Keywords

1 Introduction

Carbon nanodots (CNDs), also known as carbon dots (CDs), graphene quantum dots (GQDs), carbon quantum dots (CQDs), carbonized polymer dots, or C-dots, are zero-dimensional fluorescent carbonaceous nanomaterials with a typical size of <10 nm [1,2,3]. CNDs were first discovered from the purification of single-walled carbon nanotube fragments in 2004 [4], and they have been widely investigated since pure CNDs with multicolor emission were prepared by Sun et al. [5].

Generally, CNDs have an internal core composed of sp2-hybridized carbon atoms, and an external surface with various functional groups such as amino, carboxyl, hydroxyl, epoxy, ether, and carbonyl groups [6,7,8]. Both the conjugated carbon core and surface defects can affect the optical properties, conductivity, and catalytic activity of the CNDs [9]. Many CNDs possess interesting excitation-dependent emission properties [10,11,12,13], which means that the emission wavelengths have redshifts with the increasing excitation wavelengths. It is believed that the distinct optical properties of CNDs are highly related to their carbon core and surface defects [9].

Compared to other fluorescent materials, CNDs have several advantages that are beneficial for biomedical applications. First, most CNDs have good water-dispersity due to the abundant hydrophilic groups on their surfaces, which makes further hydrophilic modification unnecessary [14,15,16]. Second, their surface groups such as amino and carboxyl groups endow CNDs with the convenience of conjugating with functional molecules such as targeting ligands and drugs [17,18,19]. Third, CNDs have a strong anti-photobleaching property [20,21,22]. For example, it was reported that the fluorescence intensity of CNDs prepared by glycerol and (3-aminopropyl)triethoxysilane (APTES) only decreased to ~60% after 200 min ultraviolet (UV) light irradiation [23]. In contrast, other fluorescent probes including CdTe quantum dots, gold nanoclusters (Au NCs), and fluorescein isothiocyanate (FITC) were almost completely quenched under the same condition. Fourth, CNDs can be facilely prepared at a low cost. They have been synthesized from various precursors such as small organic molecules [24, 25], polymers [26, 27], bulk carbon [28], cells [29, 30], and biomass resources [3] through different approaches. Acidic oxidation, hydrothermal/solvothermal route, microwave-assisted synthesis, and electrochemical preparation are the commonly used inexpensive approaches for the preparation of CNDs [9, 31]. Fifth, the optical properties of CNDs can be tuned through changing the precursors and reaction conditions such as temperature and time [32]. For example, Jiang et al. used three isomers of phenylenediamines (including m-phenylenediamine, o-phenylenediamine, and p-phenylenediamine) as the carbon sources to obtain blue-, green-, and red-emitting CNDs, respectively [33]. Zhang et al. reported that the fluorescence color of the CNDs prepared by L-valine (as the carbon sources) and H3PO4 (as the oxidant) could be changed from green to yellow through prolonging the reaction time [34].

Owing to their superior properties, CNDs have broad applications in catalysis [35], electronics [36], sensing [37], drug delivery [38], and bioimaging [39, 40]. In this chapter, we will summarize the use of CNDs in imaging a variety of cells, including mammalian cells, microbial cells, and plant cells.

2 Preparation of CNDs

“Bottom-up” and “top-down” approaches are the two commonly used methods to prepare nanoparticles. CNDs as fluorescent nanomaterials can also be synthesized via the two strategies. The “bottom-up” approach to synthesize CNDs is realized through the carbonization of small organic molecules or the stepwise chemical fusion of small aromatic molecules, while the “top-down” approach is achieved by breaking down carbonaceous materials. To realize the “bottom-up” or “top-down” synthesis of CNDs, hydrothermal/solvothermal, microwave-assisted, electrochemical, and acidic oxidation methods have been proposed.

2.1 Hydrothermal/Solvothermal Method

Hydrothermal/solvothermal method, as the most popular “bottom-up” approach for synthesizing CNDs, only requires an inexpensive heating system and a hydrothermal reactor, and can realize the high-quality synthesis of CNDs with simple procedures [41,42,43]. The hydrothermal/solvothermal temperature used for the preparation of CNDs is usually in the range of 120–300 °C [9], and the reaction temperature and reaction time can largely affect the size and optical properties of the as-prepared CNDs [44, 45].

Zhang et al. prepared nitrogen-doped and fluorescence-tunable CNDs by a one-pot solvothermal reaction using CCl4 and NaNH2 [44]. They found that the size of the CNDs increased with increasing reaction time, and their excitation and emission wavelengths had a redshift when the reaction duration prolonged from 1 to 8 h. Li et al. demonstrated that the CNDs with different surface densities of amino groups and the excitation-dependent/excitation-independent fluorescence property could be prepared through controlling hydrothermal reaction temperature [45]. They found that the CNDs prepared at lower temperatures (e.g., 160 °C) had a higher surface density of amino groups with the excitation-independent fluorescence property. By comparison, the CNDs synthesized at higher temperatures (e.g., 240 °C) had a lower surface density of amino groups with the excitation-dependent fluorescence property.

Besides, the reaction solvent also plays an important role in the preparation of CNDs. For example, the CNDs obtained from o-phenylenediamine using the hydrothermal method had the maximum emission wavelength located at 567 nm under 420 nm excitation with a low photoluminescence quantum yield (PLQY) of 2% [46]. In contrast, the o-phenylenediamine-derived CNDs using ethanol as the reaction solvent had a much higher PLQY of 17.6% under 420 nm excitation [33].

2.2 Microwave-Assisted Method

Compared with the hydrothermal/solvothermal method, the microwave-assisted method is a much faster approach for the synthesis of nanomaterials with a reaction duration from minutes to tens of minutes [47,48,49,50]. The CNDs prepared via the microwave-assisted method usually have a narrow size distribution, which is mainly because microwave has a deep penetration depth and uniform heating performance. If the raw materials can form CNDs under hydrothermal/solvothermal conditions, they may also be used to synthesize CNDs through the microwave-assisted method. However, the CNDs prepared by the two different approaches may have some differences.

