Hydrogen-Bond Induced Emission of Carbon Dots for Wash-Free

Jun 16, 2019 - Carbon dots (CDs) are emerging as powerful tools for biosensing and bioimaging because of their intrinsic properties such as abundant ...
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Article Cite This: Anal. Chem. 2019, 91, 9259−9265

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Hydrogen-Bond-Induced Emission of Carbon Dots for Wash-Free Nucleus Imaging Haifang Liu,† Jie Yang,† Zhaohui Li,*,† Lehui Xiao,‡ Aaron Albert Aryee,† Yuanqiang Sun,*,† Ran Yang,† Hongmin Meng,† Lingbo Qu,*,† Yuehe Lin,§ and Xiaobing Zhang∥

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†

Institute of Chemical Biology and Clinical Application at the First Affiliated Hospital, Henan Joint International Research Laboratory of Green Construction of Functional Molecules and Their Bioanalytical Applications, College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, People’s Republic of China ‡ College of Chemistry, Nankai University, Tianjin 300071, People’s Republic of China § School of Mechanical and Materials Engineering, Washington State University, Pullman, Washington 99164, United States ∥ College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, People’s Republic of China S Supporting Information *

ABSTRACT: Carbon dots (CDs) are emerging as powerful tools for biosensing and bioimaging because of their intrinsic properties such as abundant precursors, facile synthesis, high biocompatibility, low cost, and particularly robust tunability and stability. In this work, a new type of CDs was prepared from mphenylenediamine and folic acid by hydrothermal method. Interestingly, the asprepared CDs show blue emission in non-hydrogen-bonding solution, whereas robust green emission in hydrogen-bonding solution. Based on this phenomenon, a novel fluorescence sensing mechanism named as hydrogen-bonding-induced emission (HBIE) was proposed. The HBIE-CDs have large Stokes shift (141 nm) in water, good biocompatibility, and ultrasmall size, which facilitates their translocation into living cells. Very importantly, the as-prepared HBIE-CDs show strong affinity toward nucleic acid without interference from other biological species. After binding with DNA/RNA through hydrogen bond, as high as 6-fold green fluorescence enhancement of HBIE-CDs was observed. Since the nucleus is rich in DNA/RNA, these HBIE-CDs were successfully used for rapid and, especially, wash-free subcellular in situ imaging of the nucleus in living cells in a fluorescence turn on mode, which has a great practicability to be used for nucleus imaging in bioanalytical studies and clinical applications.

A

for the selective and sensitive imaging of a nucleus still remains a big challenge. The hydrogen-bonding (H-bonding) network plays important roles in numerous biological fluorophores. The most wellknown example is green fluorescent proteins which have become a powerful imaging tool in recent years. The chromophore in natural green fluorescent protein is embedded in the protein pocket and stabilized by a hydrogen-bond network, which minimizes the nonirradiation transfer and results in high fluorescence quantum yields.21 Bilirubin is a yellow color metabolite of heme in our body, which shows very weak fluorescence due to its flexible molecular structure. Recently, Miyawaki et al. reported a bilirubin inducible fluorescent protein UnaG from Japanese eel in which the nonirradiation transfer of endogenous ligand, bilirubin, is restricted via many hydrogen bonds.22 Inspired by this, we proposed that the nonirradiation of CDs could be inhibited by H-bonding. Since hydrogen bonds play very important roles in

s one of the most sophisticated organelles, the cell nucleus contains most of the genetic materials and plays a vital role in numerous cellular events, including DNA replication, ribosome and mRNA synthesis, and gene expression.1 Imaging of the cell nucleus has been widely used to study cell growth, DNA quantification, drug−DNA interactions, and so on. Although many organic fluorescent nucleus probes have been reported and commercialized (such as DAPI, Hoechst, and Syto dyes), challenges still exist to visulize a live cell nucleus with high temporal and spatial resolution because of the high cost, small Stokes shift, poor photostability, and low water solubility.2 Due to the intrinsic properties such as abundance precursors, facility synthesis, high biocompatibility, low cost, tunability, and stability, carbon dots (CDs) have attracted intensive attention and been widely applied in chemo-/ biosensors, bioimaging, drug delivery, white-light-emitting diodes, catalysis, and optoelectronic devices3−14 However, it is difficult to specifically label the nucleus by pristine CDs, which generally require additional surface modifications with nucleus-targeted peptides or proteins.15−20 CDs-based probes © 2019 American Chemical Society

