Glowing graphene quantum dots and carbon dots: properties, syntheses, and biological applications.

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Graphene Quantum Dots

Glowing Graphene Quantum Dots and Carbon Dots: Properties, Syntheses, and Biological Applications Xin Ting Zheng, Arundithi Ananthanarayanan, Kathy Qian Luo, and Peng Chen*

From the Contents 1. Introduction .........................................1621 2. Properties.............................................1621 3. Synthetic Methods................................1625 4. Biological Applications .........................1627 5. Outlook ................................................1633

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The emerging graphene quantum dots (GQDs) and carbon dots (C-dots) have gained tremendous attention for their enormous potentials for biomedical applications, owing to their unique and tunable photoluminescence properties, exceptional physicochemical properties, high photostability, biocompatibility, and small size. This article aims to update the latest results in this rapidly evolving field and to provide critical insights to inspire more exciting developments. We comparatively review the properties and synthesis methods of these carbon nanodots and place emphasis on their biological (both fundamental and theranostic) applications.

© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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1. Introduction The discovery of the green fluorescence protein (GFP) has fundamentally changed the landscape of biomedical research in the past decades. However, the poor photostability of organic fluorophores makes long-term imaging difficult. And creating and expressing fluorophore-tagged molecules involve non-trivial molecular biology processes including the construction of chimeric plasmids and subsequent transfection in live cells. Semiconductor quantum dots (semi-QDs) have been regarded as the promising alternative because of their high brightness and photo-stability.[1] But semi-QDs are toxic and poorly soluble. And their blinking characteristics make single-molecule tracking difficult. In addition, because of a much-larger size than a biomolecule, they might affect the dynamics and functions of the target molecules and create artificial clusters while associating with multiple targets. Because of the intrinsic drawbacks of the current fluorophores, searching for better fluorophores has been a constant and critical effort for bio-imaging. Carbon dots (C-dots) and graphene quantum dots (GQDs) recently emerge as superior and universal fluorophores because of their unique combination of a number of key merits, including excellent photostability, small size, biocompatibility, highly tunable photoluminescence (PL) property, exceptional multi-photon excitation (up-conversion) property, electrochemiluminescence, ease to be functionalized with biomolecules, and chemical inertness. These glowing carbon nanocrystals provide unprecedented opportunities for bioimaging and optical sensing. Because of their small size and biocompatibility, they may also serve as effective carriers for drug delivery while allowing simultaneous visual monitoring of releasing kinetics. Furthermore, their unique catalytic and physicochemical properties promise various biomedical applications. There are already several excellent review articles on syntheses, properties, and application potentials of C-dots[2] and GQDs.[3] Here, we update the latest results in this rapidly evolving field and place emphases on the comparison between these two 0D cousins, providing mechanistic insights, highlighting their unique advantages, and their biological (both fundamental and theranostic) applications.

2. Properties 2.1. Structures C-dots and GQDs are structurally distinct (Figure 1). The former, which were initially reported in 2006 by Sun et al.,[4] are quasi-spherical nanoparticles usually 75%.[53b] In a comparative study, Shen et al. synthesized bare GQDs and GQDs passivated with PEG and found that QY of the latter was doubled.[49a] Surface passivation, however, complicates the synthesis process and increases the particle sizes whereby imposing limitations on applications. Attaching Chemical Moieties. Various chemical groups (e.g., diamine,[75] thiol,[75] hydrazide,[37] alkylamine)[70] have been attached onto GQDs and C-dots during or after synthesis. These electron-donating groups usually enhance QY by preventing non-radiative recombination and often cause obvious wavelength shift.[70] For example, green oxygenated GQDs become blue after replacing carboxyl with alkylamine.[70] Tetsuka et al. showed that emission wavelength of GQDs could be widely tuned (blue to yellow) by controlling the degree of amine functionalization.[76] 3.3.3. Heteroatom Doping Heteroatom doping (most commonly thus far, nitrogen doping) can be used to fine-tune or obtain new PL and other physicochemical properties of GQDs and C-dots.[2c,77] Heteroatoms can be inherited from precursors during synthesis. small 2015, 11, No. 14, 1620–1636

