and phototherapy.

Pioneering research reveals renal clearance as the predominant elimination pathway for intravenously injected CDs with peak signal from the bladder evident in small animals within an hour of injection [117]. The renal filtration cut-off of around 10 nm allows the elimination of sub-10 nm CD particulates. Analyses reveal gradual release over 12–24 hours with negligible toxicity via the fecal route, too (Fig. 11) [118]. However, surface PEGylation and charge modulation variably prolong circulatory residence time compared to anionic raw CDs, which undergo rapid opsonization. Metabolic fate through enzymatic breakdown into constituent biomolecules minimizes risks of bioaccumulation upon chronic exposures [119].

Fig. 11.

In terms of biodistribution, Yang et al. [120] investigated the toxicity and biodistribution of CDs in mice after a single inhalation exposure. CDs were synthesized by hydrothermal method and characterized. Mice were exposed by inhalation to CDs at doses of 5, 2 and 1 mg/kg. Results showed CDs caused dose-dependent mortality, with 14% mortality at the highest dose after 15 days. Blood analysis indicated CD-induced inflammation. Liver enzymes were elevated in a dosedependent manner, suggesting liver damage. The fluorescent intensity of lung and liver homogenates increased in a time-dependent fashion over 28 days, indicating accumulation of CDs over time. In contrast, kidney intensity peaked at one day and then declined, suggesting clearance of CDs. Histopathology and TEM confirmed lung and liver injury but no brain damage. Lung effects included inflammation, edema, and epithelial cell vacuolation. Liver effects included necrosis and cytoplasmic vesiculation of Kupffer cells from CD phagocytosis. Huang et al. [121] investigated how different injection routes, intravenous (IV), subcutaneous (SC), and intramuscular (IM), affected the biodistribution, clearance, and tumor uptake of fluorescent CDs in mice (Fig. 12). The CDs were labeled with the near-infrared dye ZW800 to allow tracking by optical imaging. Blood circulation analysis found that IV injection resulted in the fastest clearance, with blood levels 17-fold lower at 60 minutes post-injection than at one minute. SC and IM injection showed slower clearance, with blood levels increasing up to 30 min before plateauing. Biodistribution studies showed the majority of CDs accumulated in the kidneys by 1 h postinjection, indicating renal clearance. By 24 h, CDs were completely cleared from the body by all routes. Urine analysis confirmed IV injection had the fastest clearance, with fluorescence plateauing after 10 min. Tumor uptake was higher in the IV and SC groups compared to IM at 2 h, likely due to the interplay between circulation time, clearance rate, and particle concentration.

Fig. 12.

Surface functionalized CDs grafted with targeting ligands selectively accumulate in tumors by binding overexpressed receptors on cancer cells. Cross-recognition hence amplifies signal contrast for sensitive diagnosis. In parallel, specific oligopeptides also enable permeation across tight blood-brain barrier junctions for neural imaging.

Due to the enhanced permeability and retention effect of leaky tumor vasculature, pristine CDs display some passive uptake, too. However, conjugation with homing elements provides active facilitation via ligand-receptor binding events. Common targeting motifs used include cyclic RGD peptides for recognizing αvβ3 integrins, folic acid to interact with folate receptors, and antibodies identifying accessible cancer antigens. Liu et al. [122] successfully developed integrin αvβ3-targeted CDs nanocomposites called MB-CDs@NH-RGD. Soybean milk was used as a green carbon source, along with methylene blue and TTDDA, to fabricate the CDs. Surface modification via TTDDA coating and RGD peptide conjugation endowed the nanocomposites with superior biocompatibility and highly specific targeting to αvβ3-overexpressed breast cancer and melanoma cell lines. A significant photothermal effect was generated when targeted CD nanocomposites were irradiated with a pulsed laser, proving their potential for thermal ablation therapy. In vivo, photoacoustic imaging using nanocomposites as contrast agents showed excellent targeting sensitivity and enhancement. Cytotoxicity assays found the modified nanocomposites had better biocompatibility and lower toxicity than non-modified counterparts in cancer cell lines. Dual-channel fluorescence imaging verified targeted nanocomposite internalization and labeling of cancer cells via receptor-mediated endocytosis. Confocal microscopy imaging demonstrated that cancer cell lines overexpressing the folate receptor (HeLa, SKOV3) were strongly labeled by the folic acid CDs through folate receptor-mediated uptake [123]. Cell lines with medium or low folate receptor expression (HepG2, MCF7) showed moderate labeling, while lines lacking the receptor (CHO, A549) displayed negligible labeling. Competition experiments with free folic acid reduced cell labeling in a concentration-dependent manner.