Received: May 7, 2019 Accepted: June 17, 2019 Published: June 16, 2019 9259

DOI: 10.1021/acs.analchem.9b02147 Anal. Chem. 2019, 91, 9259−9265

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rpm for 10 min and the precipitate was discarded; then the solution was purified using silica column chromatography. The as-prepared HBIE-CDs can be collected and redispersed into deionized water with a concentration of 5 mg/mL. Cellular Imaging. Cells were seeded on confocal dishes and cultured at 37 °C with 5% CO2 in RPMI 1640 medium (including 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin). Cells were incubated for 36 h and approximately 70% confluence. Then, the medium with HBIE-CDs (15 μg/mL) was added. The confocal dish was incubated for 5 min before imaging. The cells were washed with 0.1 M PBS (pH = 7.4) for three times. Finally, the cell imaging was taken on a TCS-SP8 confocal laser scanning microscope (Leica, Germany). DNase and RNase Digest Test. Cultured cells grown in a glass bottom dish were stained with 15 μg/mL HBIE-CDs for 5 min. Then three sets of pretreated HeLa cells were fixed by precooled methanol (−20 °C) for 15 min, washed with PBS for three times. The cells were treated with 30 μg/mL DNase, or 25 μg/mL RNase, and incubated at 37 °C for 2 h. After then, the cells were washed three times by utilizing PBS and then used for imaging. Cells Counterstain Experiment. HeLa cells were stained with 2 drops of NucRed Live 647 for 10 min. After being washed with PBS, the same cells were incubated with 15 μg/ mL HBIE-CDs for 5 min and then imaged.

biological compounds, such as DNA, RNA, protein, and cellulose. etc., it is expected that this unique H-bondinginduced emission strategy can be employed in the design of CDs-based probes for the detection of such issues. In this work, we reported the rational design of DNA and RNA sensitive CDs based on the H-bonding network. The asprepared CDs show strong blue emission in non-H-bonding solution and robust green emission in H-bonding solution. This novel phenomenon was named as hydrogen-bondinginduced emission (HBIE). After binding with DNA and RNA, a 6-fold fluorescence enhancement was observed. The HBIECDs are suitable for label-free, in situ, and wash-free subcellular labeling and imaging of a nucleus in living cells. While most reported nucleus probes have strong fluorescence signals in water and culture medium, the unbound probes must be washed off to get high signal-to-noise ratio. The HBIE-CDs described here permit specific labeling without washing and are, therefore, able to continuously monitor the entire biochemistry process in the nucleus without interruption. The results presented here are expected to generate research enthusiasm for creating functional HBIE-active luminescent nanoprobes, thereby creating a bright future for nanomaterials in bioanalysis applications.

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EXPERIMENTAL SECTION Chemicals and Materials. Folic acid (FA), lysine (Lys), arginine (Arg), cysteine (Cys), tyrosine (Tyr), asparagine (Asn), tryptophan (Trp), histidine (His), threonine (Thr), glutamine (Gln), serine (Ser), leucine (Leu), alanine (Ala), isoleucine (Ile), proline (Pro), methionine (Met), glutamic acid (Glu), aspartic acid (Asp), phenylalanine (Phe), valine (Val), glycine (Gly), and glutathione (GSH) were purchased from Sigma-Aldrich (MO, United States). Bovine serum albumin (BSA), human serum albumin (HSA), histone, dsDNA (CAA TCG GAT CGA ATT CGA TCC GAT TG), ssDNA (AGG GTT GGG CGG GAT GGG AAA CCC ATC CCA GAA GAA AAC TTC), RNA (UGG CUC AGU UCA GCA GGA ACA G), and G4 (GGG TTA GGG TTA GGG TTA GGG) were purchased from Sangon Biotech (Shanghai, China). m-Phenylenediamine (m-PD), K+, Na+, Mg2+, Zn2+, Fe3+, Fe2+, Ca2+, Al3+, Ba2+, Mn2+, Co2+, and Ni2+ were bought from Aladdin Reagent Co. Ltd. (Shanghai, China). RNase and DNase were bought from Beyotime (Jiangsu, China). NucRed Live 647 was purchased from Thermo Scientific (Pittsburgh, PA, USA). Apparatus and Characterization. UV−vis absorption measurements were performed on an Agilent 8453 spectrophotometer (Agilent, USA). Photoluminescence spectra were taken on an F-4600 spectrometer (Hitachi, Japan). Measurements of FL quantum yields of CDs were carried out with a FLS 980 fluorometer (Edinburgh Instrument, Britain). Fourier transform infrared (FTIR) spectra were obtained using a Bruker Tensor 27 spectrometer (Bruker, Germany). Transmission electron microscope images were obtained on a TEM (FEI-Tecnai G2, USA). X-ray photoelectron spectroscopy (XPS) spectra were obtained on an ESCALAB 250 photoelectron spectrometer (Thermo Fisher Scientific, USA). Preparation of HBIE-CDs. The HBIE-CDs were prepared by a hydrothermal method using FA and m-phenylenediamine as precursor. FA (0.0050 g) and m-PD (0.0130 g) were dissolved in 5 mL of deionized water. Followed with sonicating for 10 min, the solution was then heated for 12 h at 200 °C. When naturally cooled, the sample was centrifuged at 10,000