Carbon nanodots are superior to the current organic and inorganic fluorophores for bioimaging due to the unique combination of a number of key merits. Although this area is still at its infant stage, a large variety of new fluorescence tags based on these nanodots shall provide unprecedented possibilities for bioimaging and therefore significantly change the landscape of biomedical research. 4.1.1. Cellular Imaging The possibility of using C-dots as fluorescent labels for cellular imaging was first demonstrated by Sun et al. who used PEG1500N passivated C-dots to non-specifically stain Caco-2 cells.[4] Since then, non-specific intracellular imaging of uptaken C-dots in other cell types have been shown, including E.coli,[86] ehrlich ascites carcinoma cells (EACs),[6] HeLa cells,[84,87] HepG2 cells,[87b] LLC-PK1,[56] NIH-3T3 fibroblast cells,[88] human lung cancer (A549) cell.[89] Hollow C-dots have also been used to stain the cytoplasm of HEK 293 cells by passive uptake.[5a] GQDs have also been used to label a variety of cell types such as T47D,[36] HeLa,[48] murine alveolar macrophage (MH-S) cells,[14] human hepatic cancer cells (Huh7),[90] MCF-7 cells,[46] and stem cells including neurospheres cells (NSCs), pancreas progenitor cells (PPCs), cardiac progenitor cells (CPCs) and neural stem cells.[38,91] The potential of C-dots (5 nm, propionylethylenimine-coethylenimine or PPEI-EI passivated) for two-photon luminescence microscopy with 800 nm excitation was first explored in MCF-7 cells.[21a] C-dots exhibited bright PL both on the cell membrane and in the cytoplasm after 2 h incubation at 37 °C. The cellular uptake of C-dots was found to be temperature-dependent, with no internalization observed at 4 °C. The ability of dimethyl amine functionalized GQDs (∼3 nm) for two-photon imaging was investigated in human cervical carcinoma (HeLa) cells under 800 nm excitation.[23] The study by Pan et al. showed that GQDs (∼3.0 nm, obtained by hydrothermal cutting of GO sheets)


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preferentially accumulated around the nucleus in HeLa cells and exhibited no obvious reduction in PL intensity after 10 min continuous excitation, suggesting the feasibility for long-term imaging.[48] It has been shown that C-Dots[6] (∼2 – 6 nm, synthesized from acidic exfoliation of carbon soot) and GQDs[46] (∼15 nm, synthesized by acidic exfoliation of carbon black) are able to penetrate and label the nucleus without being functionalized with nucleus targeting motif. The reason underlying such interesting nuclear localization is not yet clear. In most studies, however, scattered distribution of these carbon nanodots in cytoplasm is observed and their possible affinity to certain sub-cellular structures (e.g., endosomes) has not been rigorously investigated. Charge state and functional groups of GQDs are expected to exert significant influence on their cellular penetration capability and intracellular localization. As an example, a comparative study showed that photochemically reduced GQDs were up-taken more efficiently by A549 cells than chemically reduced GQDs because photochemical reduction is more effective to remove the negatively charged carboxyl groups on GQDs.[71] Conjugating C-dots/ GQDs with biofunctional species (e.g., antibodies) allows them to specifically label molecular targets in cells. For example, Li et al. coupled C-dots with human transferrin through carbodiimide chemistry and the conjugates can selectively label cancer cells over-expressing transferrin receptors such as HeLa cells.[87a]

Figure 6. (a-e) Application of insulin-GQD conjugates for real-time tracking of insulin receptors in living adipocytes. (a) Typical total internal reflection fluorescence microscopy (TIRFM) image of a 3T3-L1 adipocyte after 1 h incubation with insulin-GQDs. Scale bar = 5 µm. (b) Membrane patch consisting of insulin-GQD/insulin receptor clusters. (c) Endocytosis of fluorescent membrane patches into a vesicle. (d) Exocytosis of a vesicle containing insulin-GQD/insulin receptor complexes. (e) Transient approaching and retrieval of insulin-GQD/insulin receptor containing vesicle. Scale bars = 0.2 µm. Reproduced with permission.[67] Copyright 2013, American Chemical Society. (f) Dual color fluorescence images of subcutaneously injected C-dots (top) and ZnS-doped C-dots (bottom) in mice (excitation and emission wavelength are indicated). Reproduced with permission.[92] Copyright 2009, American Chemical Society. (g) Subcutaneously injected NIR-emitting C-dots under the 704 nm excitation. Green is the background autofluorescence. Reproduced with permission.[42] Copyright 2012, Wiley-VCH.

4.1.2. Real-Time Molecular Tracking in Live Cells Real-time molecular tracking of dynamic cellular processes requires fluorescent tags that are bright, photo-stable, biocompatible, and of molecular size to minimize physical hindrance. Recently, Zheng et al. demonstrated that oxygenated GQDs (∼2 nm and ∼2 kDa) can be readily conjugated (oneto-one pairing without spacer) with any proteins, without impairing the functionalities of the protein or largely altering the size, weight, and charge state.[67] A number of proteins (including neuropeptide Y, bovine serum albumin, immunoglobulin G, concanavalin A, insulin, and nerve growth factor) were tested. In this seminal work, they used insulinconjugated GQDs to reveal the real-time dynamics (distribution, internalization, and recycling) of insulin receptors in adipocytes (Figure 6a-e). This study discovered, for the first time, that internalization and recycling of insulin receptors in adipocytes are oppositely regulated by apelin and TNFα. The finding helps to understand how these cytokines regulate