Huang et al. prepared the CNDs using the mixture containing glycerol and (3-aminopropyl)triethoxysilane (APTES) under microwave irradiation within a short reaction period of 30 min [23]. After the incubation of these CNDs with HeLa cells, some dot-like fluorescence signals were observed in the cytoplasms. By contrast, the CNDs prepared using similar raw materials by the solvothermal method targeted the mitochondria after incubation with the HeLa cells, as demonstrated by our group [51].

2.3 Electrochemical Method

Compared with the above-mentioned “bottom-up” methods, the electrochemical method for the preparation of CNDs has some distinct advantages, such as low cost, low energy consumption, the capability of real-time monitoring of the CND formation, and large-scale production [52, 53]. Moreover, both thermally stable and unstable raw materials can be used to synthesize CNDs using the electrochemical method. Besides, the size and properties of the as-prepared CNDs can be tuned through changing the reaction conditions, such as electrode potential, electrolyte type, electrolyte concentration, and reaction time. For example, Deng et al. developed a simple method to prepare CNDs through the electrochemical carbonization of low-molecular-weight alcohols [54]. The size of the CNDs could be tuned by changing the electrode potential, and the fluorescence emission of the resultant CNDs was excitation- and size-dependent.

Besides its use for the “bottom-up” synthesis of CNDs, the electrochemical method can also serve as a “top-down” way to produce CNDs [55, 56]. It is proposed that OH• and O• radicals formed from the anodic oxidation of water can oxidize bulk carbon sources, leading to the formation of small hydroxylated carbon particles [57]. To introduce functional groups or heteroatoms into the CNDs, choosing different electrolyte solutions is considered as an available way [58, 59]. For instance, to prepare nitrogen (N)-doped CNDs, Li et al. used N-containing tetrabutylammonium perchlorate in acetonitrile as the electrolyte to introduce N atoms into the resultant CNDs [58].

2.4 Acidic Oxidation Method

Besides the electrochemical exfoliation, the acidic oxidation method is also a common “top-down” way to produce CNDs. The acidic oxidation method has been successfully used to exfoliate CNDs from various carbon sources, such as carbon fibers [60], carbon nanotubes [61], nanodiamonds [62], graphite oxide [63], plant soot [64], coal [65], carbon black [66], and activated carbon [67]. Large-quantity synthesis of CNDs from bulk carbon sources can be realized by this method. Furthermore, acidic treatment usually endows CNDs with negatively charged groups on their surfaces, making them hydrophilic and easy to be further modified. For example, Zhang et al. synthesized water-dispersible CNDs with tunable photoluminescence via the one-pot acidic oxidation of nanodiamonds using H2SO4, HNO3, and KMnO4 [62]. During the oxidation process, many functional groups such as carboxyl and hydroxyl groups were introduced on the surface of the CNDs. In addition, the CNDs were successfully used in cytoplasm imaging due to its excellent water dispersibility and biocompatibility.

3 CNDs for Mammalian Cell Imaging

Owing to their superior fluorescence properties and excellent biocompatibility, CNDs show great potential in biomedical applications. Imaging cells especially mammalian cells using CNDs is a hot research topic [68,69,70]. CNDs have been used to light up the whole cells or some parts of the cells, and monitor the locations and concentrations of the molecules/ions in the cells. Besides, thanks to the presence of many reactive groups on their surfaces, CNDs can be modified with targeting moieties and realize selective imaging of the cells especially the cancer cells.

3.1 CNDs for Subcellular Imaging

In mammalian cells, various subcellular structures such as the nucleus, mitochondrion, lysosome, Golgi apparatus, endoplasmic reticulum, ribosome, and plasma membrane are essential components of the cells. Each subcellular structure performs its specialized role to support the fundamental cellular functions. Thus, visualization and monitoring of subcellular components are of great importance. Compared with other fluorescent probes, CNDs have many functional groups on their surfaces, enabling subcellular structure-targeting moieties to be easily conjugated with the CNDs. On the other hand, because a variety of carbon sources can be used for the preparation of CNDs, the as-prepared CNDs have different surface properties, and even have intrinsic subcellular structure-targeting ability without further modifications. Besides, CNDs have excellent photostability, making them suitable for the long-term monitoring of subcellular components. Additionally, CNDs can be facilely prepared with low cost, making them much cheaper than the commercial subcellular imaging probes.

3.1.1 CNDs for Nucleus/Nucleolus Imaging

Cell nucleus which contains the majority of cellular genetic materials acts as the brain of the cell. Thus, many efforts have been devoted to visualizing cell nuclei [71, 72]. However, the probes with large sizes are hard to enter the nuclei because of the presence of the small nuclear pores [73]. Thanks to their ultrasmall size and excellent fluorescence properties, CNDs show great potential in imaging cell nuclei.

As shown in Fig. 1, Yang et al. synthesized the amine group-containing CNDs, and functionalized the CNDs with nuclear localization signal (NLS) peptide through carboxyl–amine reaction for realizing cell nucleus imaging [74]. Besides, it has been reported that zwitterionic CNDs may have the intrinsic nucleus-targeting ability [75, 76]. For example, Jung et al. prepared zwitterionic CNDs using citric acid and β-alanine as raw materials [75]. The CNDs had excitation-dependent photoluminescence, which could monitor the cell nuclei in blue, green, and red fluorescence channels. Similarly, ascorbic acid and polyethyleneimine were used as raw materials to fabricate nitrogen-doped zwitterionic CNDs [76]. The as-prepared CNDs could also enter the cell nucleus without further modification.

Fig. 1
figure 1

Confocal microscopic images showing the nuclear imaging performance of NLS-conjugated CDs (NLS-CDs). Reprinted with permission from Ref. [74]. Copyright © 2015 Royal Society of Chemistry

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As the largest structure in a nucleus, the nucleolus is the site of ribosome biogenesis, and plays an important role in the formation of signal recognition particles and the cellular response to stress [77, 78]. Several studies have reported that CNDs can enter the cell nuclei and even target the nucleoli [79,80,81,82].

