Controllable Fabrication, Photoluminescence Mechanism and Novel

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Characterization, Synthesis, and Modifications

Controllable Fabrication, Photoluminescence Mechanism and Novel Application of Green-Yellow-Orange Fluorescence Carbon-Based Nanodots Xiaojuan Gong, Li Zhang, Yang Liu, Huiping Wang, Xinyan Tracy Cui, Qin Hu, Shengmei Song, Shaomin Shuang, and Chuan Dong ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b01153 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 23, 2019

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ACS Biomaterials Science & Engineering

Controllable Fabrication, Photoluminescence Mechanism and Novel Application of Green-Yellow-Orange Fluorescence Carbon-Based Nanodots Xiaojuan Gong,†,* Li Zhang,† Yang Liu,† Huiping Wang,† Xinyan Cui,‡ Qin Hu,§ Shengmei Song,† Shaomin Shuang,† and Chuan Dong† †Institute

of Environmental Science, and School of Chemistry and Chemical Engineering,

Shanxi University, Taiyuan, Shanxi, 030006 (P. R. China) ‡Bioengineering,

University of Pittsburgh, Pittsburgh, PA 15260,

U.S.A. §College

of Food Science and Engineering, Jiangsu Key Laboratory of Dairy Biotechnology

and Safety Control, Yangzhou University, Yangzhou, Jiangsu, 225001 (P. R. China) *Corresponding

author. E-mail: [email protected].; Tel: +351-7011011.

ABSTRACT Carbon-based nanodots (CBNs), as spick-and-span carbon nanomaterials, have been widely studied and applied in numerous fields. However, the controlled fabrication and photoluminescence (PL) mechanism remains an incompletely understood and widely debated topic. Herein, green, yellow, and orange light-emitting CBNs were fabricated by a one-step hydrothermal process with PABA and 1,3-DB, 1,2-DB, and 1,4-DB as precursors, respectively; the resulting CBNs were named gCBNs, yCBNs, and oCBNs, respectively. By adjusting the reaction conditions, including precursor, solvent, atmosphere, and dissolved solvents, the controllable fabrication of CBNs can be realized. We speculate that the PL of CBNs is dominated by the degree of oxidation and amidation, which, together, cause the differences in density of N-states and quantum size, ultimately manifesting as changes in three CBNs’ fluorescence behaviors. Finally, the CBNs were used as agents to image four model cells, demonstrating that CBNs are potentially useful in biological labeling and multicolor 1 ACS Paragon Plus Environment

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bioimaging. More importantly, CBNs can be conjugated to targeted micromolecule, DNA, RNA, and/or anticarcinogen groups to construct nanocomposite materials, which could be applied to identify target materials and/or execute the sustained release of drugs. We want to offer the guide for controllable fabricating of CBNs, and further expand application depth and breadth in the biomedical field. KEYWORDS controllable fabrication; photoluminescence mechanism; novel application; green-yellow-red fluorescence; carbon-based nanodots

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1. INTRODUCTION Over the past few decades, new carbon-based nanomaterials with unique and extraordinary physicochemical properties have been discovered, among which fluorescent carbon nanotubes, nanodiamonds, fullerene, graphene, as well as an endless variety of their combinations, have drawn considerable attention.1 A novel type of carbon-based nanomaterial, carbon-based nanodots (CBNs), has recently attracted considerable attention among chemists and materials scientists because of their exceptional advantages, including excellent water solubility, high photoluminescence

(PL)

