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WIREs Nanomed Nanobiotechnol
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Fluorescent carbon dots as intracellular imaging probes

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Abstract Fluorescent carbon dot has emerged as promising alternative of conventionally known quantum dot or molecular probe as potential intracellular imaging probe. In particular, <10 nm size, tunable and bright fluorescence of carbon dot deserve the application potential as intracellular imaging probe. However, synthesis of carbon dot with narrow particle size distribution, preparation of high‐quality red/near‐infrared emitting carbon dot and appropriate design of functional carbon dot for subcellular targeting are most critical issues. This advanced review focus on the application potential of fluorescent carbon dot as intracellular imaging probe. At first, we briefly discuss different types of fluorescent carbon dots and origin of their fluorescence. Next, we focus on surface chemistry and functionalization which are relevant to intracellular probe development. Finally we have summarized various types of intracellular nanoprobes that are developed from fluorescence carbon dot. This article is categorized under: Diagnostic Tools > in vitro Nanoparticle‐Based Sensing
Carbon dot‐based imaging of intracellular ions (Fe3+, Ca2+, Cu2+, and superoxide ions). (i) Fluorescence microscopy imaging of intracellular Fe+3 (A, B) with respect to corresponding bright‐field image (C, D) of HeLa cells. (Reprinted with permission from Liu, Y., Duan, W., et al. (2017). Copyright © 2017, American Chemical Society. (ii) (A) Real‐time fluorescence imaging of intracellular cytosolic Ca2+ under histamine stimulation in T24 cells. The scale bars are 15 μm. (B) Real‐time fluorescence intensity under histamine stimulation. (Reprinted with permission from Chen et al. (). Copyright © 2018, American Chemical Society. (iii) Fluorescence imaging of intracellular Cu+2 in HeLa cells using CdSe‐carbon dot‐based nanoprobe. Panel (a) is the overlay of bright‐field and fluorescence image of cells under control condition and panels (b) and (c) are the fluorescence images of cells before (b) and after (c) the exogenous Cu source treatment. A bar graph on amount of Cu+2 is shown in panel (d). Scale bar: 25 μm. (Reprinted with permission from Zhu et al. (). Copyright © 2012, John Wiley and Sons). (iv) (a) Pseudocolored ratiometric imaging of superoxide in HeLa cells using carbon‐based nanoprobe. Fluorescence (a), bright‐field (b), overlay (c) image of cells along with the intracellular fluorescence scan (d, I–IV) at different time interval of measuring superoxide. (Reprinted with permission from Gao et al. (). Copyright © 2014, American Chemical Society)
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Schematic representation of fluorescent carbon dot synthesis, surface chemistry, and functionalization. Chemical structures of important precursors are shown in top box. Functionalization approach for hydrophilic carbon dot via conjugation chemistry is shown in middle left of the scheme and functionalization approach for hydrophobic carbon dot via coating chemistry and conjugation chemistry is shown in middle right of the scheme. Chemical structures of selected biomolecules are shown in lower box that are conjugated with carbon dots. The highlighted functional groups (red circle) are used for conjugation chemistry
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Origin of fluorescence from carbon‐based different nanomaterials. (a) Schematic representation of the energy levels associated with size‐dependent fluorescence from the graphene‐based quantum dots. The nonbonding (n) energy states remain undisturbed by the change in the particle size, while the energy levels of the π, π*, and σ* orbitals changes with the size. The relaxation of electrons from σ* to the π* is essential for short‐wavelength excitation‐induced fluorescence. The fluorescence color varies from orange‐red (for QD79) to blue (for QD10). (Reprinted with permission from Yeh et al. (). Copyright © 2016, American Chemical Society. (b) Schematic representation of excitation‐dependent and excitation‐independent fluorescence from carbon dot via surface engineering. (Reprinted with