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RESULTS AND DISCUSSION Design Rationale. Folic acid (FA), which is also known as vitamin B9, plays an essential role in a one-carbon metabolism and methionine cycle.23 FA contains versatile functional groups, such as -OH, -NH2, -COOH, and pterin, which can serve as both H-bonding donor and acceptor. Recent works reported that FA can bind nucleobases at multiple sites through H-bonding, hydrophilic, and hydrophobic contacts. The FA binding does not break the DNA duplex and does not alternate DNA and RNA conformations. Molecular modeling indicated that the complexes between FA and DNA or RNA are stabilized by the H-bonding network.24 Therefore, FA was selected as the precursor for the preparation of nucleobase− target CDs. By using FA as the sole precursor, we synthesized high emissive CDs. However, both the absorbance and emission of this CDs are in the UV region, which is basically not suitable for bioimaging.25 Since the first m-PD-based CDs reported in 2014,26 m-PD has been widely used in preparing CDs. On the basis of this result, m-PD and FA were chosen as the precursors. The amino group in m-PD can react with the carboxylic group and pterin in FA to form extended πconjugations, which shift the absorption and emission of the CDs to the visible range. Moreover, FA can act as H-bonding donor and acceptor; thus it is expected that the resulted CDs can inherit the H-bonding donor and acceptor ability and, most importantly, the binding ability toward RNA and DNA. The nonirradiation process of CDs would be restricted when they bind to the grooves of DNA or RNA and thereby result in the restoration of the CDs’ fluorescence, which could serve as the basic sensing mechanism for RNA and DNA. Synthesis and Characterizations of HBIE-CDs. The HBIE-CDs were prepared by folic acid (FA) and mphenylenediamine through hydrothermal method and purified by silica column chromatography (Scheme 1). The as-prepared HBIE-CDs were characterized with TEM images, FTIR, and XPS. As shown in Figure 1a, the morphology and size 9260

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3390 cm−1 in the FTIR spectrum (Figure 1b) is attributed to C−OH stretching and -OH bending vibration. The absorptions at 1250 and 1310 cm−1 are attributed to C−N and CN of the pterin ring. The absorption peaks at 1731 and 1425 cm−1 can be assigned to the CO stretching vibrations and C−O bending vibration, respectively. The peaks at 1648 and 1533 cm−1 are related to the bending vibration of N−H. In the XPS broad spectrum of HBIE-CDs, three peaks at 284.1, 398.7, and 531.7 eV were attributed to C 1s, N 1s, and O 1s (Figure 1c). Figure 1d shows the high-resolution XPS spectra of N 1s. The N 1s spectrum was deconvolved into two peaks at 398.5 and 399.5 eV, corresponding to pyridine-like N and pterin-like N, respectively. These results demonstrated that the N atoms were doped as the form of pyridine-like N in the HBIE-CDs. The results fit well with the FTIR data and demonstrate that the functional groups, including -COOH, -OH, and -NH2 exist in the HBIE-CDs’ surface. All of the results confirmed the formation of HBIE-CDs. The HBIE-CDs show a green color under a hand-held UV lamp, as shown in Figure S2. Figure 2a shows the optical

