Nitrogen-Rich D-Ï-A Structural Carbon Quantum Dots with a Bright

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Nitrogen Rich D-#-A Structural Carbon Quantum Dots with Bright Two-Photon Fluorescence for Deep-Tissue Imaging Fanglong Yuan, Yunchao Li, Xiaohong Li, Jia Zhu, Louzhen Fan, Shixin Zhou, Yiran Zhang, and Jiangbing Zhou ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00276 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 30, 2018

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ACS Applied Bio Materials

Nitrogen Rich D-π-A Structural Carbon Quantum Dots with Bright Two-Photon Fluorescence for Deep-Tissue Imaging Fanglong Yuan,a Yunchao Li,a Xiaohong Li,a Jia Zhu,* a Louzhen Fan,* a Shixin Zhou,b Yiran Zhang,c and Jiangbing Zhou* c ABSTRACT: Two-photon fluorescent (TPF) probes, wh ich allo w imaging of bio logical events in high spatiotemporal resolution, are in great demand. Recently, carbon quantum dots (CQDs) have emerged as a promising class of TPF probes. Unfortunately, the use of the existing CQDs has been limited by their weak TPF capacities. Herein, we report the first facile and large-scale synthesis of nitrogen rich CQDs (NRCQDs) based on a donor-π-acceptor (D-π-A) strategy. The resulting NRCQDs demonstrated a tremendous TPF capacity with two-photon absorption cross-section (TPACS) and quantum y ield (QY) up to 61200 Göppert-Mayer (GM ) units and 63%, respectively, which is greater than those that could be achieved by the existing TPF carbon probes. Structural and optical analyses of NRCQDs revealed that the great TPF capacity is contributed by the nitrogen rich D-π-A structure as well as the high crystallinity, large plane, rig id, graphitic n itrogen doped π-conjugated system. We further demonstrated that NRCQDs allo w imaging of live cells as well as live liver tissues at depths of up to 440 μm. Our results suggest NRCQDs as a robust TPF probe that can be potentially used for a variety of biological a pplications. KEYWORDS: two-photon, carbon quantum dots, nitrogen rich, donor-π-acceptor, absorption cross-section, biological applications

Introduction Due to their great penetration depth, limited background signal, and reduced photodamage and photobleaching , two-photon fluorescent (TP F) probes with high two-photon absorption cross-section (TPACS) and quantum yield (QY) have great potential in studying biological events in living cells and tissues. 1 -3 Over the past many years, a variety of TP F probes, most of which are derived from organic dyes , semiconductor quantum dots (QDs) or rare earth element doped QDs, have been developed. 4-6 Unfortunately, a majorit y of the existing probes suffer from low TP F capacity, high photobleaching rate, and/or high toxicity due t o the presence of heavy metals, and may not be suitable for biologica l applications. 4-6 Therefore, the development of novel probes with great TP F capacity, biocompatibility and photostability, and limited toxicity is in high demand. Recently, carbon quantum dots (CQDs) have emerged as new alternatives to the traditional organic dye - and semiconductor QD- based TP F probes. Compared to the traditional probes, CQDs have many advantages in that the y typically have greater optical properties, biocompatibility and photostability, and lower toxicity. In addition, CQDs contain plenty of active groups, which allow for easy chemica l functionalization for biomedical applications.7-2 6 CQD-base d TP F probes were first reported by Sun and colleagues. 2 7 I n that study, the authors prepared CQDs through laser ablation of graphite powder followed with surface passivation, and evaluated them for TPF imaging in human breast cancer MCF-7 cells. In another study, Gong and colleagues prepare d CQDs through hydrothermal treatment of graphene oxide (GO)

in dimethylformamide, and applied them for cellular imaging as well as for deep-tissue imaging with intralipid as moc k tissue.2 8 However, despite being promising, the CQDs documented in these studies have low TP F capacity, wit h TP ACS and QY limited to 48000 Göppert-Mayer (GM) units and 20%, respectively. In comparison, TP ACS and QY for well-developed semiconductor QDs are ~60000 GM and >50%, respectively. 27 -3 3 In addition, in both studies, the preparation of CQDs involves multiple steps, which limits the yields to a milligram scale. Therefore, the CQDs documente d in the literature remain unideal for wide biologica l applications. Herein, we report the first facile and large -scale synthesis of nitrogen rich CQDs (NRCQDs) with bright TP F based on a donor-π-acceptor (D-π-A) strategy. NRCQDs were synthesized through a one-pot hydrothermal approach using citric acid (CA) and guanidine carbonate (GC) as carbon and nitrogen sources, respectively. The yield of NRCQDs is up t o 65%, which is suitable for industrial-scale production. The NRCQDs had a TPACS of about 61200 GM units and a QY of 63%, both of which are greater than those of existing TP F carbon mate rials. Structural and optical characterizations revealed that the nitrogen rich D-π-A structure and the high crystallinity, large plane, rigid, graphitic nitrogen dope d π-conjugated system are key to the bright TP F. We further demonstrated that a large imaging depth of up to 440 μm for live rat liver tissues could be achieved by NRCQD-based TP F imaging.