Barbosa et al. found that the surface modification of the CNDs with ethylenediamine could endow the CNDs with the nucleolus-targeting ability [79]. Besides, CNDs with intrinsic nucleolus-imaging ability were also reported. For example, Kong et al. synthesized highly bright fluorescent CNDs by refluxing polyethylene glycol in the presence of sodium hydroxide, which were used to selectively stain cell nucleoli [80]. The nucleolus-targeting ability of the CNDs may be attributed to the large number of oxygen-containing functional groups on their surfaces, which make the CNDs easy to interact with the weakly alkaline chromatin of the nuclei rather than other subcellular structures. Our group reported the one-pot hydrothermal synthesis of nucleolus-targeting CNDs using m-phenylenediamine and L-cysteine [81]. Different from the commercial nucleolus-imaging probe SYTO RNASelect which can only be used to stain the nucleoli of the fixed cells, the as-prepared CNDs could realize nucleolus imaging in both fixed and living cells (Fig. 2), making it possible for the in situ monitoring of nucleolus-related biological behaviors. For the nucleolus-targeting mechanism of the CNDs, we found that the CNDs could selectively bind to RNA instead of DNA after entering the nucleus. Furthermore, the fluorescence intensity of the CNDs was markedly increased after interaction with RNA, which makes the RNA-bound CNDs much brighter than free CNDs. Recently, we also found that the mixture of metal ions and p-phenylenediamine could form red-emitting CNDs after hydrothermal treatment, and the as-prepared CNDs could be used for the real-time and high-resolution fluorescence imaging of the nucleoli of living cells [82].

Fig. 2
figure 2

Confocal microscopic images showing the nucleolus-imaging performance of CDs and SYTO RNASelect in unfixed and fixed cells. Reprinted with permission from Ref. [81]. Copyright © 2018 American Chemical Society

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3.1.2 CNDs for Mitochondrial Imaging

Besides the cell nucleus, the mitochondrion is also an important organelle for the cell, because it is the “powerhouse” of the cell and is related to various cellular functions such as energy conversion, storage of calcium ions, and regulation of cellular metabolism [83]. Owing to the large membrane potential gradient of the mitochondrion, mitochondrion-targeting probes are usually positively charged, which enable the interaction of these probes with mitochondria.

Triphenylphosphonium (TPP), a commonly used mitochondrion-targeting ligand, was successfully conjugated with the CNDs derived from citric acid and urea [84]. The TPP-functionalized CNDs were used for both one- and two-photon mitochondrial imaging in living cells. Our group synthesized fluorescent CNDs through the hydrothermal treatment of chitosan, ethylenediamine, and mercaptosuccinic acid [85]. The as-prepared CNDs were endocytosed by the cells through the caveolae-mediated pathway and then specifically targeted mitochondria without further modification. We presumed that the raw materials mercaptosuccinic acid and chitosan/ethylenediamine could form delocalization structures and positively charged surfaces, respectively, which endowed the CNDs with excellent mitochondrion-targeting ability. Different from the commercial mitochondrial imaging dye Mito-Tracker which cannot be used to image mitochondria for a long time, the CNDs could firmly attach to mitochondria and realize long-term mitochondrial tracking for more than 24 h. Besides, our group prepared a series of CNDs with inherent mitochondrial targeting/imaging capability using the solvothermal treatment of glycerol and silane molecules (Fig. 3) [51]. The cationic (3-aminopropyl)trimethoxysilane (APTMS) CDs were easy to accumulate in mitochondria rather than in other organelles due to the large membrane potential of the mitochondria. Interestingly, the CNDs could effectively distinguish cancerous cells from normal ones due to the differences in the mitochondrial membrane potentials and uptake efficiencies of the two types of cells.

Fig. 3
figure 3

(a) Confocal microscopic images showing the colocalization of APTMS CDs with Mito-Tracker, Lyso-Tracker, Golgi-Tracker, or ER-Tracker. (b) Fluorescence distribution of APTMS CDs and Mito-Tracker. (c) Three dimensional merged confocal microscopic images of Mito-Tracker- and APTMS CD-costained HeLa cells viewed via multiple cross sections. Reprinted with permission from Ref. [51]. Copyright © 2017 Royal Society of Chemistry

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3.1.3 CNDs for Lysosomal Imaging

Lysosomes are the waste disposal system of cells and essential in various physiological processes (e.g., autophagy and protein degradation) [86,87,88]. Generally, acidotropic dyes and some large molecules are the two types of lysosome-targeting reagents. The former type usually includes weakly basic amines, such as morpholine and commercial lysosomal imaging agents Lyso-Tracker/Lyso-Sensor probes. They can target the lysosomes due to their acidic environment. The latter one (i.e., Alexa Fluor 594-conjugated dextran and BODIPY-conjugated bovine serum albumin) can be internalized by cells and enter the lysosomes through the endo-lysosomal pathway.

Taking advantage of the lysosome-targeting ability of morpholine derivatives, Wu et al. modified the CNDs (prepared by citric acid and polyethylenimine) with morpholine groups for long-term lysosomal imaging [89]. Similar to the morpholine derivatives, the ruthenium (II) complex (Ru1) is also a ligand that can target lysosomes. Based on this, Zhang et al. constructed the nanohybrids composed of CNDs and Ru1 for one- and two-photon imaging of lysosomes [90]. Besides, it was reported that some amine-containing CNDs have the intrinsic lysosome-targeting ability. For example, E et al. found that amine group-functionalized CNDs prepared from citric acid and urea could image the lysosomes [91]. Zhang et al. used p-benzoquinone and ethanediamine to prepare highly photoluminescent lysosome-targeting CNDs [92]. They believed that the lysosome-targeting ability of the CNDs was attributed to their abundant hydrophilic groups, especially the amine groups. Liu et al. reported the synthesis of lysosome-targeting CNDs using dexamethasone and 1,2,4,5-tetraaminobenzene as the raw materials [93]. The authors attributed the lysosomal targeting performance of the CNDs to the acidotropic effect of the amine groups on the surface of the CNDs. Chen et al. found that the surface amine groups could improve the lysosome-targeting specificity of the CNDs that were prepared from methionine and citric acid and further modified by a naphthalimide derivative [94]. Zhao et al. synthesized lysosome-targetable CNDs using 1,3,6-trinitropyrene and NaOH without further modification [95]. They also believed that the amine groups endowed the CNDs with lysosome-targeting ability. Recently, Qin et al. developed a novel fluorescent probe based on CNDs prepared from N-methyl-1,2-phenylenediamine hydrochloride [96]. The CNDs were non-fluorescent in water, but emitted strong yellow fluorescence in cells. Besides, the CNDs had good lysosome-targeting ability due to the presence of amine groups, making them suitable for fast imaging lysosomes without washing steps. Singh et al. reported a simple and facile hydrothermal method to synthesize the CNDs using neem root extracts as the raw materials [97]. The as-prepared CNDs with strong lysosomal specificity were suitable for structured illumination microscopy and two-photon microscopy (Fig. 4). The authors presumed that the presence of the ether, carboxyl, and amino groups on the surface of the CNDs might ensure the appropriate lipophilicity for lysosomal targeting. Similarly, Guo et al. used a facile microwave-assisted method to prepare green-emitting CNDs with intrinsic lysosome-targeting ability using citric acid and N,N-dimethylaniline as the raw materials [98]. The CNDs could specifically monitor the lysosomes in various cell lines for more than 48 h, and even stain the lysosomes in apoptotic cells and fixed cells.