Scheme 1. (a) Schematic Illustration of the Synthesis of HBIE-CDs and Their Bioanalysis Application for Wash-Free Cell Nucleus Imaging. (b) Synthesis Route of HBIE-CDs from Folic Acid and m-Phenylenediamine

distribution of HBIE-CDs illustrated their uniform dispersity. The particle diameters mainly focused on 1.6−4 nm with an average size of 2.6 ± 0.1 nm. As shown in Supporting Information Figure S1, results from XRD reveal one diffraction peak of HBIE-CDs at 23.66°, which corresponds to the (002) graphite lattice of HBIE-CDs. The broad absorption peak at

Figure 2. (a) UV/vis absorption (black line), PL excitation (green line), and emission (red line) spectra of HBIE-CDs in aqueous solution. (b) Excitation-independent PL of HBIE-CDs, starting from 310 to 470 nm with a 20 nm increment. (c) Fluorescence lifetime decay spectrum of HBIE-CDs: decay times, τ1 = 3.75 ns and τ2 = 12.00 ns; average lifetime, τ = 10.60 ns.

Figure 1. (a) TEM image and statistic analyzed size distribution of HBIE-CDs based on statistics of the TEM image. (b) FTIR spectra of HBIECDs. (c) XPS spectra of HBIE-CDs. (d) High-resolution XPS spectra of N 1s of HBIE-CDs. 9261

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Figure 3. (a) Absorption and (b) PL spectra of HBIE-CDs (0.1 mg/mL) in different solvents: λex = 405 nm. (c) Photographs of HBIE-CDs in different solvents taken under irradiation of 365 nm UV light.

response of HBIE-CDs in water with λex/λem = 394/535 nm, thus making them far better suited for use with a commercial confocal microscope (405 nm laser). The UV−vis absorption spectrum characterized, at 390 nm, the fluorescent band of HBIE-CDs that was largely red-shifted from the main absorption band with a large Stokes shift (∼141 nm), which is very useful for fluorescence imaging and could avoid the selfabsorption effect and inner filter effect. The emission wavelength of HBIE-CDs is independent of the excitation wavelength (Figure 2b and Figure S3). The HBIE-CDs exhibit good photostability (Figure S4). The PL decay curve of the HBIE-CDs exhibits a two-exponential function, and the fluorescence decay is 10.60 ns (Figure 2c). The fluorescence QY was measured directly on a FLS 980 fluorometer, the QY of the HBIE-CDs in water is 20.41%. Hydrogen-Bonding-Induced Emission of HBIE-CDs. The absorption and fluorescence spectra of the HBIE-CDs were then tested in different organic solvents and water. The main absorption of HBIE-CDs in nonprotic media such as DMF and acetone are around 355 nm; by contrast, the main absorption wavelength of the HBIE-CDs in protic solvents, such as H2O, EtOH, and MeOH, shifts to 395−416 nm (Figure 3a). As shown in Figure 3b, the HBIE-CDs exhibit strong blue emission in nonprotic media, such as DMF, acetone, acetonitrile, and dioxane. To our delight, the HBIECDs showed green emission with enhanced fluorescence intensity in protic solvents, such as H2O, EtOH, and MeOH. These results demonstrate that the hydrogen-bond network between HBIE-CDs and protic solvent not only inhibits the nonirradiation of HBIE-CDs but also increases the intramolecular charge transfer effect from the electron donating -OH and -NH2 groups to electron withdrawing -COOH, pyridine, and pterin groups, thereby causing a bigger Stokes’ shift in protic solvents.27 This interesting hydrogen-bond network-triggered fluorescence enhancement phenomenon could be ascribed to the formation of distinguished intermolecular interactions between water and HBIE-CDs. Further experiments to assess the effect of water content on the fluorescent spectra of HBIE-CDs were