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insulin sensitivity. This work demonstrates the great potential of GQDs as universal and superior fluorescent tags for realtime molecular imaging in live cells. 4.1.3. Optical Imaging in Vivo Ideally, fluorescent probes for in vivo imaging should be bright, biocompatible and able to absorb/emit in the long wavelength range that is transparent to biological tissues.[21b] Injecting C-dots subcutaneously, intradermally or intravenously into mice, Yang et al. reported the first in vivo study using PEG-passivated C-dots (emission at 650 nm) and ZnS-doped C-dots (emission at 510 nm) for dual-color fluorescence imaging with good contrast (Figure 6f).[92] More recently, Tao et al. subcutaneously injected C-dots into nude mice for in vivo imaging.[42] Under red excitation, NIR emitting C-dots could be clearly distinguished from the


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autofluorescence background (green) with good contrast (Figure 6g). It was reported by Cao et al. that ZnS doped C-dots (QY∼60%) are comparable to the well-established CdSe/ZnS QDs in vivo imaging performance.[93] Yang et al. applied PEG1500N passivated C-dots for ex vivo imaging in mice models.[35] The two-photon fluorescence images (green emission under 800 nm excitation) were obtained in sliced liver and spleen harvested from mice 6 h after intravenous injection. Wu et al. subcutaneously or intramuscularly injected highly luminescent (QY ∼54.5%) GQDs prepared by pyrolysis of L-glutamic acid (showing excitation dependent emission from blue to NIR) into nude mice and obtained clear in vivo fluorescence images.[14] Nurunnabi et al. demonstrated visual localization of intravenously injected polydopaminecoated GQDs (emission 550 nm) in different organs of mice and showed that the particle distribution depended on GQD size and polydopamine coating.[94] In the interesting work by Liu et al., dimethyl amine functionalized GQDs with high two-photon absorption cross section (48000 GM) were synthesized.[23] These GQDs permit a large imaging penetration depth in tissue specimens and display little photobleaching and photothermal effects under repeated femtosecond NIR (800 nm) laser irradiation, promising long-term deep-tissue imaging.

4.2. Biosensing The PL, electronic and electrochemical properties of C-dots/ GQDs are sensitive to minute perturbations, rendering them of great potential for sensing applications. Moreover, with a size comparable to a biomolecule, these nanodots can intimately interact with biomolecules, leading to improved detection sensitivity and selectivity. 4.2.1. PL Sensors The exceptional PL properties of C-dots/GQDs have been utilized to detect biologically relevant ions, sugars, and proteins in solutions or inside cells, based on either PL turn-on or turn-off mechanisms. Cu2+, as a cofactor for numerous enzymes, is critical in physiological functions. Abnormalities in its metabolism may cause Wilson disease, Alzheimer’s disease, infant liver damage and childhood cirrhosis.[95] Branched poly(ethylenimine) (BPEI)-functionalized C-dots have been used to develop a rapid, reliable and selective Cu2+ sensing system with a detection limit of 6 nM.[74] The selective interaction of Cu2+ ions with the amino groups of BPEI is responsible for the quenching of C-dot PL. This method was improved by Lin et al. who encapsulated BPEI-C-dots into zeolitic imidazolate framework materials (ZIF-8) to form highly fluorescent metal-organic frameworks (MOFs).[96] Detection signal is amplified due to enhanced PL and selectively accumulation of Cu2+ ions by MOF, resulting in a ultra-low detection limit (80 pM) and a wide response range (2–1000 nM). A drawback of MOFs is their large size (∼400 nm), which hinders their cellular entry and intracellular sensing applications. small 2015, 11, No. 14, 1620–1636