Fig. 4
figure 4

Evaluation of the colocalization of the CNDs (carbon dots) with Lyso-Tracker Deep Red probes in RAW cells. Reprinted with permission from Ref. [97]. Copyright © 2019 Royal Society of Chemistry

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3.1.4 CNDs for Golgi Apparatus Imaging

The Golgi apparatus is a crucial eukaryotic organelle for biogenesis, secretion, and intracellular distribution of a wide range of macromolecules [99]. The morphological change of the Golgi apparatus is related to external stimuli, which can thus effectively reflect the physiological state of cells. As a result, it is urgently needed to develop new fluorescent probes capable of real-time monitoring the morphology of the Golgi apparatus.

Li et al. developed a pyrolysis method to prepare chiral CNDs using citric acid and L-cysteine as the carbon sources [100]. The chiral CNDs (termed LC-CQDs) had a high PLQY of 68% and showed excellent photostability. Moreover, as shown in Fig. 5, long-time Golgi apparatus targeting and monitoring was realized using the CNDs. The authors believed that the Golgi apparatus-targeting ability of the CNDs was attributed to the presence of the cysteine residues on their surfaces. In another work, Wang et al. reported a simple molecular fusion route for industrial production of sulfonated CNDs from 1,3,6-trinitropyrene [101]. The 1,3,6-trinitropyrene molecules were completely converted into the CNDs without byproducts after a green sulfonation reaction at a low hydrothermal temperature of 130 °C. Then, the CNDs were used to target and visualize the Golgi apparatuses in living cells.

Fig. 5
figure 5

Evaluation of the Golgi apparatus-targeting ability of the LC-CQDs. (a–d) Fluorescence images of the LC-CQDs (blue), Golgi-GFP (green, Golgi apparatus-specific green fluorescent protein), and Bodipy ceramide (red, a commercial dye for Golgi apparatus) in a HEp-2 cell. (e–h) Fluorescence images showing the colocalization of the LC-CQDs and the cis-Golgi. (i–l) Fluorescence images showing the colocalization of the LC-CQDs and the trans-Golgi. (m) Photographs of the Au-doped LC-CQDs under illumination by white light (left) and UV (365 nm) light (right). (n) TEM image of the Au-doped LC-CQDs. (o, p) TEM images of the Golgi apparatus of the cells incubated with (o) and without (p) Au-doped LC-CQDs. Reprinted with permission from Ref. [100]. Copyright © 2017 Royal Society of Chemistry

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3.2 CNDs for Monitoring the pH and Ions/Molecules in the Cells

3.2.1 CNDs for Intracellular pH Sensing

Intracellular pH modulates the functions of many organelles and plays a pivotal role in biological systems, such as cell proliferation and apoptosis, ion transport, and muscle contraction [102]. As a result, it is essential to monitor the pH distribution and change in living cells. CNDs with good water-dispersity usually have numerous pH-sensitive groups such as amino groups and carboxyl groups on their surfaces, endowing the fluorescence of CNDs with pH responsiveness [10, 14, 92, 103,104,105,106].

Zhang et al. developed a simple method to prepare highly photoluminescent CNDs by mixing p-benzoquinone and ethanediamine under ambient conditions [92]. The as-synthesized CNDs had massive amino groups (came from ethanediamine) and lysosomal targeting ability, and their fluorescence emission could sensitively respond to the pH in the lysosomes. Moreover, the CNDs were successfully used to monitor the lysosomal pH dynamics during the apoptosis of living cells. Ye et al. fabricated red-emitting CNDs with two-photon fluorescence excitation ability by one-pot hydrothermal method using p-phenylenediamine, o-phenylenediamine, and dopamine [106]. The CNDs exhibited a broad pH-sensitive range from 1.0 to 9.0 due to the aggregation and disaggregation of the CNDs. Owing to the excellent fluorescence properties and pH-responsive ability, the CNDs could be used as a fluorescent agent to sense and visualize pH fluctuation in cells, tissue, and zebrafish.

Besides, ratiometric probes with more accurate sensing results have also been designed based on CNDs. Nie et al. prepared a ratiometric probe based on CNDs and pH-sensitive fluorescein isothiocyanate (FITC) [10]. Similarly, CNDs with dual emissions located at 475 and 545 nm under single-wavelength excitation were prepared by the one-pot hydrothermal treatment of citric acid and basic fuchsin (Fig. 6) [105]. Taking advantage of the pH sensitivity of the as-obtained CNDs at the two emissions, ratiometric detection of intracellular pH was successfully realized using the CNDs.

Fig. 6
figure 6

Schematic illustrating the synthesis of the CNDs (carbon dots) and their application for extracellular and intracellular pH sensing. Reprinted with permission from Ref. [105]. Copyright © 2016 American Chemical Society

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3.2.2 CNDs for Intracellular Metal Ion Sensing

Various metal ions are present in the cells. Some of the metal ions are essential for living cells, while others are toxic to the cells [107]. Therefore, the in situ detection of these metal ions is important for the diagnosis of metal ion-related diseases. Up till now, CNDs have been used to detect many kinds of metal ions (such as Zn2+, Al3+, Fe3+, Cu2+, Ag+, and Hg2+) in living cells.