carried out in different ratios of water−THF mixture. As shown in Figure S5, the emission of HBIE-CDs at 460 nm gradually red shifts to 535 nm with the increment of water content. When the water content is above 30%, the green emission increases with increasing water content, indicating the vital role of the water molecules. To understand the underlying reaction mechanism, differential scanning calorimetry (DSC) experiments were carried out. As shown in Figure S6, the endothermic melting peak gradually increases and moves to lower temperatures with the water content, indicating the presence of both nonfreezing and freezing bound type water.28 The hydrogen-bonded network induced by nonfreezing water and freezing water can construct robust bridgelike hydrogen-bonded networks, which restrict the structure of HBIE-CDs and result in the enhancement of fluorescence. Furthermore, the Raman spectrum (Figure S7) shows the peak of N−H at 2966 cm−1 gradually red-shifted with the increment of water content, which corroborates the presence of hydrogen-bonding interaction between HBIE-CDs and water molecules. The average hydrodynamic diameters of HBIE-CDs and tetraphenylethylene (TPE; typical fluorophore with aggregation-induced emission property) were further examined. As shown in Figure S8, the average hydrodynamic diameters of HBIE-CDs are almost unchanged irrespective of the water, indicating that the emission changes in different water content were not a result of aggregations of HBIE-CDs. This is the novel phenomenon named hydrogen-bondinginduced emission. By combining a suitable fluorophore with a suitable sensing mechanism, rationalized design of fluorescent probes for the specific recognition of an important analyte can be achieved.29 A fluorescence sensing mechanism, such as fluorescence resonance energy transfer, intramolecular charge transfer, photoinduced electron transfer, excited state intramolecular proton transfer, or aggregation-induced emission (AIE), has been widely employed in the design of fluorescent probes.30,31 For example, Tang’s group reported AIE which is another photophysical phenomenon associated with chromophore aggregation.32 In the AIE process, aggregate formation of 9262

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fluorescence enhancement of HBIE-CDs should not be induced by the electrostatic interaction. The photophysical properties of HBIE-CDs thus highlight the great potential in wash-free imaging. Fluorescent Imaging of Nucleus in Living Cells. The HBIE-CDs show very low toxicity toward living cells (Figure S11). The fluorescence imaging application of HBIE-CDs was investigated in living HeLa cells. After incubation with HBIECDs, HeLa cells showed strong fluorescence in the nucleus upon excitation at 405 nm with no significant background signal, which can be attributed to large amount of nucleic acids (dsDNA, RNA) in the nucleus (Figure 5a). The HBIE-CDs

chromophore could induce non-emissive luminogens to emission. Very recently, Yuan et al. reported a novel hydrogen-bond-induced enhanced emission (HIEE) sensing mechanism, in which the fluorophore was restricted in the fluorescent state by reducing the rotational and vibrational energy loss.27 Despite the vigorous progress of small-moleculebased fluorescent probes, the development of CDs-based fluorescent probes lacked vigor, probably due to the lack of suitable fluorescence sensing mechanisms which could perform effectively toward CDs. Therefore, we hope the novel HBIE fluorescence sensing mechanism presented here would show wide applications in the design of CDs-based probes for the detection of biological compounds. Fluorescence Response of HBIE-CDs toward Nucleic Acids. Next, the response of HBIE-CDs toward nucleic acids was tested. After addition of dsDNA into the buffer solution of HBIE-CDs, the fluorescence intensity of HBIE-CDs at 535 nm was progressively increased (Figure 4a). Thereby, the weak

Figure 5. HeLa cells were stained with 2 drops of NucRed Live 647 for 10 min and then stained with 15 μg/mL HBIE-CDs for 5 min as a counterstain experiment: (a) green channel, HBIE-CDs; (b) red channel, NucRed Live 647; (c) bright field; (d) merge channel. The images were collected in 480−580 (a) and 650−720 nm (b) with the excitation wavelengths 405 nm for CDs and 638 nm for NucRed Live647. Scale bar = 10 μm.

Figure 4. (a) Fluorescence intensities of HBIE-CDs and nucleic acids complex at 535 nm as a function of [dsDNA], [ssDNA], [RNA], or [G4] in 10 mM PBS (pH = 7.4). (b) Fluorescence spectra of HBIECDs (15 μg/mL; λex = 405 nm) in water, dsDNA solution, and RNA solution and then treated with DNase or RNase.