Detection of Cu2+ was demonstrated in NIH-3T3 cells using amine functionalized C-dots (∼12 nm).[22b] Upon the addition of Cu2+ to cell medium, the PL of intracellular C-dots decreased due to Cu2+ uptaking. C-dot based ratiometric detection of Cu2+ has recently been demonstrated.[95] C-dots were first synthesized by pyrolysis of N-(β-aminoethyl)-γaminopropyl methyldimethoxysilane (AEAPMS) and then covalently linked to Rhodamine B coated silica nanoparticles. The residual ethylenediamine groups on the surface of C-dots serve as the Cu2+ recognition sites. Upon Cu2+ binding, the blue PL of C-dot was quenched whereas the red PL of Rhodamine B remained constant (as the reference signal). The ratio of fluorescence intensity (F467/F585 or C-dots/Rhodamine B) linearly decreased with increasing Cu2+ concentration in the range of 0 to 3 µM and a detection limit of 35.2 nM was achieved. Notably, this nanosensor was successfully applied for intracellular imaging of Cu2+ in MCF-7 cells. Ratiometric detection provides advantages over single wavelength sensing as the latter is compromised by various uncertainties such as drift of light source or detector, variation in cell uptake, environmental influences. Intracellular pH plays a vital role in cell physiology including receptor-mediated signal transduction, calcium regulation, ion transport and homeostasis.[21c] Nie et al. constructed a ratiometric pH sensor using fluorescein isothiocyanate (FITC)-modified C-dots.[97] The PL of FITC is pH-dependent while that of C-dot is not. The fluorescence ratio (FITC/C-dot) shifted linearly in the range from pH 5 to 8 and the sensor was employed to report the intracellular pH of HeLa cells (5.2 ± 0.4). Based on two-photon PL of C-dots, Kong et al. measured physiological pH in living cells and tissues at depth of 65–185 µm.[21c] A specific H+ receptor, 4′-(Aminomethylphenyl)-2,2′: 6′,2″-terpyridine (AE-TPY) molecule, was bound to electrochemically synthesized C-dots (∼6 nm) for pH sensing (Figure 7a). Under two-photon excitation, the PL intensity showed good linearity with pH variation from 6.0 to 8.5 (in vivo physiological range). Furthermore, real-time cytosolic pH change (upon induced cellular acidification and subsequent recovery) was imaged using confocal microscope (Figure 7b-i). Boronic acid modified C-dots (∼4.5 nm) have been fabricated from one-step hydrothermal carbonization of phenylboronic acid for nonenzymatic blood glucose sensing.[98] Based on the specific covalent binding between the cis-diols of glucose and the boronic acid moieties on the C-dot surface, glucose addition leads to aggregation of C-dots and consequently fluorescence quenching. Glucose level can be quantified in the range of 9–900 µM with a detection limit of 1.5 µM. The plasma glucose concentration determined by this method is in good agreement with the values measured by a commercial blood glucose monitor. The versatility of C-dot based PL sensors is evidenced by their use for a range of detection targets including DNA,[99] phosphate,[100] acetylcholine,[101] α-fetoprotein,[102] thrombin,[103] melamine,[81] or metal ions.[22b,74,104,105] Fe3+ ion is a critical co-factor of various regulatory proteins. On the other hand, excess Fe3+ ions can lead to overproduction of free radicals and hence cytotoxicity. High Fe3+ concentration in neurons is considered as a key indicator for



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to detect biothiols using GQDs as fluorescence indicators.[106] In presence of Ag+ ions, addition of biothiols causes PL reduction of GQDs because of thiol-catalyzed reduction of Ag+ ions into Ag nanoparticles (AgNPs) on GQDs and bridging AgNPs on GQDs through Ag-S bonds. A linear relationship between quenching percentage and the concentration of Cys over the range of 0 – 100 nM was observed with a detection limit of 6.2 nM. The performance is similar for detection of Hcy and GSH. Using GQDs and boronic acid substituted bipyridinium salt (BBV), a fast and convenient label-free fluorescence assay for glucose and other monosaccharides was reported by Li et al.[107] Initially, GQD PL is quenched by electrostatically interacting BBV because of transfer of excited-state electrons from GQD to bipyridinium. With addition of glucose, the boronic acids are converted to tetrahedral anionic glucobornate esters, which effectively neutralize the net charge of the cationic bipyridinium, thus greatly diminishing its quenching efficiency (therefore recovering GQD fluorescence). The linear relationship between the recovered PL and the glucose concentration ranges from 1 to 60 mM, suitable for clinical measureFigure 7. (a) Schematic illustration of C-dot-TPY nanoprobe for two-photon pH sensing. ment of blood glucose level. Blue fluorescent glutathione-func(b–h) Na+-H+ exchange dependent two-photon confocal fluorescence images of A549 cells. False colors from blue to red indicate increase of PL intensity (decrease in pH). (b) Two- tionalized GQDs (GSH-GQDs) with a photon confocal fluorescence image and (c) Overlapped images of A549 cells incubated in QY of 33.6% were used to measure the NaCH3SO3-Ringer’s solution containing 0.04 mg mL−1 C-dot-TPY probe for 30 min at 37 °C. adenosine triphosphate (ATP) level in cell (d-f) Two-photon confocal fluorescence of ouabain-treated A549 cells in choline CH3SO3lysate and human blood serum.[108] ATP, Ringer’s solution (cellular acidification triggered) for (d) 2, (e) 4, and (f) 8 min at 37 °C. known as universal energy source, is also (g,h) Two-photon confocal fluorescence images of Na+-dependent real-kalinization of ouabaina critical signaling mediator in many biotreated A549 cells by adding 100 mM NaCH3SO3 for another (g) 4 and (h) 8 min at 37 °C. + + logical processes and an indicator for cell i) Mean fluorescence intensity induced by Na -H exchange of A549 cells. Data represent the mean fluorescence intensity of ROI 1–3. Reproduced with permission.[21c] Copyright viability and cell injury. The fluorescence 2012, Wiley-VCH. of GSH-GQDs is initially quenched by Fe3+ ions. The detection of ATP is ena[ 20c ] + Parkinson’s disease. BMIM -functionalized GQDs were bled by the recovery of fluorescence, as Fe3+ ions are released 3+ due to the high binding affinity of the used to detect Fe from GQDs after complexing with ATP molecules. The linear imidazole ring of BMIM+ to Fe3+.[20c] Fe3+ acts as a coordi- relationship between PL recovery and ATP concentration nation center to bridge several GQDs together, consequently was observed in the range of 25–250 µM with the detection leading to fluorescence quenching. A detection limit of limit ∼22 µM. GQDs emit light when they react with oxidants. 7.22 µM was achieved. In another study, N-doped GQDs Based on this interesting chemiluminescence phenomenon, a were used for label-free detection of Fe3+ based on the coor- sensor with a detection limit of 0.5 µM has been developed dination between Fe3+ and nitrogen atoms.[59] Linear cor- to detect uric acid.[109] It can accurately determine uric acid relation between PL quenching and Fe3+ concentration was concentration in human plasma or urine samples. obtained in a wide range from 1 to 1945 µM with a detection A fluoroimmunoassay has been developed for sensitive limit of 90 nM. detection of human immunoglobulin G (IgG) in serum or Biological thiols (e.g., cysteine – Cys, homocysteine – Hcy cell culture media with a detection limit of 10 ng mL−1.[110] and glutathione – GSH) play key roles in biological processes Anti-IgG functionalized GQDs are first quenched by modand are implicated in various pathological conditions such as erately-reduced graphene oxide (rGO) sheets because of cancer, AIDS, cardiovascular diseases, and pregnancy com- luminescence resonance energy transfer (LRET). Specific plications. A novel, rapid and label-free assay was developed interaction between IgG and antigen increases the distance