Zinc (Zn), an essential element in the human body, is an indispensable component of many enzymes. Thus, sensitive detection of Zn2+ is important to understand these enzyme-related physiological processes. Yang et al. designed Zn2+-passivated CNDs whose fluorescence could be effectively quenched by ethylenediaminetetraacetic acid disodium salt (EDTA) [108]. Moreover, taking advantage of the complexation between Zn2+ and EDTA, the fluorescence of the Zn2+-passivated CND/EDTA complex could be recovered by the addition of external Zn2+ with a detection of limit (LOD) of 5.1 × 10−7 M, making the system suitable for monitoring the intracellular Zn2+ concentration.

Al3+ ion has been widely investigated in the etiology of neurological disorders, such as Parkinson’s disease, Alzheimer’s disease, and dialysis encephalopathy [109]. Hence, the accurate detection of Al3+ is crucial for understanding the progression of neurological diseases. Kong et al. synthesized amphiphilic blue-emitting CNDs from citric acid and methionine, which could be quenched by morin (MR) through electrostatic interaction [109]. After the addition of Al3+, the system could emit strong green emission due to the Förster resonance energy transfer (FRET) process between the CNDs and the MR-Al3+ complex. Besides, the designed system could be used to visualize the Al3+ distribution in human umbilical vein endothelial cells.

Ferric ion (Fe3+) can regulate cellular metabolism and oxygen transport in hemoglobin in the human body. The fluorescence of many CNDs shows excellent responsiveness to Fe3+ [110,111,112,113,114,115]. For example, it was reported that the N-doped CNDs derived from a popular antibiotic aminosalicylic acid showed excellent sensitivity to Fe3+ in living cells [113]. Similarly, a CND-based fluorescent probe with the “on-off-on” property was fabricated by our group via a one-pot solvothermal method using glycerol and N-[3-(trimethoxysilyl)propyl]ethylenediamine (DAMO) as the raw materials (Fig. 7) [115]. The fluorescence of CNDs could be selectively and sensitively quenched by Fe3+ with a low LOD of 16 nM. The potential Fe3+ detection mechanism of the CNDs was attributed to the presence of the amino groups on the surface of the CNDs. The amino groups may have strong interaction with Fe3+, making the electrons in the excited state transfer to Fe3+ and quenching the fluorescence of CNDs.

Fig. 7
figure 7

Schematic illustrating the preparation of the CNDs and their applications for Fe3+ detection and cancer/normal cell differentiation. Reprinted with permission from Ref. [115]. Copyright © 2018 Elsevier Ltd. All rights reserved

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Besides Fe3+ ions, Cu2+ ions also play essential structural roles in many proteins and enzymes and are involved in many physiological behaviors. However, high concentrations of intracellular Cu2+ are toxic to organisms. To detect the Cu2+ concentration in the cells, Salinas-Castillo et al. developed a microwave-assisted strategy to prepare the CNDs with both down- and up-conversion fluorescence properties using citric acid and polyethylenimine as the precursors [116]. The CNDs showed low cytotoxicity and were successfully used for imaging intracellular Cu2+ with high selectivity and sensitivity.

Silver has a long and intriguing history as an antibiotic for fighting against bacterial infections, but high concentrations of Ag+ can elicit high toxicity [117]. Therefore, sensitive detection of Ag+ is highly required. Zuo et al. prepared fluorine (F)-doped CNDs using 4,5-difluoro-1,2-benzenediamine and tartaric acid as the raw materials, and found that the F-doped CNDs could selectively bind to Ag+, making the F-doped CNDs suitable for the detection of Ag+ in various biological systems including mammalian cells [118].

Hg2+ as one of the most hazardous and toxic ions to the environment and human health is responsible for many fatal diseases such as nervous system damage and nephritic syndrome even at a low concentration of 2 mg/kg per day [119]. To detect the intracellular Hg2+, two kinds of CNDs were prepared using “citric acid + 1,2-ethylenediamine” and “citric acid + N-(β-aminoethyl)-γ-aminopropyl methyldimethoxysilane (AEAPMS)” as the starting materials [120]. Both of the two types of the CNDs could be effectively quenched by Hg2+, and even trace the Hg2+ in living cells. The excellent Hg2+ detection selectivity of the CNDs was possibly due to the fact that Hg2+ ions have a stronger affinity for the electron-rich surface (due to the presence of amino and carboxyl groups) of the CNDs than other metal ions.

Besides the above-mentioned metal ions, CNDs have also been used to detect other ions, such as Fe2+ [121], Sn2+ [122], Sn4+ [123], Mo6+ [123], Pb2+ [124], Cr6+ [125], and even anions, including I− [126] and phosphate [127]. However, only a few studies have reported that the CNDs can be used for detecting these ions in the presence of cells or in physiological conditions. In the future, the feasibility of using CNDs for in vitro and in vivo ion detection should be extensively investigated.

3.2.3 CNDs for Intracellular Molecule Sensing

Besides pH and metal ions, CNDs are also a powerful tool for the detection of intracellular molecules. As fluorescent probes, CNDs can detect and image molecules through “turn-off”, “on-off-on”, and ratiometric ways.

Hydrogen peroxide (H2O2) is a prominent member of the reactive oxygen species (ROS) family and necessary in biological systems, and hence the detection of H2O2 and the elucidation of its biological functions have become an important research topic in the biological and medical fields. Du et al. designed a CND-based nanoprobe for detecting mitochondrial H2O2 by conjugating the CNDs with TPP and 3-oxo-3′,6′-bis(4,4,5,5-tetra-methyl-1,3,2-dioxaborolan-2-yl)-3H-spiro[isobenzofuran-1,9′-xanthene]-6-carboxylic acid (PFl) for mitochondrial targeting and H2O2 recognition, respectively, in which the CNDs served as the carrier and the FRET donor [128].

Hydrogen sulfide (H2S) is an endogenous gaseous signaling compound generated in cells via the enzymatic or non-enzymatic pathway, which can regulate cardiovascular, neuronal, and immune systems [129]. Yu et al. designed a CND-based ratiometric fluorescent probe for intracellular H2S detection [130]. The blue-emitting CNDs (em = 425 nm) were conjugated with green-emitting naphthalimide-azide (em = 526 nm). No FRET process occurred between the CNDs and naphthalimide-azide without H2S. After the addition of H2S, the naphthalimide-azide could be reduced to an energy acceptor naphthalimide-amine by H2S. As a result, the fluorescence ratio (I526/I425) increased with increasing H2S concentrations.