fluorescence of HBIE-CDs is substantially enhanced by dsDNA (3.4-fold; QY is 36.13%), leading to bright green fluorescence, which can be attributed to the inhibition of the nonirradiation process by H-bond network among the FA residues and m-PD residues on the surface of HBIE-CDs and the bases of the nucleic acids. Similarly, ssDNA causes similar fluorescence enhancement, and the fluorescence intensity of HBIE-CDs saturated at [ssDNA] = 3.5 μM (4.3-fold; QY is 48.52%). Moreover, RNA can also induce emission enhancement of HBIE-CDs. The fluorescence intensity of HBIE-CDs for [RNA] = 4 μM increased 6.2-old; QY is 63.11%. For G4, the fluorescence enhancement can reach up to 7.3-fold at [G4 DNA] = 5 μM; QY is 76.92%. In contrast, after addition of histone, the fluorescence intensity of HBIE-CDs only enhances slightly (1.3-fold, Figure S9). Upon addition of RNase and DNase to the HBIE-CDs−nucleic acids complex, fluorescence of the HBIE-CDs−nucleic acids complex decreased sharply (Figure 4b). It is revealing that the HBIE-CDs can be employed as a fluorescence turn on probe for the detection of DNA or RNA. Moreover, the HBIE-CDs show high selectivity over amino acids and proteins as shown in Figure S9. Furthermore, the interaction of HBIE-CDs with dsDNA was examined by circular dichroism. As shown in Figure S10, the circular dichroism spectra of dsDNA are almost unchanged before and after addition of HBIE-CDs. These results indicate that the DNA duplex was not broken by HBIE-CDs. The ζ potential of HBIE-CDs was determined to be −16.0 ± 2.2 mV (n = 11). Because the ζ potential of DNA is negative, the

showed high affinity toward the nucleus with a much lower concentration (15 μg/mL) and a much shorter time (5 min) than other reported nucleus staining probes.33,34 The nucleus were further labeled with commercial organic dyes of NucRed Live 647 (λex = 638 nm and λem = 686 nm, which stains DNA of the live cells, Figure 5b). As further revealed in the merged channel (Figure 5d), the overlaps of green (HBIE-CDs) and red fluorescence (NucRed Live 647) were just from the location of the nucleus DNA. Importantly, good correlation was revealed from the Pearson’s correlation coefficient (0.88) determined by Leica software, which demonstrated the specific targeting property of HBIE-CDs in DNA and RNA in the cell nucleus. To classify the nucleus-targeting mechanism of HBIE-CDs, digest experiments of the cellular nucleo acids by using deoxyribonuclease (DNase) and ribonuclease (RNase) were employed to investigate the impacts of nucleo acids on the fluorescent imaging capability of HBIE-CDs. Figure 6 shows that the green fluorescence in a nucleus decreases obviously after being digested with DNase and RNase, which is in good agreement with the results in Figure 4. The fluorescence was located in the nucleoli of the DNA-digested and in the nucleus of RNA-digested, indicating the different localizations of RNA and DNA in the nucleus. After the cells were digested by DNase and RNase, the whole cell became dark (Figure 6d). On the basis of these results, it can be concluded that the 9263

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nonirradiation by a H-bond network. The bases of nucleic acid can be used as the donor of H-bond. Therefore the green fluorescence of HBIE-CDs would increase dramatically after binding with DNA or RNA. The strong green fluorescence switch-on response of HBIE-CDs toward nucleic acids makes the HBIE-active CDs suitable as a wash-free bioimaging regent for cellular nucleus since they contain plenty of DNA/RNA species. Furthermore, the HBIE-CDs exhibited low cytotoxicity and efficient nucleus permeability. These light-up HBIECDs have great potential in clinical diagnosis and biological studies.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b02147. Photographs, FL spectra, confocal images, stability assay, and cell viabilities (PDF)

Figure 6. HeLa cells stained with 15 μg/mL HBIE-CDs (a) and then incubated with 25 μg/mL RNase (b), 30 μg/mL DNase (c), and both 25 μg/mL RNase and 30 μg/mL DNase (d): λex = 405 nm; λem = 480−580 nm. Scale bar = 20 μm.

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nucleus staining is due to the binding of HBIE-CDs to DNA and RNA after entering into the cells. Wash-Free Fluorescent Imaging of Nucleus in Living Cells. The HBIE-CDs have great potential to be utilized for nonwash bioimaging applications. Figure 7 and Figure S12

AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-371-67780037. E-mail: [email protected] (Z.L.). *Tel.: +86-371-67780037. E-mail: [email protected] (Y.S.). *Tel.: +86-371-67780037. E-mail: [email protected] (L.Q.). ORCID

Zhaohui Li: 0000-0003-3946-0656 Lehui Xiao: 0000-0003-0522-2342 Hongmin Meng: 0000-0003-0723-1242 Yuehe Lin: 0000-0003-3791-7587 Xiaobing Zhang: 0000-0002-4010-0028 Notes

The authors declare no competing financial interest.