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Figure 8. (a) Schematic representation of the CK2 kinase assay based on the aggregation and PL quenching of phosphorylated peptide-GQD conjugates via Zr4+ ion linkages. (b) PL quenching of peptide-GQDs treated with various amounts of CK2 followed by the addition of 0.2 mM Zr4+. The concentrations of CK2 were 0, 0.05, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, and 6.0 units mL−1 for curves 1 to 12, respectively. The concentration of ATP was 50 µM. The inset shows the PL quenching of peptide-GQD solutions illuminated with a UV lamp at the homologous CK2 concentrations of (left to right) 0, 0.1, 0.4, 0.8, 1.0, 2.0, and 6.0 units mL−1. (c) Plot of the relative PL intensity [(I0 − I)/I0] at 445 nm vs CK2 concentration. I and I0 are the PL intensities in the presence and absence of CK2, respectively. Reproduced with permission.[112] Copyright 2013, American Chemical Society.

between rGO and GQD, hence alleviates LRET-induced quenching. This immunosensing scheme is universal and can be modified to specifically detect other antigens. Similar PL turn-on strategy has been used to detect target-DNA strands which relieve the LRET between probe-DNA functionalized GQDs and GO sheets (detection limit of 75.0 pM).[111] A novel PL sensing strategy for profiling kinase activity was designed by Wang et al.[112] When the substrate peptides attached on GQDs are phosphorylated by casein kinase II (CK2), extensive aggregation of the GQDs and effective PL quenching occur due to coordination interaction between Zr4+ ions and phosphorylated sites of phosphopeptides (Figure 8a-b). The PL quenching of peptide-GQD conjugates is linearly dependent on CK2 concentration in the range from 0.1 to 1.0 unit mL−1 with a detection limit of 0.03 unit mL−1 (Figure 8b-c). The proposed method may also be used for kinase inhibitor screening in human serum. In comparison to existing fluorescent kinase assays, this method has advantages such as simplicity, low cost, high sensitivity and selectivity. 4.2.2. Electrochemiluminescence (ECL) Sensors ECL is a unique and sensitive analytical method. As the light emitting species are generated in situ close to electrode small 2015, 11, No. 14, 1620–1636

surface, ECL has nearly zero background and allows temporal and spatial control over the reaction. The intense and stable ECL of GQDs has been utilized for ultrasensitive detection of ATP (as low as 1.5 pM).[30] ATP molecules enhance ECL signal in dose-dependent manner because they facilitate the attachment of complementary ssDNA-functionalized GQDs onto probe ssDNA functionalized electrode. A DNA sensor with a detection limit of 13 nM has also been devised.[29] Specifically, ECL signal from GQDs on electrode is first quenched by probe-ssDNA conjugated gold nanoparticles (AuNPs) due to ECL resonance energy transfer; and detection is then reported by ECL recovery upon release of AuNPs by hybridization with target ssDNAs. Ratiometric ECL measurement on C-dots (one peak depends on target concentration; the other serves as stable internal reference) has been used to detect Fe3+ ions with good sensitivity (0.7 µM) and reproducibility.[28] 4.2.3. Electrochemical Sensors Large specific surface area, abundant edge sites, and intrinsic catalytic activities make GQDs advantageous for electrochemical sensing. In addition, owing to their small sizes, GQDs may be able to access the catalytic centers of enzymes and facilitate electron transfer. As an example, a glucose