stability,

easy

functionalization,

low

cytotoxicity,

good

biocompatibility, and resistance to metabolic degradation in bioapplications.2-6 These superior properties of CBNs distinguish them from traditional fluorescent materials. Specifically, the advantage of inexpensive one-step synthesis5-8 renders CBNs suitable for various applications, such as analyte detection,6,9 catalysis,10 light-emitting devices,11,12 drug delivery,8,13 biomedical imaging,6,8,13-15 and flexible electroluminescent devices.16 Given that CBNs possess outstanding properties and exhibit broad application prospects, many efforts have been exerted to develop diversified preparation methods, which can be divided into two major categories, i.e., the “top-down approach” and the “bottom-up approach”. The top-down approach involves laser ablation of a carbon target,17 electrochemical oxidation of graphite18 and multi-walled carbon nanotubes,19 and chemical acid oxidation of soot,20 while the bottom-up approach includes microwave-assisted carbonization of chitosan,6 hydrothermal cutting of waste microorganism,21 and carbonization of organic molecules.22 Although abundant CBNs have been fabricated and researchers have focused on understanding the fundamental mechanism of their PL, the details of the basic photophysical and spectroscopic properties of CBNs remain unclear. Several patterns, such as quantum effect,23,24 surface passivation/defect,23,25-27 edge state and carbon-core state effect,23,25,26 radioactive recombination nature of excitation,5,23,28 surface state effect,5,23,26-29 and heteroatom doping23,28 are known to be sensitive to surface passivation and solvent pH.30 Ensemble emission spectroscopic studies on CBNs indicate that these quantum dots possess excitation wavelengthdependent emission behavior,1,6,8 which suggests that fluorescent CBNs possess multichromophoric units. However, whether multiple emissive centers are present within a single particle or originate from separate particles remains unknown. This information cannot be gained from ensemble measurements, which only capture the average information of CBN size and shape or the presence of chemical defects. Thus, the PL origin of CBNs remains a research topic of increasing interest.31 Given this background, the detailed PL mechanism



should be further investigated since the knowledge gained from such studies would be beneficial for further development of controllable synthesis methods of CBNs. In this paper, we propose a new strategy to controllably prepare green, yellow, and orange fluorescent CBNs with precise fluorescent emission peaks %Jem) via a one-pot hydrothermal treatment method. 4-aminobenzoic acid (PABA) and diaminobenzene [three isomers: 1,3diaminobenzene (1,3-DB), 1,2-diaminobenzene (1,2-DB), and 1,4-diaminobenzene (1,4-DB)] serve as precursors and produce CBNs with similar light-emitting color and various Jem by simple adjustment of their proportions. Compared with a previously published paper,22 we introduce PABA as a new precursor to fabricate CBNs by amide reaction and carbonization. The effects of reaction conditions and solvent on CBNs fabrication are discussed in detail. The cytotoxicity of the as-synthesized CBNs was determined by apoptotic and JC-1 determination, which is a novel method to explain cell toxicity from CBNs. These CBNs can be successfully applied for in vitro bioimaging because of their advantages, such as high FQY, stable FL, and low toxicity. Furthermore, the ratio of carboxyl (-COOH) and amino (-NH2) groups was controlled in order to let CBNs efficiently react with small organic molecules, e.g., fluorescent dyes, triphenylphosphine or folic acid; biomacromolecules, e.g., aptamer, DNA, or protein; and/or functional drugs, e.g., doxorubicin, camptothecin, cis-platinum, or aspirin, by amidation and/or the reaction between amidogen and hydrosulphonyl. All the as-fabricated CBNs possess favorable fluorescent quantum yields (FQY), and their PL behaviors demonstrate a direct relationship with the quantum size effect, the degree of surface oxidation and amidation of the CBNs, as well as the density of the surface states of the CBNs. Based on these findings, we propose a PL mechanism. More importantly, we demonstrate that a series of CBNs exhibiting various Jem can be fabricated by varying their size, their degree of surface oxidation and amidation, or their surface states. 2. MATERIALS AND METHODS 2.1. Materials. Reagent grades of 4-aminobenzoic acid (PABA), 1,3-diaminobenzene (1,3DB), 1,2-diaminobenzene (1,2-DB), 1,4-diaminobenzene (1,4-DB), and absolute ethyl alcohol were purchased from Sigma Aldrich Reagents Company (Milwaukee, WI, U.S.A.). All biological reagents were obtained from Solarbio Reagents Company (Beijing, China). The other chemical reagents were from Beijing Reagents Company (Beijing, China). All reagents were used as received without further purification unless otherwise specified. Deionized water (DIW) was obtained from a Millipore Milli-Q-RO4 water purification system with a resistivity higher than 18 ;PQ)

(Bedford, MA, U.S.A.) and used throughout this study. 4 ACS Paragon Plus Environment

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2.2. Fabrication of gCBNs, yCBNs and oCBNs. 0.05 g PABA and 0.05 g 1,3-DB, 0.05 g PABA and 0.05 g 1,2-DB, and 0.05 g PABA and 0.05 g 1,4-DB were first dissolved in 10 mL absolute

ethyl

alcohol,

respectively.