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Figure 7. Fluorescent images of HeLa cells which were stained with 15 μg/mL HBIE-CDs for 5 min with (first line) or without (second line) washing steps: λex = 405 nm; λem = 480−580 nm. Scale bar = 10 μm.

ACKNOWLEDGMENTS This work was supported in part by the National Natural Science Foundation of China (Grant Nos. 21605038 and 21708035), the Foundation for University Key Teacher by Henan Province (Grant No. 2017GGJS007), the Key Scientific Research Project in Universities of Henan Province (Grant No. 19A150048), and the Outstanding Young Talent Research Fund of Zhengzhou University (Grant No. 1421316038).

show the incubation of cells with HBIE-CDs for 5 min and subsequently imaged with or without a wash step. The HeLa cells without a wash step also show strong fluorescence in the nucleus and almost non-background fluorescence in cytoplasm, which can be attributed to the unique phenomenon in which HBIE-CDs show weak fluorescence in water but exhibit strong fluorescence after binding with DNA and RNA (Figure S13). The unique fluorescent properties of the HBIE-CDs make them a useful choice for cellular imaging with negligible background fluorescence from the culture medium and cytoplasm. A wash-free labeling method is particularly suitable to monitor the nucleus in living cells, since it allows continuous monitoring of the entire biological process of a nucleus in living cells without interruption.

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REFERENCES

(1) Raška, I. Trends Cell Biol. 2003, 13, 517−525. (2) Gao, T.; Wang, S.; Lv, W.; Liu, M.; Zeng, H.; Chen, Z.; Dong, J.; Wu, Z.; Feng, X.; Zeng, W. Chem. Commun. 2018, 54, 3578−3581. (3) Hassan, M.; Gomes, V. G.; Dehghani, A.; Ardekani, S. M. Nano Res. 2018, 11, 1−41. (4) Shi, W.; Li, X.; Ma, H. Angew. Chem., Int. Ed. 2012, 51, 6432− 6435. (5) Lou, J.; Liu, S.; Tu, W.; Dai, Z. Anal. Chem. 2015, 87, 1145− 1151. (6) Pu, K. Y.; Li, K.; Zhang, X.; Liu, B. Adv. Mater. 2010, 22, 4186− 4189. (7) Shen, P.; Xia, Y. Anal. Chem. 2014, 86, 5323−5329. (8) Liu, H.; Sun, Y.; Yang, J.; Hu, Y.; Yang, R.; Li, Z.; Qu, L.; Lin, Y. Sens. Actuators, B 2019, 280, 62−68. (9) Sun, Y. P.; Zhou, B.; Lin, Y.; Wang, W.; Fernando, K. A. S.; Pathak, P.; Meziani, M. J.; Harruff, B. A.; Wang, X.; Wang, H.; Luo, P.

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CONCLUSION HBIE-CDs were easily obtained by hydrothermal strategy with FA and m-PD as the precursor. The resulted HBIE-CDs have ultrasmall size, large Stokes shift (141 nm) in water, and good biocompatibility. Interestingly, the HBIE-CDs show blue fluorescence in nonprotic solvents and switch to strong green fluorescence in protic solvents due to the inhibition of 9264