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biosensor with good stability and sensitivity (1.73 µM) has been developed using enzyme (glucose oxidase) and GQD modified electrode.[113] Zhao et al. designed an electrochemical sensor for DNA or protein detection and demonstrated that GQDs can greatly enhance the sensitivity by promoting electron transfer to electrode.[114] The intrinsic catalytic activity of GQDs towards H2O2 has led to the development of H2O2 sensor wherein GQDs are covalently modified onto Au electrode.[33a] The sensor is highly stable with a detection limit of 0.7 µM. The as-prepared electrode has also been used to monitor the dynamic H2O2 release from human breast adenocarcinoma MCF-7 cells. Shao et al. used C-dots (∼6 nm) to improve the detection limit (to ∼100 nM) of electrochemical detection of cerebral Cu2+ in rat brain.[115] 4.2.4. Colorimetric Detection GQDs possess intrinsic peroxidase-like catalytic activity towards H2O2 (an important bio-signaling molecule and byproduct of many biological processes), probably attributable to sp2 carbon nanodomains on the basal plane and carboxyl groups at the edges. Using GQDs as peroxidase and ABTS as the redox mediator and color reporter, colorimetric detection of H2O2 has been realized with fast detection time (2 min), low detection limit (20 µM) and large linear range (0.1 to 10 µM).[14] More sensitive colorimetric detection of H2O2 (33 nM) has been demonstrated using AgNPs as the color reporter.[116] The absorbance peak of AgNPs decreases because of size reduction while AgNPs are oxidized by H2O2 with GQDs as the catalysts. Combining such GQDs/AgNPs system with glucose oxidase, which specifically catalyzes the oxidation of glucose in the presence of oxygen to form H2O2, a colorimetric sensor for glucose is obtained (detection limit of 170 nM).

4.3. Drug/Gene Delivery Micrometer-sized graphene sheets are proven to possess excellent drug / gene loading capability due to their large specific surface area and stable interaction with various molecules through π-π stacking, hydrophobic interaction, electrostatic attraction, or physisorption. While inherited with these merits, GQDs are smaller to allow readily cell uptake and more biocompatible to minimize cytotoxic effects. Both GQDs and C-dots are expected to be safe, effective, and visible delivery vectors. As a pioneering work, PEGylated nanographene oxide (NGO-PEG, 5–50 nm) was applied by Dai group to deliver insoluble aromatic drug SN38.[117] The soluble NGO-PEGSN38 complexes are highly potent in cancer cell killing (1000 fold more potent than a FDA approved SN38 prodrug for clinical colon cancer treatment). They also demonstrated the efficient physisorption of doxorubicin onto anti-CD20 conjugated NGO-PEG via π−π stacking for targeted killing of Raji B-cell lymphoma.[118] As expected, GQDs passivated with PEG (∼15 nm) has been shown to possess good drug loading capacity (2.5 mg/ mg of anticancer drug doxorubicin at pH 7.4).[72b] To achieve targeting specificity, hyaluronic acid (HA) moieties were


anchored to GQDs for targeted delivery to CD44-overexpressed tumors.[41] HA-GQDs are able to load ∼75% of doxorubicin (DOX) initially applied, indicating them as excellent agents for loading hydrophobic drug molecules via π−π interaction. The release of DOX quickly reached 42% within the first 6 h, and almost complete release was achieved within 48 h at pH 5.5. In contrast, drug release at pH 7.4 was much slower with only 20% release after 48 h. This pH dependent release behavior is advantageous since tumor tissues commonly have a slightly acidic environment. Moreover, the specific targeting on tumor tissues and cancer cells was confirmed by in vivo and in vitro imaging HA-GQDs in balb/c female mice and A549 cells. Wang et al. showed that GQDs (∼30 nm) can efficiently accelerate DOX accumulation to the nucleus and markedly enhance the DNA cleavage activity of DOX, leading to a dramatic improvement in the cytotoxicity of DOX.[119] Notably, DOX/GQD conjugates are effective to the drug-resistant MCF-7 cancer cells. Lee et al. demonstrated DOX delivery in vitro and in vivo using blue luminescent C-dots.[120] The positively charged DOX was loaded onto negatively charged C-dots via electrostatic interactions with 95% loading efficiency. The DOXloaded C-dots (∼14.9 nm) induced death of HepG2 and MCF-7 cancer cells as well as tumor in mice, indicating enhanced anticancer efficacy as compared to free DOX injection. The cell uptake and intracellular distribution of bare C-dots and DOX loaded C-dots were visualized by two-photon confocal imaging. It was observed that free C-dots preferably labeled the nucleus whereas DOX-loaded C-dots were mainly distributed in the cytoplasm. Wang et al. used hollow C-dots for the delivery of DOX.[5b] DOX was loaded via π−π interaction with a loading ratio of 6 wt%. The intracellular distribution and release was monitored by confocal microscopy. It was observed that the C-dots and DOX complexes were first transported into the lysosomes where the drug was released due to low pH and subsequently localized to the nucleus.[5b] Other than drug delivery, gene delivery has been successfully demonstrated with PEI-functionalized C-dots (CD-PEI) by Liu et al.[73] The positively charged CD-PEI is able to condense DNA molecules and transfect COS-7 and HepG2 cells with higher efficiency and lower cytotoxicity as compared with the polymer control (PEI25k). The DNA transfection was lightened by C-dots under confocal. Kim et al. demonstrated real time monitoring of gene delivery by combining C-dots with metal nanoparticles.[121] Blue-emitting CD-PEI and PEI functionalized Au-nanoparticles (Au-PEI) were first complexed with TOTO-iodide (red emission) labeled plasmid DNA (pDNA) via electrostatic interaction, leading to initial fluorescence quenching. The complex of CD-PEI/pDNA/Au-PEI was up-taken into the cytosol as shown by co-localization of pDNA signal and moderately quenched CD-PEI signal. Gene dissociation induced by low-pH inside cancer cells was evidenced by the recovery of CD-PEI blue fluorescence localized in the cytosol and pDNA fluorescence localized in the nucleus. GQDs have also been explored as fluorescent tracers in drug delivery. Jing et al. developed a multifunctional core-shell capsule with GQDs encapsulated as fluorescent labels.[122] This system was composed of olive oil for drug loading, a TiO2 shell to suppress the initial burst release of