Then,

the

solutions

were

transferred

into

poly(tetrafluoroethylene)-lined autoclaves and heated at 180oC in an oven for 12 h. After thr reaction, the reaction products were cooled down to room temperature naturally. The obtained orange, brown, and brown-dark suspensions were handled in an orderly manner by filtering, evaporating, dissolving in DIW, and centrifuging, respectively. Then, the pellucid orange gCBNs, brown yCBNs, and brown-dark oCBNs aqueous solutions were dialyzed through a dialysis membrane with a molecular weight cutoff (MWCO) of 500-1000 Da against DIW for 72 h with changing DIW every 6 h to remove the small molecular and then filtered, respectively. Finally, gCBNs in the orange aqueous solution, yCBNs in the brown solution, and oCBNs in the brown-dark solution were collected and lyophilized to obtain the dry gCBNs product, yCBNs product, and oCBNs product, respectively. 2.3. Characterizations. The UV-Vis absorption and PL spectra were performed on a Lambda 950 spectrophotometer (PerkinElmer, Llantrisant, U.K.) and Varian Cary Eclipse spectrofluorometer (Palo Alto, CA, U.S.A.), respectively. Nanosecond fluorescence lifetime and partial PL spectra were acquired with a FLS 920 spectrofluorometer (Edinburgh, Livingston, UK). Transmission electron microscopy (TEM) and atomic force microscope (AFM) observations were carried out with a JEOL JEM-2100 transmission electron microscopy (Tokyo, Japan) and a Multimode 8 AFM (Bruker AFM Probes, Worcestershire, UK), respectively. Elemental analysis (EA), Fourier Transform Infrared Spectra (FTIR) and X-ray photoelectron spectra (XPS) were obtained on a Vario EL cube elemental analyser (Hanau, Germany), Bruker Tensor II FTIR spectrometer (Bremen, Germany), and AXIS ULTRA DLD X-ray photoelectron spectrometer (Kratos, Tokyo, Japan), respectively. 2.4. Fluorescent quantum yield (FQY) measurement. The FQY of the CBNs samples was determined by a relative method,32 which compares the integrated photoluminescence (PL) intensities and optical density (OD) between the sample and the reference. Especially, rhodamine 6G (FQY = 95% in absolute ethyl alcohol) was selected as the reference for gCBNs and yCBNs, and rhodamine B (FQY = 56% in absolute ethyl alcohol) was chosen as the reference for oCBNs. The FQY of the as-prepared CBNs was calculated according to the following equation (1): FQYS = FQYR(GradS/GradR(%VS2=VR2)

(1)

where the subscripts S and R refer to the sample CBNs and references, respectively. Grad is the gradient from the plot of integrated PL intensity against OD. The V is the refractive index %VS 5 ACS Paragon Plus Environment


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and VR = 1.33). In order to minimize the self-absorption effect, the OD in the 10-mm pathlength fluorescence cuvette was kept under 0.10 at different excitation wavelengths (Figure S1 and Table S1). 2.5. Cytotoxicity assay. Measurement of cell viability was carried out by reduction of CCK8 relative to control cells with the same volume of Dulbecco's phosphate-buffered saline (DPBS, Gibco) incubated. Typically, SMMC7721 cells were seeded at a density of 5 ×104 cells/mL into a 96-well cell culture plate and were cultured at 37oC for 24 h. Afterwards, the medium was changed with 100 Y of different concentrations of CBNs in DMEM, and then the cells were incubated for another 24 h. After removal of the supernatant, 90 µL of fresh DMEM culture medium and 10 µL of CCK-8 were added. After 0.5 h incubation at 37oC and 5% CO2, the CCK-8 assay was performed. Absorbance at 450 nm was measured, and the assay was repeated at least three times. Cell viability was estimated as follows: Cell viability (%) = (ODTreated/ODControl) × 100%