In parallel, crossing the tightly packed bloodbrain barrier (BBB) endothelium remains a huge challenge for conventional imaging agents and therapies against aggressive cancers like glioblastoma. Unique oligopeptides conjugated over CDs facilitate transport across this barricade to neural cells. For instance, the glucose-based CDs (GluCDs) and conjugated them with fluorescein (GluCDs-F) to test their ability to cross the BBB in vivo [124]. They first showed that the cellular uptake of GluCDs in yeast cells requires glucose transporters, suggesting the GluCDs enter cells through glucose transporter proteins. They then injected GluCDs-F intravascularly into the hearts of zebrafish and observed accumulation in the central nervous system, indicating successful BBB passage. Similarly, intravenous injection of GluCDs-F in rats led to its presence in the brainstem and spinal cord grey matter 4 h later, consistent with neuronal localization. The absence of GluCDs-F in white matter suggested limited axonal transport. Together, these findings demonstrated that GluCDs can cross the BBB without additional targeting ligands and carry small molecule cargo into the central nervous system in both zebrafish and mammalian models (Fig. 13). As neuron targeting drug delivery platforms, GluCDs may have promising therapeutic potential for neurodegenerative diseases, spinal cord injuries, and brain malignancies.

Fig. 13.

Leveraging their long circulation and tumorhoming abilities, theranostic CDs platforms loaded with chemotherapeutic payloads enable tracking delivery to improve efficacy and safety. Light-triggered unloading of drugs/proteins additionally grants spatiotemporal control for site-specific therapy with imaging feedback.

By exploiting the enhanced permeability and retention effect, nanoparticles passively permeate and accumulate in tumor interstitium enhancing local concentrations of cargo like doxorubicin (DOX) and paclitaxel for greater apoptosis. Yuan et al. [125] developed a drug delivery system using CDs synthesized from milk to carry the chemotherapy drug DOX. The CDs were characterized as small (less than 10 nm), hydrophilic particles with oxygen and carboxyl surface groups. DOX was successfully loaded onto the CDs through electrostatic interactions with 87% efficiency. In vitro tests showed the CDs-DOX complexes released more DOX in acidic conditions similar to the tumor microenvironment compared to neutral pH. Cell studies found the CDs were non-toxic, while the CDs-DOX complexes showed enhanced toxicity against human adenoid cystic carcinoma cells compared to free DOX. The CDs-DOX complexes are localized in the cell nucleus due to interactions between the CDs and the nuclear membrane. Flow cytometry indicated the CDs-DOX complexes had slower uptake than free DOX but increased apoptosis of cancer cells (Fig. 14). Chowdhury and Das [126] developed a biotin-modified iron-doped CD (FCDsb) that can selectively sense, activate, and deliver anticancer drugs to cancer cells. FCDsb was synthesized via a hydrothermal method and surface functionalized with biotin to target biotin receptors overexpressed in cancer cells. FCDb exhibited blue fluorescence and could sense H₂O₂ in cancer cells by fluorescence quenching. In the presence of FCDsb, H₂O₂ generated reactive oxygen species that caused oxidative DNA damage. FCDsb showed higher cellular uptake and H₂O₂-mediated fluorescence quenching in cancer cells compared to normal cells. The anticancer drug paclitaxel was loaded onto FCDsb (FCDsb-PTX). FCDsb-PTX exhibited 2.7-3.5 fold higher killing of melanoma cancer cells compared to normal cells through a combination of H₂O₂-induced oxidative damage and paclitaxel’s anticancer effects. Cancer cell death occurred via early and late-stage apoptosis.