Formaldehyde (FA) is an important intermediate in cellular metabolism in mammals, and is related to Alzheimer’s disease, cancer, and other diseases. Thus, to develop approaches for detecting FA with high sensitivity and selectivity in living cells is highly required. To this end, as reported by Liu et al., dexamethasone and 1,2,4,5-tetraaminobenzene (TAB) were used to synthesize the lysosome-targeted CNDs for the ratiometric fluorescence detection of FA (Fig. 8) [93]. The residual o-diamino groups in the synthesized CNDs could react quickly and selectively with FA, leading to the ratiometric fluorescence response to FA through altering the intramolecular charge transfer (ICT) process from the amino groups (electron donors) to the carbonyl groups (electron acceptors) of the CNDs. Chen et al. synthesized naphthalimide derivative (ND)-conjugated CNDs for monitoring lysosomal FA, in which the fluorescence intensity of green fluorescent ND (em = 535 nm) and blue-fluorescent CNDs (em = 414 nm) served as the response signal and the reference signal, respectively [94]. The fluorescence intensity ratio (F535/F414) of the probe was linearly dependent on the FA concentration with a LOD of 3.4 × 10−7 M. Combined with its lysosome-targeting ability, the probe was suitable for the ratiometric fluorescence imaging of FA in lysosomes.

Fig. 8
figure 8

Schematic illustrating the synthesis of the CNDs (CDs) and their possible mechanism for FA detection. Reprinted with permission from Ref. [93]. Copyright © 2019 Royal Society of Chemistry

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Glutathione (GSH) is an important antioxidant that prevents the cells from being damaged by ROS and heavy metals. Besides, it also participates in many physiological processes such as cell metabolism. As we stated above, our group reported that Fe3+ could quench the fluorescence of the CNDs made from glycerol and DAMO [115]. More interestingly, we found that GSH could recover the fluorescence of the CNDs quenched by Fe3+. Based on the different GSH contents in normal cells and cancerous cells [131], we successfully distinguished cancerous cells from normal ones using the mixture of CNDs and Fe3+. Sun et al. also designed an “on-off-on” probe based on the CNDs prepared from citric acid and diethylenetriamine [132]. The fluorescence of the CNDs could be significantly quenched by Hg2+, and could be recovered after the addition of GSH. Then the CND-based fluorescent agent was successfully applied in monitoring the Hg2+ and GSH in living cells.

Ascorbic acid (AA), as an essential nutrient for humans, functions as a cofactor in many enzymatic reactions, and is related to many diseases. As a result, to develop a sensitive method for detecting the intracellular AA level is of great importance [133]. Feng et al. prepared near-infrared-emitting CNDs which could be quenched by cobalt oxyhydroxide (CoOOH) nanoflakes by energy transfer [134]. After the addition of AA, CoOOH was reduced to Co2+, resulting in the “turn-on” of the CND fluorescence. The as-prepared nanosystem had high AA detection selectivity and sensitivity with an LOD of 270 nM. Furthermore, the nanosystem was successfully employed for two-photon imaging of endogenous AA in living cells and deep tissues.

Sensitive and selective glucose sensing is highly needed because glucose detection is incredibly important to the patients suffering from diabetes. Kiran et al. found that the boronic acid-functionalized CNDs could enter the cells and form aggregates due to the presence of glucose in the cells, leading to the fluorescence quenching of the CNDs [135]. As a result, intracellular glucose detection could be realized using the CNDs.

Besides small molecule imaging, CNDs can be used for macromolecule sensing, such as DNA [136] and RNA [137]. Han et al. synthesized carboxyl- and hydroxyl-containing CNDs through acidic oxidation of conductive carbon nanoparticles, and modified the CNDs with p-phenylenediamine and 4-carboxybutyl triphenylphosphonium to endow the CNDs with positive charges [138]. Interestingly, the fluorescence intensity of the as-prepared green-emitting CNDs could be enhanced by double-stranded DNA (dsDNA) and single-stranded RNA (ssRNA), and both the absorption and emission profiles of the CNDs had a bathochromic shift after the addition of ssRNA but not dsDNA. Besides, the CNDs could penetrate through various biological barriers, and could emit spectrally distinguishable fluorescence when they bound to dsDNA and ssRNA in living cells, thus realizing real-time visualization of dsDNA and ssRNA in situ.

In addition, CNDs have been successfully used for detecting other molecules such as dopamine [139], tetracycline [140], amino acids [141, 142], and even proteins [143]. Similar to ions, most of these molecules are detected by CNDs without cells. As a result, novel CNDs that are suitable for in vitro and in vivo molecule detection are urgently needed.

3.3 CNDs for Selective Cell Imaging

Owing to their flexibility of surface functionalization, CNDs can be conjugated with targeting motifs for targeted cell imaging. Based on this, Li et al. designed a type of transferrin-modified CNDs for imaging transferrin receptor-overexpressed cancer cells [144]. Wang et al. connected the CNDs with DNA aptamers by carboxyl–amine reaction, and the resultant aptamer-modified CNDs maintained both the bright fluorescence of the CNDs and the recognition ability of the DNA aptamer [18]. The authors also demonstrated that the aptamer-conjugated CNDs could sensitively and selectively image human breast cancer cells (MCF-7 cells).

Besides, it has been reported that some CNDs can selectively image the cells without further modifications. Zheng et al. developed a pyrolysis strategy to synthesize the tumor-targeting CNDs (termed CD-Asp) without the modification of any extra targeting ligands using D-glucose and L-aspartic acid as the raw materials (the tumor-targeting performance of the CNDs was shown in Fig. 9) [145]. The self-targeting ability of the CNDs was attributed to the presence of D-glucose and L-aspartic acid residues, which helped the CNDs to cross the blood–brain barrier through the GLUT-1 and ACT2 transporters. Bhunia et al. used tumor-targeting folate molecules as the carbon source to prepare CNDs [146]. The as-prepared CNDs could target folate receptor-overexpressed cancer cells as they expected.