permission from Li, Zhang, Kulinich, Liu, and Zeng (). Copyright © 2014, Springer Nature. (c) Schematic illustration of the excitation‐independent fluorescence mechanism of nanodiamonds. (Reprinted with permission from Xiao, Liu, Li, and Yang (). Copyright © 2015, American Chemical Society. (d) Schematic representation of excitation‐dependent fluorescence in nanodiamond. The OH, ketone C═O, and ester C═O groups are denoted by blue, green, and yellow emissions, respectively. The essence of the excitation‐dependent fluorescence lies in the relative intensity of these three groups and lowest unoccupied molecular orbital (LUMO) of the blue band changes more than those of the green and yellow bands. (Reprinted with permission from Xiao et al. (). Copyright © 2015, American Chemical Society
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Representative structures of different carbon nanomaterials. Transmission electron microscopy (TEM) image of fluorescent carbon nanoparticle, schematic structure of fluorescent nanodiamond, chemical structure of graphene‐based fluorescent carbon dot, schematic structure of fullerene, schematic structure of single‐walled carbon nanotube (SWCNT), and TEM image of graphene
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Carbon dot‐based imaging of intracellular organelles. (i) Carbon dot‐4‐(2‐aminoethyl) morpholine‐based lysosome imaging in HeLa cells (Reprinted with permission from Wu et al., ). Copyright © 2017, American Chemical Society. (ii) Malic acid‐derided carbon dot for mitochondria imaging in epithelial cells (Reprinted with permission from Zhi et al., ). Copyright © 2018, American Chemical Society. (iii) Folic acid and m‐phenylenediamine‐derived carbon dot for nucleus imaging in HeLa cells (Reprinted with permission from Liu et al., ). Copyright © 2019, American Chemical Society. (iv) Citric acid and urea‐derived carbon dot for nucleolus imaging in HeLa cells (Reprinted with permission from Khan et al., ). Copyright © 2018, American Chemical Society
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Carbon dot‐based imaging for intracellular molecules. (i) o‐Phenylenediamine‐derived carbon dot for imaging of lysine in HeLa cells under red and blue channels. Scale bar is 50 μm. (Reprinted with permission from Song, W., Duan, W., et al. (2017). Copyright © 2017, American Chemical Society. (ii) Fluorescence imaging of histidine in HeLa cells. (A) Stained with nanoprobe, (B) stained with nanoprobe and Cu (II) ions, and (C) stained with nanoprobe‐Cu (II) ions and histidine. Scale bar: 20 μm. (Reprinted with permission from Zhu et al. (). Copyright © 2016, the Royal Society of Chemistry. (iii) Fluorescence imaging of cysteine in A549 cells incubated with nanoprobe. Cells are incubated with N‐methylmaleimide (a) or cysteine first and then with nanoprobe (c) or incubated with the nanoprobe and cysteine together (b). (Reprinted with permission from Tang et al. (). Copyright © 2017, American Chemical Society. (iv) Confocal laser scanning microscopy (CLSM) image of HeLa cells pretreated with glutathione (GSH) scavenger (N‐ethylmaleimide: NEM) or GSH enhancer (alpha lipoic acid: ALA) along with MnO2–carbon quantum dot (CQD) nanocomposites. The top row shows fluorescence microscopy images; the bottom row shows the overlap of fluorescence and bright‐field images. The scale bar is 20 μm. (Reprinted with permission from He et al. (). Copyright © 2015 the Royal Society of Chemistry)
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Carbon dot‐based imaging of intracellular pH. Fluorescence imaging of intracellular pH in HeLa cells using carbon dot‐FITC/rhodamine B isothiocyanate (RBITC). The images of the first row (FITC channel) and second row (RBITC channel) are collected in the ranges of 510–550 nm and 570–610 nm, respectively. The third row shows the corresponding differential interference contrast images. The fourth row corresponds to the ratiometric image. The bottom color strip represents the pseudocolor change with pH. Scale bar, 20 μm. (Reprinted with permission from Shi et al. (). Copyright © 2012, John Wiley and Sons)
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Diagnostic Tools > Biosensing
Diagnostic Tools > In Vitro Nanoparticle-Based Sensing

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