Fig. 14.

Despite extensive strides, the adoption of CDs for practical applications faces roadblocks like batch-to-batch variability and a lack of robust, scalable green production routes. As an emerging field, reproducibility issues plague the domain, with results heavily contingent on exact experimental conditions. The complex interplay of factors like precursor nature, process conditions, and workup protocols, unlike small organic dyes, leads to intricacies in duplicated production.

Moreover, current laboratory-scale syntheses demand extensive inputs of solvents, time, and infrastructure, precluding commercial adoption. For instance, hydrothermal methods employ corrosive acids and bases alongside high temperatures and pressures. Microwave and ultrasonication routes require specialized instrumentation, too [127]. Biocompatible production hence necessitates clean, high-yielding assembly strategies. Some beginnings have been made, like flow chemistry as well as sustainable bio-mediated and photochemical set-ups [128].

The intricacies mean products need extensive benchmarking and custom protocols developed by individual groups, unlike off-the-shelf dyes. Developing robust, environmentally benign scalable chimies for consistent, shape-controlled CDs with lasting usable fluorescence remains an important goal [129]. Taming the complex parametric interplays is vital to cement reproducibility.

While most research corroborates the general biocompatibility of CDs, nagging concerns about long-term toxicity, especially for therapeutic applications like drug delivery, persist [130]. Troubling environmental health reports on graphene and carbon nanotubes, despite early promise, warrant cautionary adoption.

Issues like oxidative stress induction, bioaccumulation upon chronic exposures, and activation of inflammatory pathways need careful assessments across cell and animal models before clinical translation [131]. Unlike transient labeling applications, particle carriage of drugs and tenure in phototherapy mandates safety vetting with well-designed longitudinal trials and multi-parametric bioanalyses for formal toxicological profiling. Transport across BBB also warrants neuroimmune appraisal [132]. Terrestrial toxicology through simulated environmental releases also merits attention prior to manufacturing scale up to avoid unintended contamination similar to microplastics.

Standardized toxicity analysis frameworks, building risk-benefit matrixes spanning vector design variables, and emphasizing safe-by-design principles need community adoption prior to widespread endorsements for biomedical uses. Regulations need to balance innovation possibilities against ethical prerogatives.

While fluorescence-based optical imaging constitutes the predominant implementation of CDs currently, other modalities like Raman spectroscopy [133], photoacoustics [134], radiolabeling [135], and integration with magnetic platforms offers uncharted possibilities.

For instance, surface-enhanced Raman scattering (SERS) tags based on gold-CD nanocomposites provide ultra-detection sensitivity and multiplexing capabilities for intracellular mapping unviable through fluorescence alone. Radio-labeled CDs similarly enhance penetration depths for wholebody analyses, as opposed to optical means limited by light scattering. Ultrasound and magnetic resonance interfacing also improve in vivo imaging capabilities in opaque anatomical niches.

In parallel, diverse clinical adoption avenues beyond common targeting, drug delivery, and phototherapy continue to emerge. Usage as contrast enhancers for surgical resection, theranostic dentistry, plant imaging, and food safety analysis illustrates expanding utility. Further clinical solutions for challenging applications like multiple sclerosis, cardiovascular imaging, immunotherapy, and central nervous system drug delivery will catalyze impact.

Building such multimodal, cross-domain partnerships by interfacing CDs with varied modalities, analysis tools, and application landscapes promises to usher in the next wave of biomedical solutions, fully harnessing their nanoscale versatility.