Fig. 9
figure 9

(a) In vivo fluorescence images of C6 glioma-bearing mice after the injection of CD-Asp. (b) Three-dimensional reconstruction of CD-Asp distribution in the brain 20 min after injection. (c) Ex vivo fluorescence image of the brain 90 min after the injection of CD-Asp. Reprinted with permission from Ref. [145]. Copyright © 2015 American Chemical Society

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4 CNDs for Microbial Cell Imaging

Microbial infection induced by bacteria, fungi, and viruses is one of the major public health problems worldwide [147]. To fight against microbial infections, early, sensitive, and accurate detection of microorganisms is of great importance. The fluorescence-based methods are considered as a powerful tool for microbial detection. Owing to the outstanding optical properties of CNDs, recent decades have witnessed considerable research progress in their applications in microbial imaging and sensing [148].

Mehta et al. reported the synthesis of Saccharum officinarum juice-derived CNDs [149]. The CNDs were proven to be suitable for bacterial and fungal imaging. Nandi et al. synthesized hydrocarbon chain-functionalized amphiphilic CNDs for bacterial detection and imaging [150]. The fluorescence intensity and spectral position of the CNDs were dependent on the bacterial species, and could be used for distinguishing bacteria.

4.1 CNDs for Gram-Positive and Gram-Negative Bacterial Distinguishment

The Gram staining method is a standard diagnostic method to classify bacteria into Gram-positive and Gram-negative ones. Gram-negative bacteria but not Gram-positive ones are sensitive to some antibiotics such as streptomycin and gentamicin, while Gram-positive bacteria but not Gram-negative ones are sensitive to other antibiotics such as penicillin. Thus, differentiating Gram-positive and Gram-negative bacteria is very important.

Our group prepared quaternized CNDs using the reaction between carboxyl group-containing lauryl betaine and amine group-containing CNDs [151]. The as-synthesized quaternized CNDs (termed CDs-C12) had polarity-sensitive fluorescence emission property, leading to the significantly enhanced fluorescence when the CNDs interacted with the Gram-positive bacteria. Moreover, owing to the presence of both hydrophobic hydrocarbon chains and positively charged quaternary ammonium groups on their surfaces, the CNDs could selectively attach to Gram-positive bacteria (Fig. 10), realizing the bacterial differentiation. Recently, we developed a simpler method for the one-step synthesis of quaternized CNDs via the solvothermal treatment of glycerol and dimethyloctadecyl[3-(trimethoxysilyl)propy]ammonium chloride (termed Si-QAC) [152]. Similarly, the as-obtained CNDs had the bacterial contact-enhanced fluorescence emission property and could be used for fast Gram-type identification.

Fig. 10
figure 10

(a) Confocal microscopic images of Gram-positive bacteria (Micrococcus luteus (M. Luteus) and Bacillus subtilis (B. subtilis)) and Gram-negative bacteria (Pseudomonas aeruginosa (P. aeruginosa) and Proteus vulgaris (P. vulgaris)) after incubation with the quaternized CNDs (CDs-C12) for 1 h under 405, 488, and 552 nm laser excitations. (b) Corresponding flow cytometric data. Reprinted with permission from Ref. [151]. Copyright © 2016 American Chemical Society

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Besides Gram-positive bacterial imaging, the CNDs have been also used for selective Gram-negative bacterial imaging. Colistin, a well-known antibiotic against Gram-negative bacteria, was reacted with diammonium hydrogen citrate to prepare CNDs [153]. Owing to the excellent specificity of colistin to Gram-negative bacteria, selective imaging of Gram-negative bacteria over Gram-positive ones was realized using the CNDs. Similarly, amikacin, an antibiotic with strong bactericidal activity against most Gram-negative bacteria, and diammonium hydrogen citrate were used to prepare CNDs [154]. The amikacin-functionalized CNDs were successfully used for the selective detection of Gram-negative bacteria with an LOD of 552 colony forming units (CFU)/mL.

4.2 CNDs for Microbial Live/Dead Differentiation

To assess the microbial inactivation performance of antimicrobial agents, the distinguishment between live and dead microbial cells is of great importance. Thus, various methods and techniques have been developed for microbial live/dead differentiation, such as the plate counting method, atomic force microscopy, and fluorescence-based methods. Among them, the fluorescence labeling method is one of the most commonly used approaches for rapidly and sensitively distinguishing live/dead microbial cells [155].

Our group prepared bacteria-derived CNDs with a highly negative zeta potential of −42 mV [29]. The CNDs were hard to be internalized by live bacterial cells due to the strong electrostatic repulsion between the CNDs and the bacterial surfaces, but could enter the dead cells with damaged cell surfaces. As a result, the CNDs could selectively image the dead microbial cells (Fig. 11). Compared to propidium iodide (PI) which is a commercial dye for dead cell imaging, the as-synthesized CNDs showed low toxicity to microbial cells and excellent photostability. Nitrogen- and phosphorus-doped CNDs with tunable surface potentials were prepared using a facile hydrothermal method [156]. Similarly, the obtained CNDs selectively stained dead bacterial cells but not live ones due to the electrostatic repulsion between the negatively charged CNDs and the negatively charged bacterial walls.

Fig. 11
figure 11

Confocal microscopic images (a) and flow cytometric results (b) of live and dead S. aureus stained with the CNDs (CDs-S. aureus) for 1 h. Reprinted with permission from Ref. [29]. Copyright © 2017 Royal Society of Chemistry

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4.3 CNDs for Microbial Biofilm Imaging

Microbial biofilms are surface-attached communities of microbes encased in an extracellular matrix of biomolecules and display high levels of antibiotic tolerance, because most antibiotics cannot penetrate the biofilms effectively [157]. Similar to antibacterial agents, fluorescent materials could also be blocked by the sticky extracellular polymeric substance (EPS) matrix, which hinders their successful biofilm imaging.

Ritenberg et al. showed that the amphiphilic CNDs could readily bind to the EPS scaffold of the bacteria P. aeruginosa, making them suitable for the fluorescence microscopic visualization of the EPS structural features [158]. Lin et al. developed a one-step hydrothermal carbonization method to synthesize the CNDs from the bacteria Lactobacillus plantarum (L. plantarum) [30]. The as-prepared CNDs were found to be capable of imaging the microorganisms within the biofilms. Our group prepared Si-QAC/glycerol-derived CNDs by a solvothermal method [152] and used the obtained CNDs with ultrasmall size (~3.3 nm) and strong positively charged surfaces (zeta potential: ~33 mV) for excellent fluorescence imaging of the S. aureus biofilms [159] (Fig. 12).