(2)

where ODTreated and ODControl were obtained in the absence and presence of CBNs, respectively. 2.6. Apoptotic detection by Annexin V Assay. SMMC7721 cells were seeded into each well of a six-well plate at a density of 1×104 cells/mL and were incubated overnight. Medium was changed with different concentrations of gCBNs, yCBNs, and oCBNs and then respectively incubated with the cells for 2 h. Afterwards, the cells were trypsinized, washed, and centrifuged at 1,000 rpm for 3 min, and then the supernatants were removed. The cells were resuspended in 1× Annexin V binding buffer at a density of 1×104 cells/mL. For 100 Y of cell suspension, 5 Y of Alexa®Fluor 488 Annexin V and 1 Y of 100 Y =

propidium iodide (PI) in a working

solution were added, and the suspension was incubated for 15 min at room temperature. Following incubation, 400 Y of 1 × Annexin-binding buffer were added to each sample. The samples were gently mixed and kept on ice. Then, the samples were immediately analyzed by flow cytometry (BD FACS Verse; Becton, Dickinson and Company, USA). 2.7. JC-1 determination by flow cytometry. SMMC7721 cells were seeded at a density of 1 × 104 cells/mL in each well of a six-well plate and were cultured overnight. The JC-1 working solution was prepared by mixing 500 µL of 1 × CBNs solution (in pH 7.4 PBS) with 5 µL JC1. Medium was changed with different concentrations of JC-1 working solution and then respectively incubated with the cells for 2 h. Afterwards, the cells were trypsinized, washed, and centrifuged at 1,000 rpm for 3 min, followed by removal of the supernatants. The cells were resuspended in JC-1 working solution at a density of 1 × 104 cells/mL. For 100 Y of cell suspension, 500 µL of 1 × CBNs solution was added, and the suspension was incubated for 15 min at room temperature. The samples were gently mixed and kept on ice. Then, the samples 6 ACS Paragon Plus Environment

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were immediately analyzed by flow cytometry (BD FACSVerse; Becton, Dickinson and Company, USA). 2.8. In vitro cellular imaging. Human breast cancer A549 cells, human hepatocarcinoma SMMC7721 cells, human pulmonary epithelial normal BEAS-2B cells and human pheochromocytoma PC 12 cells were respectively cultured in the culture dish at 37oC containing DMEM supplemented with 10% FBS. The filtered CBNs aqueous solution (100 µL, 1.0 mg/mL) was mixed with the culture medium (0.9 mL) and then added to the culture dish. After incubation for 1 h, the A549, SMMC7721, BEAS-2B and PC 12 cells were harvested using 0.25% trypsin/0.020% EDTA, washed three times (1.0 mL each) with pH 7.4 phosphatebuffered saline (PBS, which is comprised of 137 mM NaCl, 2.7 mM KCl, 8.0 mM Na2HPO4, and 2.0 mM KH2PO4) and kept in PBS for LSCM (laser scanning confocal microscopy) imaging by a ZEISS LSM 880 with Airyscan super-solution confocal microscope (Germany) at 20 and 40 × objective. 3. RESULTS AND DISCUSSION The hydrothermal method has been used to fabricate the fluorescent CBNs, which can be attributed to its advantages, such as low preparation requirements, relatively mild reaction conditions, moderate reaction time, or high degree of carbonization.5,7,14,21,22 However, in the fabrication process of CBNs, the functionality and effect of the hydrothermal method were rarely reported. Many reports have pointed out that the reaction precursor is of utmost importance in the controllable fabrication of CBNs.4-6,8,22 However, when choosing the hydrothermal method to synthesize CBNs, solvent and atmosphere are important factors potentially affecting the optical performance of CBNs. Twelve types of CBNs have been prepared by varying the precursor, solvent and atmosphere, and the results were shown in Table S2. These CBNs show significant differences under visible and UV light (365 nm). With the same reaction precursors, we could prepare different luminous CBNs with different solvents (EtOH or DIW) and atmosphere (air or N2). The results illustrate that the controllable fabrication of CBNs can be realized by adjusting the reaction precursor, solvent and atmosphere. Among these twelve kinds of CBNs, the first, fifth, and ninth CBNs have attracted our interest since they demonstrate unique photoluminescence performance, high yield and perfect fluorescence efficiency. Therefore, we choose them as representative CBNs for further detailed study.