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paclitaxel, and Fe3O4/GQDs inside the oil core for magnetic targeting and fluorescence imaging, respectively. GQDs act as fluorescent tracers for capsule targeting and indicators for drug release upon breaking of the capsules, whereby providing important optical information for in situ monitoring of drug release process. Nigam et al. similarly used GQDs as fluorescent reporters in drug delivery.[123] They conjugated GQDs to hyaluronic acid-functionalized human serum albumin (HSA) for simultaneous bioimaging and targeted delivery of gemcitabine to pancreatic cancer. Chen et al. coated GQDs onto drug-loaded mesoporous silica nanoparticles for DOX delivery to human lung carcinoma cells.[124] GQDs serve as both capping agents to allow pH-triggered drug release and imaging agents to monitor the release. Wang et al. used GQDs as both drug carrier and imaging agent to monitor drug release.[125] They conjugated folic acid to GQDs (GQD-FA) for targeted delivery of DOX to cancer cells (Hela), wherein the red fluorescence of DOX was quenched by GQDs upon loading. GQD-FA-DOX nanoassembly was distributed in the cytoplasm initially as indicated by green GQDs. Subsequent release of DOX was indicated by its recovered fluorescence observed in both cytosol and nucleus.

4.4. Anticancer Agents A novel in vivo theranostic platform based on photosensitizer (Ce6) conjugated C-dots is designed for NIR fluorescence imaging guided photodynamic treatment for gastric cancer.[126] The synergy between Ce6 and C-dots is attributed to the enhanced photodynamic effects. An electrochemically prepared GQD has been found to generate reactive oxygen species (ROS) such as singlet oxygen (1O2) under the irradiation of blue light, likely attributable to the defects on GQD surface.[127] The increased cellular ROS level caused by uptaken GQDs led to severe oxidative stress, and consequently apoptosis and autophagy in human glioma U251 cells. This phenomenon could be exploited for photodynamic therapy. Very recently, Ge et al. synthesized red luminescent GQDs that outperformed other photodynamic therapy agents due to their high 1O2 quantum yield (1.3).[128] The in-vivo application of these GQDs was demonstrated in mice, wherein the tumors were destroyed within 17 days of treatment. Zhou et al. discovered that ∼90% supercoiled DNA was converted into nicked DNA using GQDs and Cu2+.[129] They attributed the high efficiency to the ability of GQDs to intercalate with DNA. Zheng et al. carried out further systematic investigations on this cleavage mechanism.[130] They proposed that electron rich GQDs transfer electrons to the metal complex of Cu2+, which in turn produces reactive oxygen species upon reduction. The produced ROS are responsible for oxidative DNA cleavage. Such GQD assisted DNA cleavage promises applications in anti-cancer therapeutics.