Fig. 12
figure 12

Three-dimensional confocal microscopic images of an S. aureus biofilm stained with the CNDs (Si-QAC CDs). These images include the ones taken under different excitation wavelengths and the merged one. Reprinted with permission from Ref. [159]. Copyright © 2019 Royal Society of Chemistry

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As stated above, CNDs have become a powerful tool for microbial imaging, including Gram-positive and Gram-negative bacterial distinguishment, microbial live/dead differentiation, and biofilm imaging. Although many studies have demonstrated that CNDs can image bacteria and fungi, few studies have reported virus imaging using CNDs. Besides, taking advantage of the excellent optical properties of CNDs and super-resolution fluorescence imaging techniques [151, 160], the imaging of subcellular structures in microbial cells may also be realized; however, relevant studies are still lacking.

5 CNDs for Plant Cell Imaging

Besides mammalian and microbial cells, CNDs also show potential in imaging plant cells. CNDs usually have ultrasmall sizes, enabling their penetration into the walls and membranes of the plant cells.

Zhang et al. found that the chiral CNDs fabricated from D-cysteine and citric acid could be absorbed by mung beans during the period of seed germination, and were detected in the vascular system of the root, stem, and leaf after 5-day incubation [161]. Wang et al. synthesized bright CNDs using L-glutamic acid with nitric acid as a carbonization agent and ethylene glycol as a passivation agent [162]. The CNDs with negligible cytotoxicity could be endocytosed by intact plant cells with 10 min and even directly stain the nuclei of these cells (Fig. 13). Li et al. found that the CNDs prepared from p-phenylenediamine have a strong affinity for cellulose through hydrogen bonding, and the PLQY of the CNDs dramatically increased from 8% to 44% when the CNDs were dispersed in cellulose matrices [163]. The authors demonstrated that the affinity of the CNDs for binding cellulose endowed them with the capability of imaging the cellulosic plant cell walls.

Fig. 13
figure 13

Confocal microscopic images of HeLa cells incubated with CNDs (a) and acridine orange (AO) (b), and onion epidermal cells incubated with CNDs for 10 and 60 min (c, d) and AO (e). Reprinted with permission from Ref. [162]. Copyright © 2014 Elsevier Ltd. All rights reserved

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Recent studies have reported that some CNDs are able to enhance the production of cereal crops [164] and promote carbohydrate accumulation in mung bean plants [161]. However, the detailed interaction mechanisms between CNDs and plants remain largely unknown. Using CNDs for plant cell imaging will help the researchers to investigate the interactions between CNDs and plants, and study the biodistribution of the CNDs in the plants. Besides, whether the various CNDs synthesized by the researchers can be used for plant cell imaging should be tested to enrich the applications of the CNDs and foster the advancement of the CND-related plant research. Further, to get a deep understanding of the interaction between CNDs and plant cells, the evaluation of the CNDs for organelle-specific imaging in plant cells is highly desirable.

6 Conclusion and Future Perspective

With their ultrasmall size, ease of preparation and modification, good anti-photoblinking and anti-photobleaching properties, and excellent biocompatibility, CNDs have become popular probes for bioimaging. Up to now, CNDs have been successfully used in imaging various cells, including mammalian cells, microbial cells, and plant cells. Subcellular distributions and selective/sensitive detection of ions/molecules using CNDs can be achieved by controlling their size, charge, and surface chemistry.

Although many efforts have been devoted to the preparation of CNDs and cell imaging using CNDs, there are still several issues that need to be addressed in the future. First, most CNDs have broad and excitation-dependent emissions, limiting their imaging applications when they are used in combination with other fluorescent probes. Hence, novel CNDs with bright and narrow emissions are highly desired. Second, a large number of CNDs with novel optical properties such as two-photon excitation [165,166,167], long-wavelength emission [168,169,170], room-temperature phosphorescence [50, 171,172,173], photo-activation [174], piezochromic luminescence [174,175,176], up-conversion properties [177,178,179], and electrochemiluminescence [143], have been synthesized, and their applications in bioimaging also need to be investigated. Third, many CNDs have poor water-dispersity and low cellular uptake efficiency. Thus, developing suitable surface modification strategies for these CNDs is in urgent need for improving their cell imaging performance. Fourth, the structures of most CNDs are not clear, and the mechanisms of the interactions between CNDs and cells, organelles, or intracellular ions/molecules need to be further investigated. It is challenging to rationally design CNDs with suitable compositions, sizes, and surface chemistries for monitoring the cells, organelles, ions, and molecules with high selectivity and sensitivity. Fifth, although CNDs have been used for imaging many organelles (including nuclei, mitochondria, lysosomes, and Golgi apparatus), ions, and molecules, new kinds of CNDs need to be synthesized to image other subcellular structures (such as plasma membrane, cell wall, endoplasmic reticulum, ribosome, centrosome, lipid droplet, and peroxisome) and intracellular ions/molecules. Sixth, owing to their surface modifiability, the diverse CNDs can be conjugated with specific targeting ligands to be ideal probes for imaging various cells (such as cancer cells, inflammation-related cells, stem cells, and immune cells), subcellular structures, and intracellular ions/molecules. Hence, more efforts should be devoted to the modification of the CNDs for realizing the selective imaging of these cells and cellular structures/compositions. Seventh, most of the CNDs can be quenched by the ions/molecules, thus realizing molecule/ion detection. However, the design of CND-based “turn-on” probes for intracellular ion/molecule imaging is still lacking. Eighth, recent studies regarding the use of CNDs for bioimaging mainly focus on mammalian cell imaging, but the applications of CNDs in microbial cells and plant cells are still insufficient. The imaging performance of the developed CNDs for microbial and plant cell imaging should be extensively evaluated. Last, due to their excellent optical properties and good biocompatibility, CNDs may have potential in the selective imaging of cellular biomacromolecules such as the protein antigens on the cell surfaces. The feasibility of using CNDs for monitoring the biomacromolecules should be tested.