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(E)

(F)

Figure 2. The UV-Vis absorption, excitation and emission spectra of (A) gCBNs, (B) yCBNs, and (C) oCBNs. The insets are the representative electronic photographs of the CBNs under daylight (left) and UV irradiation (right). The emission spectra of (D) gCBNs, (E) yCBNs, and (F) oCBNs were excited at different excitation wavelengths.

The PL lifetimes of the as-synthesized CBNs were measured. As depicted in Figure S4 and Table S5, the PL decays of the gCBNs, yCBNs, and oCBNs in DIW solution were fitted by a bi-exponential function with average lifetimes of 2.01, 4.91, and 7.74 ns, respectively. The photostability of the as-prepared three CBNs in DIW solution was investigated, and, as shown in Figure S5A-S5C, gCBNs, yCBNs, and oCBNs are highly stable; only slight (< 10%) and little (< 20%) decays are found under continuous illumination at Jex = 365 nm for 2 h and 4 h, respectively. The as-fabricated gCBNs, yCBNs, and oCBNs exhibit superior resistance stability under Xe lamp irradiation for 2 h, showing fluorescence decays of only 12%, 10%, and 3%, respectively (Figure S5D-S5F). These results indicated that the as-prepared CBNs possess good photostability, excellent luminescent properties, and stable fluorescence behaviors. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to characterize the morphology and size of CBNs. As depicted in Figure 3A-3C, the CBNs 11 ACS Paragon Plus Environment



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To probe the chemical composition, content, chemical bonds, and functional groups of the CBNs, FTIR spectroscopy and XPS measurements were carried out. As displayed in Figure S7, the CBNs show similar FTIR spectra, indicating similar chemical composition. In comparison with those of the starting materials, some new peaks are observed in all as-prepared CBNs at approximately 2987-2905, 1243-1265, and 1049-1074 cm`1 (Figure S8A-S8D), which can be attributed to aliphatic C-H, C-N, and C-O stretching vibrations, respectively.22 These observations imply that decomposition, intermolecular cyclization, and condensation reactions occur during the formation of CBNs. Other peaks at 3685-3662, 2116-2127, 1930-1938, 13731411, and 884-895 cm`1 are associated with O-H/N-H stretching, -C=C=O stretching, aNH3+, cC-H, and cN-H, respectively. The absorption peak at ca. 1641 cm`1 observed in the spectra of the yCBNs and oCBNs represents the vibrations of C=O, implying an increase in their degree of oxidation, which could be related to the PL red-shift of these CBNs.34,35 The survey scan spectra presented in Figure S9A-S9C demonstrate three typical XPS surveys of the as-synthesized CBNs confirm the peaks: C(1s) (283 eV), N(1s) (396 eV), and O(1s) (530 eV), indicating that the CBNs consist of the same elements (i.e., C, H, N, and O), consistent with the FTIR results. In high-resolution spectra of the CBNs (Figure 4A-4C), the C(1s) XPS could be fitted into four peaks corresponding to sp3 carbons (C–N, 287.7, 287.8, or 288.6 eV), sp3 carbons (C–C/C–O, 285.9, 285.8, or 286.2 eV), carboxyl carbons (R–O–C(O)–R, R = H, CH3, C6H5, and so on, 285.0 eV), and carbonyl carbons (C=O, 284.2 or 284.0 eV).8,35 Figure 4D, 4E, and 4F show the N(1s) spectra of three CBNs; the spectra can be divided into four peaks at ca. 400.4, 399.7, 399.2, and 398.7 eV, representing N(1s) states in pyrrolic-N, amido-N/C-N, amino-N, and pyridinic-N, respectively.8,35 As shown in Figure S9D-9F, the O(1s) peaks depict the N-O, C6H4-(OH)2, *O=C-O, C=O, and C-O-C of the CBNs, which are associated with peak locations at ca. 534.7, 533.5, 532.5, 531.7, and 530.9 eV, respectively.8,22,35 The content of C=O (284.2 eV) in the CBNs gradually increases from gCBNs to oCBNs (Table 1), which is consistent with the results of FTIR. Collectively, the FTIR and XPS results clearly demonstrate that the three CBNs consist of ]")

$

domains in their carbon cores and amorphous regions on their

surface and that the degree of oxidation of the as-synthesized CBNs increases concurrently with their PL red-shift. (A)

(B)



(C)

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(D)

(E)

(F)

Figure 4. The C(1s) XPS spectra of (A) gCBNs, (B) yCBNs, and (C) oCBNs. The N(1s) XPS spectra of (D) gCBNs, (E) yCBNs, and (F) oCBNs.