4.5. Antibacterial and Antioxidant Activity Mycoplasma is a common contamination source in cell culture and clinical samples, and causes pneumonia and other small 2015, 11, No. 14, 1620–1636

respiratory disorders. Jiang et al. found that amine-functionalized GQDs were able to rectify the inhibition of M. urealyticum on HeLa cells proliferation.[131] The cytoprotection mechanism may be related to the peroxidase-like activity of amine-GQDs. It is worth noting that a small dose of GQDs (10 µgmL−1) is as effective as the commercial mycoplasma removal agent. Taking advantages of both peroxidase-like activity and biocompatibility of GQDs, Sun et al. applied GQDs and low dose of H2O2 to band-aids for wound disinfection on mice.[33b] The GQDs convert H2O2 with low antibacterial activity into ·OH radicals with high antibacterial activity, and avoid adverse accumulation of H2O2. This system is effective to a broad spectrum of both gram-negative and gram-positive bacteria. The photodynamic effect of GQDs has also been used to kill methicillin resistant Staphylococcus aureus and Escherichia coli.[132] As the consequence of mild traumatic brain injury, increase of ROS level prevents the autoregulation of dilation in brain vasculature resulting in hypotension. A study on a rat model found that PEG-functionalized hydrophilic C-dots (30–40 nm long, 2–3 nm wide, with both graphitic and oxidized domains) could be a solution to this problem owing to their antioxidant activity.[133] It is proposed that ROS can be annihilated at the graphitic domains of C-dot. We speculate that GQDs, which also have sp2 carbon nanodomains may also exhibit such antioxidant properties.

5. Outlook In spite of various proven and anticipated advantages over conventional fluorophores, the full potential of GQDs and C-dots has yet to be exploited because of a number of current problems. Firstly, the emission spectrum of these carbon nanodots is usually too wide due to the large heterogeneity of the synthesized products. And most of the current nanodots are either green or blue. Excitation and emission at long wavelengths are particularly desired for deep tissue imaging. Moreover, most current nanodots are of low quantum yield and produced by low-yield methods. With a better understanding of their tunable photoluminescence properties, controllable and scalable synthesis or surface engineering methods will be developed to produce GQDs or C-dots with well-defined properties tailored towards specific applications. Although the research is still at the early stage, GQDs and C-dots have already demonstrated enormous potential in bioimaging, optical sensing, drug/gene delivery, and theranostics. As the family of carbon nanodots is rapidly growing, their application scope will greatly expand beyond what is covered in this article. For example, nanoelectronic biosensors may be devised using GQDs.[134] Decorated with magnetic nanoparticles, these carbon nanodots could be used for magnetic-directed drug delivery or magnetic resonance imaging. And various novel applications devised upon other carbon nanomaterials (e.g., micro-sized graphene sheets, carbon nanotubes) may be further improved by GQDs or C-dots. We envision that these carbon nanodots may revolutionarily change the landscape of biomedical research. Although we focus here on the biological applications, GQDs



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and C-dots are also promising candidates for novel applications in other areas including energy storage and conversion, photocatalysis, and display technologies. Undoubtedly, they are the next big small things.

Acknowledgements X.T. Zheng and A. Ananthanarayanan contribute equally to this work. This work was supported by the Singapore Ministry of Education under the AcRF Tier 2 grants (MOE2011-T2–2–010, MOE2014T2–1–003), and National Research Foundation-Environment and Water Industry Development Council-Incentive for Research and Innovation Scheme (NRF-EWI-IRIS, 1102-IRIS-05–02).

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Received: September 3, 2014 Revised: October 12, 2014 Published online: December 17, 2014

small 2015, 11, No. 14, 1620–1636

Graphene quantum dots, graphene oxide, carbon quantum dots and graphite nanocrystals in coals.

Carbon quantum dots and their applications.

Carbon quantum dots and applications in photocatalytic energy conversion.

Surface functionalization of quantum dots for biological applications.

Pharmaceutical and biomedical applications of quantum dots.

Photodynamic antibacterial effect of graphene quantum dots.

Photoresponse of polyaniline-functionalized graphene quantum dots.

Mind your P's and Q's: the coming of age of semiconducting polymer dots and semiconductor quantum dots in biological applications.

Fluorescent carbon nanomaterials: "quantum dots" or nanoclusters?

Graphene nanosheets and quantum dots: a smart material for electrochemical applications.

Tailoring 10 nm scale suspended graphene junctions and quantum dots.

On the upconversion fluorescence in carbon nanodots and graphene quantum dots.

The permeability and transport mechanism of graphene quantum dots (GQDs) across the biological barrier.

Surface engineering of graphene quantum dots and their applications as efficient surfactants.

CdS Quantum Dots and Their Applications as Fluorescent Biological Labels.

Tunable amphiphilicity and multifunctional applications of ionic-liquid-modified carbon quantum dots.

Synthesis and photochemical applications of processable polymers enclosing photoluminescent carbon quantum dots.

Quantum dots in imaging, drug delivery and sensor applications.

A time-dependent DFT study of the absorption and fluorescence properties of graphene quantum dots.

Correction: Fluorescent carbon dots from mono- and polysaccharides: synthesis, properties and applications.

Preparation of carbon quantum dots based on starch and their spectral properties.

Reduced Graphene Oxide Supercapacitors.

Fluorescent carbon dots from mono- and polysaccharides: synthesis, properties and applications.

Luminescent graphene quantum dots fabricated by pulsed laser synthesis.