DOI: 10.1021/acs.analchem.9b02147 Anal. Chem. 2019, 91, 9259−9265

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Analytical Chemistry G.; Yang, H.; Kose, M. E.; Chen, B.; Veca, L. M.; Xie, S. Y. J. Am. Chem. Soc. 2006, 128, 7756−7757. (10) Liu, S.; Cao, H.; Wang, Z.; Tu, W.; Dai, Z. Chem. Commun. 2015, 51, 14259−14262. (11) Yang, S. T.; Cao, L.; Luo, P. G.; Lu, F.; Wang, X.; Wang, H.; Meziani, M. J.; Liu, Y.; Qi, G.; Sun, Y. P. J. Am. Chem. Soc. 2009, 131, 11308−11309. (12) Cai, Q. Y.; Li, J.; Ge, J.; Zhang, L.; Hu, Y. L.; Li, Z. H.; Qu, L. B. Biosens. Bioelectron. 2015, 72, 31−36. (13) Liu, H.; Sun, Y.; Li, Z.; Yang, J.; Aryee, A.; Qu, L.; Du, D.; Lin, Y. Nanoscale 2019, 11, 8458−8463. (14) Liu, H.; Sun, Y.; Li, Z.; Yang, R.; Yang, J.; Aryee, A. A.; Zhang, X.; Ge, J.; Qu, L.; Lin, Y. SciFinder-guided rational design of fluorescent carbon dots for ratiometric monitoring intracellular pH fluctuations under heat shock. Chin. Chem. Lett. 2019, DOI: 10.1016/j.cclet.2019.06.012 (15) Hövelmann, F.; Gaspar, I.; Ephrussi, A.; Seitz, O. J. Am. Chem. Soc. 2013, 135, 19025−19032. (16) Wang, X.; Wang, Y.; He, H.; Ma, X.; Chen, Q.; Zhang, S.; Ge, B.; Wang, S.; Nau, W. M.; Huang, F. ACS Appl. Mater. Interfaces 2017, 9, 17799−17806. (17) Sun, S.; Zhang, L.; Jiang, K.; Wu, A.; Lin, H. Chem. Mater. 2016, 28, 8659−8668. (18) Ding, P.; Wang, H.; Song, B.; Ji, X.; Su, Y.; He, Y. Anal. Chem. 2017, 89, 7861−7868. (19) Datta, K. K. R.; Kozák, O.; Ranc, V.; Havrdová, M.; Bourlinos, A. B.; Š afárǒ vá, K.; Holá, K.; Tománková, K.; Zoppellaro, G.; Otyepka, M.; Zbořil, R. Chem. Commun. 2014, 50, 10782−10785. (20) Kang, Y. F.; Fang, Y. W.; Li, Y. H.; Li, W.; Yin, X. B. Chem. Commun. 2015, 51, 16956−16959. (21) Piatkevich, K. D.; Subach, F. V.; Verkhusha, V. V. Chem. Soc. Rev. 2013, 42, 3441−3452. (22) Kumagai, A.; Ando, R.; Miyatake, H.; Greimel, P.; Kobayashi, T.; Hirabayashi, Y.; Shimogori, T.; Miyawaki, A. Cell 2013, 153, 1602−1611. (23) Litwack, G. Folic acid and folates; Academic Press: San Diego, CA, USA, 2008. (24) Bourassa, P.; Tajmir-Riahi, H.A. Int. J. Biol. Macromol. 2015, 74, 337−342. (25) Liu, H.; Li, Z.; Sun, Y.; Geng, X.; Hu, Y.; Meng, H.; Ge, J.; Qu, L. Sci. Rep. 2018, 8, 1086. (26) Jiang, K.; Sun, S.; Zhang, L.; Lu, Y.; Wu, A.; Cai, C.; Lin, H. Angew. Chem., Int. Ed. 2015, 54, 5360−5363. (27) Ren, T. B.; Xu, W.; Zhang, Q. L.; Zhang, X. X.; Wen, S. Y.; Yi, H. B.; Yuan, L.; Zhang, X. B. Angew. Chem., Int. Ed. 2018, 57, 7473− 7477. (28) Li, Q.; Zhou, M.; Yang, M.; Yang, Q.; Zhang, Z.; Shi, J. Nat. commun. 2018, 9, 734. (29) Vendrell, M.; Zhai, D.; Er, J. C.; Chang, Y. T. Chem. Rev. 2012, 112, 4391−4420. (30) Wu, J.; Liu, W.; Ge, J.; Zhang, H.; Wang, P. Chem. Soc. Rev. 2011, 40, 3483−3495. (31) Wang, P.; Liu, J.; Lv, X.; Liu, Y.; Zhao, Y.; Guo, W. Org. Lett. 2012, 14, 520−523. (32) Hong, Y.; Lam, J. W. Y.; Tang, B. Z. Chem. Commun. 2009, 29, 4332−4353. (33) Datta, K. K. R.; Kozák, O.; Ranc, V.; Havrdová, M.; Bourlinos, A. B.; Š afárǒ vá, K.; Holá, K.; Tománková, K.; Zoppellaro, G.; Otyepka, M.; Zbořil, R. Chem. Commun. 2014, 50, 10782−10785. (34) Kang, Y. F.; Fang, Y. W.; Li, Y. H.; Li, W.; Yin, X. B. Chem. Commun. 2015, 51, 16956−16959.

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DOI: 10.1021/acs.analchem.9b02147 Anal. Chem. 2019, 91, 9259−9265