Table 1. Data analysis of the C(1s) XPS spectra of the as-fabricated CBNs. Type of CBNs gCBNs yCBNs oCBNs

C-N 22.11% 16.39% 5.98%

C-C/C-O 23.16% 27.32% 26.12%

R-O-C(O)-R 36.84% 37.16% 47.77%


C=O 17.89% 19.13% 20.13%

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The effects of ionic strength (the concentration of KCl) and pH on PL stability of the asprepared CBNs were investigated. The PL intensity and spectral features of the CBNs show 2040 % changes at high concentration of KCl (3.0 mol/L, Figure S10), demonstrating that CBNs could not tolerate the high salt environment. However, the K+ concentration in cell imaging in vitro and bioimaging in vivo is lower than 20 mmol/L. The PL intensity of the CBNs hardly unchanged in low concentration (< 0.5 mol/L), which is beneficial for applications of CBNs in the presence of low salt concentration. Figure S11 reveals that the PL intensities of the gCBNs and oCBNs gradually decrease as pH increases from 2.0 to 13.0; by contrast, the PL intensities of the yCBNs decrease significantly at low (< 5.0) or high (> 11.0) pH, but remain unchanged at pH 5.0-11.0. These variations are likely related to the differences in the content of surface functional groups and the degree of oxidation of the as-prepared CBNs. The prominent PL properties of the CBNs make them excellent candidates for potential applications in the fields of biomedicine and bioimaging. Since the first discovery of CBNs, researchers have sought to determine the origin of luminescence. A chief problem that must be addressed is the difficulty associated with correctly depicting and evaluating the complete PL mechanism of a variety of CBNs. Thus far, impressive advances have been made through the continuous efforts of motivated scientists in this research area. Four popular PL models have been proposed for CBNs: quantum dimension effect,22-24,28 band gap transitions in conjugated ]"

# 14,28 recombination radiation of

electron-hole pairs,5,28 and surface defects.24-28,36 Red-shifting of the PL of the CBNs is attributed to the quantum size effect; that is, the PL wavelength gradually red-shifts as the quantum dimensions of the quantum dots increase (gCBNs, 2.48 nm × 0.5-1.5 nm; yCBNs, 3.56 nm × 1.0-2.5 nm; oCBNs, 4.2 nm × 4.0-5.0 nm) (Figure 5A). Our structural characterization results confirm that the degrees of oxidation and amidation of the as-prepared CBNs surfaces increase with the red-shifting of their PL (Table 1 and Table S6). Surface defects are primarily formed through surface oxidation and amidation and have been reported to serve as capture centers for excitons, resulting in surface-state-related fluorescence.25,37 More surface defects can be found when a higher degree of oxidation and amidation on the CBNs surface occurred,5,34 resulting in red-shifting of the PL of the as-fabricated three CBNs (Figure 5B). For excluding the quantum size effect to the study of the degree of oxidation and amidation, gCBNs, yCBNs, and oCBNs with the same size were fabricated by adjusting the synthesis atmosphere (air or N2). N2 atmosphere could dramatically decrease the degree of oxidation and amidation. As depicted in Fig. S12A-C, the Jem of gCBNs, yCBNs, and oCBNs fabricated under air and N2 atmospheres are 435 and 512 nm, 393 and 566 nm, and 440 and 596 nm, respectively, 15 ACS Paragon Plus Environment


demonstrating that high degree of oxidation and amidation will be conductive to fabricate the PL red-shifted CBNs. Also, as shown in Table S6, the nitrogen contents of the gCBNs, yCBNs, and oCBNs gradually increase, which may be correlated with the surface-state (labeled as Nstates) intensity. The N-state intensity of the as-prepared CBNs is in the order of oCBNs > yCBNs > gCBNs, meaning that the highest yield of radiative recombination occurs in the oCBNs. Such recombination may explain the PL red-shift of the three CBNs (Figure 5C). Chen et al. reported that sp2-hybridized carbons of carboxyl groups could induce significant local distortions, resulting in narrower energy gaps.38 Herein, the observed fluorescence can be attributed to quantum dimension, surface defects, and surface-state intensities of the asfabricated CBNs. Given that the PL features of the as-synthesized CBNs are similar to the specific molecular fluorescence,31 we hypothesize that the fluorescence centers of the CBNs surfaces are predominantly composed of conjugated carbon and nitrogen atoms and bonded oxygen atoms. The band gap between the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) strongly depends on the incorporated oxygen species,24,39 which were used to display the complete delocalization of the electrons. We speculated the structure models of gCBNs, yCBNs, and oCBNs according to the results of XPS, FTIR, and UV-Vis and PL spectra, which were shown in Figure S13. The theoretical calculations on three CBNs were performed to elucidate excited state behaviors. The theoretical band gaps were 4.559 eV (absorption peak 272 nm), 2.870 eV (absorption peak 432 nm), and 2.398 eV (absorption peak 517 nm) for gCBNs, yCBNs, and oCBNs (Figure 5D), respectively. Gauss-assisted software was used to calculate the band gaps of the inferred structure models (Figure S13), which are 3.91 eV (absorption peak 317 nm), 2.694 eV (absorption peak 460 nm), and 2.546 eV (absorption peak 487 nm) respectively for gCBNs, yCBNs, and oCBNs. The differences are 45 nm (gCBNs), 28 nm (yCBNs), and 30 nm (oCBNs), which are within the margin of error (< 50 nm). These calculated results are consistent with the proposed orbital theory. The band gap is decreased when the numbers of oxygen atoms join the structure was increased, meaning that an increase in the degree of surface oxidation will be lead to a PL redshift (Figure 5D). Based on our detailed analysis of the obtained data and discussion of the related mechanism, we speculate that the PL behavior of our CBNs may be attributed to the synergistic effects of quantum size, degree of oxidation and amidation, as well as density of the N-state, all of which involve the quantum dimension effect, surface defects and states, energy recombination, and orbital theory. However, the definite modes of action of several theories remain unclear, and further in-depth study and discussion are required to clarify the remaining issues. We think that the fundamental reason behind the observed different PL characteristics 16 ACS Paragon Plus Environment

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(F) oCBNs with different concentration. SMMC7721 cells (1 × 104 cells/mL) were treated with the as-synthesized three CBNs and analyzed by JC-1 staining. The R2 represents the percentage of the mitochondrial damaged SMMC7721 cells.

The high PL stability and low cytotoxicity of the CBNs confirm their suitability for cellular imaging application. Four model cells, i.e., human breast cancer A549, human hepatocarcinoma SMMC7721, human pulmonary epithelial normal BEAS-2B, and human pheochromocytoma PC 12, were used to investigate the cellular uptake of the as-prepared CBNs through laser scanning confocal microscopy. Here, A549, SMMC7721, BEAS-2B, and PC 12 cells were incubated with the as-synthesized CBNs at a concentration of 100 µg/mL for 1 h. As shown in Figure S16, all model cells emit blue, green, yellow, and red fluorescence at Jex of 408, 458, 488, and 543 nm, respectively, thereby illustrating the Jex-dependent fluorescence of the CBNstained cells. The as-fabricated CBNs, especially gCBNs and yCBNs, are internalized and welldecentralized into the cytoplasm region, and most of the quantum dots enter the cell nucleus, demonstrating that gCBNs and yCBNs can efficiently escape from lysosomes, which is crucial for potential biological applications. The nuclei of A549, SMMC7721, and PC 12 cells incubated with gCBNs and yCBNs emit a bright fluorescence, which implies that gCBNs and yCBNs are excellent reagents for cellular nuclear imaging. The merged images of all of the channels are shown in Figure S16. As no morphological change was observed in the cells, the low cytotoxicity of the CBNs to living cells was further confirmed. The as-fabricated CBNs do not show any sign of blinking, photobleaching, or toxicity, which means that they can serve as potential substitutes for organic dyes and semiconductor QDs in bioimaging and biomedical applications. Unique fluorescent property and excellent stability of CBNs make them as potential agents, applied for cellular imaging, which have been studied in this manuscript and on many reports based on other CBNs.2,6,13-15 Herein, we want to endow the novel applications for CBNs, attributing to the presence of abundant carboxyl (-COOH) and amido (-NH2) activity groups on the surface of CBNs. First, CBNs could be conjugated to folic acid (FA), triphenylphosphine (TPP), morpholine group (MG) and/or p-toluenesulfonamide (PTSA) by the formation of -CONH- (the reaction of -COOH and -NH2) and/or "' fff

" (the reaction of -NH2 and -HS by

SMCC-SNa) to fabricate specifically functionalized materials, which could be used for targeting cancer cells, mitochondria, lysosome and/or endoplasmic reticulum, respectively (Figure 7A). Also, the ratio fluorescent probes/sensors can be constructed by FRET between CBNs and fluorescent dyes/small organic molecule or fluorescent nanomaterials (Au 19 ACS Paragon Plus Environment



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4. CONCLUSIONS We report the controllable fabrication of green, yellow, and orange fluorescent CBNs featuring high photostability and excellent biocompatibility using a facile hydrothermal approach. The synthesis condition (precursors, solvent, and atmosphere) could control the fabrication of CBNs, and dissolving solvent could control the dissolution of CBN, giving it unique properties. Detailed characterization suggests that differences in the PL emissions of the quantum dots could be attributed to differences in their quantum size, degree of oxidation and amidation, as well as density of N-states. We proposed a new model of the PL mechanism of the CBNs and suggested a possible method for controllably generating the PL features of CBNs through incorporation of heteroatoms. More importantly, we propose the novel applications of the asprepared CBNs, including apoptotic detection, JC-1 determination, in vitro cellular imaging, conjugation with target molecules, construction of FRET systems and formation of nanocomposite materials, broadening the application ranges in specific biomedical probes, targeted recognition of cancer cells and controlled drug delivery to tumor.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomater. Plots of integrated PL intensity against absorbance of the three CBNs; the quantum yields of the three CBNs dissolved in different solvent; the reaction conditions for fluorescent CBNs; electronic photographs of the three CBNs dissolved in different solvents; UV-vis absorption spectra of the three CBNs under different solvents; peak positions and FQYs of the three CBNs under various solvents; UV-Vis absorption spectra of 1,3-DB, 1,2-DB, 1,4-DB, and PABA; fluorescence decay of the three CBNs; double-exponential fitting of the three CBNs decay curves; effect of UV excitation time and Xe lamp irradiation time on the fluorescence intensity of the three CBNs; particle size distribution histogram and 3D AFM images of the three CBNs; elemental analysis of the three CBNs; FTIR spectra of the three CBNs, PABA, 1,3-DB, 1,2DB, and 1,4-DB; XPS survey scan of the three CBNs; O(1s) XPS spectra of the three CBNs; effect of ionic strengths and pH on the fluorescence intensity of the three CBNs; the optimal excitation and emission spectra of the three CBNs fabricated under air or N2; the inferred structure models of three CBNs; effect of the three CBNs on SMMC7721 cell viability; the JC-1 results of SMMC7721 cells treated with different concentration of the three CBNs; LSCM images of A549, SMMC7721, BEAS-2B and PC 12 cells. 21 ACS Paragon Plus Environment


Acknowledgments This work was supported by the National Natural Science Foundation of China (21705101 and 21575084), China Postdoctoral Science Foundation (No. 2018M642969), Natural Science Foundation of Shanxi Province (No. 201801D121040). We want to express our sincere thanks to Dr. Ying Zuo and Juanjuan Wang of the Scientific Instrument Centre at Shanxi University for taking the AFM images and LSCM measurements.

AUTHOR INFORMATION ORCID Xiaojuan Gong: 0000-0002-2152-2639 Notes The authors declare no competing financial interest.


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Controllable Fabrication, Photoluminescence Mechanism and Novel

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