Experimental section Materials and cell culture

a

College of Chemistry, Beijing Normal University, Beijing, 100875, China.

b

Department of Cell Biology, School of Basic Medicine, Peking University

Health Science Center, Beijing, 100191, China c

Department of Neurosurgery, Yale University, New Haven, CT, 06510, USA.

E-mail: [email protected], [email protected], [email protected]

Human cervix carcinoma cell line, HeLa, was obtained from American Type Culture Collection (ATCC) and cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 units ml-1 penicillin, and 100 μg ml-1 streptomycin in a 37°C incubator containing 5% CO 2. All chemicals were purchased from Sigma -Aldrich unless otherwise noted.

Synthesis of NRCQDs

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Paper For a typical synthesis, 1g CA and 1g GC (mass ratio 1:1) were dissolved in 10 mL deionized water. The mixed solution was then transferred to a poly (tetrafluoroethylene) (Teflon)-lined autoclave (25 mL) and heated at 200 ºC for 4 h. After the reaction, the reactant was cooled down to room temperature. The resulting brown-black product was diluted t o 100 mL, and subjected to dialysis in deionized water for tw o days to obtain NRCQDs. The production yield for NRCQDs was 65%. Other different kinds of NRCQDs were prepare d with different mass ratios of CA to GC from 1:0 to 1:1.5.

Measurement of QYs QYs were determined using a Varian FLR025 spectrometer equipped with a 120 mm integrating sphere (Edinburgh Instruments). Briefly, NRCQDs were resuspended in aqueous solution and placed into a UV quarts cuvette with a light pat h of 10 mm. The same amount of water without NRCQDs was used as a reference control. The spectral correction curves were determined, based on which QYs were calculated as a ratio of photons emitted to photons absorbed.

Calculation of TPACS TP ACS was measured using rhodamine B as a reference base d on equation σ2 =σ 1 ×(I 2/I1)×(ϕ 1/ϕ2 )×(C1 /C2), where I, ϕ, C represent observed integrated TP F intensity, quantum yields , and concentration, respectively, and subscripts 1 and 2 denote values for rhodamine B and a given NRCQDs, respectively.

Physical characterization of NRCQDs Atomic force microscopic (AFM) images were captured using a MultiMode V SP M (VEECO). Morphology of NRCQDs was visualized by a JEOL JEM 2100 transmission electron microscope (TEM). X-ray diffraction (XRD) patterns were determined by X-ray diffraction using Cu-Ka radiation (XRD , PANalytical X’Pert P ro MP D). Fluorescence spectra of NRCQDs were acquired using a PerkinElmer-LS55 luminescence spectrometer with slit width at 2.5-2.5 nm. Absorption spectra were recorded using a UV-2450 spectrophotometry. Fourier transform infrared (FTIR) spectra were determined using a Nicolet 380 spectrograph. X-ra y photoelectron spectroscopy (XP S) was carried out by a n ESCALab220i-XL electron spectrometer from VG Scientific using 300 W Al Ka radiation. Raman spectrum was acquire d using a Laser Confocal Micro-Raman Spectroscopy (LabRA M Aramis). Fluorescence lifetimes of NRCQDs were determine d with a time -correlated single -photon counting (TCSP C) system under right-angle sample geometry. A 345 nm picosecond diode laser (Edinburgh Ins truments EP L375, repetition rate 2 MHz) was used to excite NRCQDs in aqueous solution.

Cytotoxicity evaluation Toxicity of NRCQDs was determined according to the standard dimethyl thiazolyl diphenyl tetrazolium salt (MTT) assay. Absorption was measured us ing a Victor3 V Multilabe l reader (P erkinElmer, U.S.A). The percentage of cell viabilit y was expressed relative to the control (untreated) cells.

TPF imaging

For TP F imaging in cell culture, HeLa cells were used. Twenty-four h after plating, cell culture medium was replace d with fresh DMEM containing NRCQDs at 0.04 mg/ml. Six h later, cells were washed three times with PBS and subjected t o TP F imaging. For TPF imaging in rat liver tissues , immediately after euthanization, the livers were isolated and sliced using a vibrating-blade microtome. Liver slices were incubated with NRCQDs at 0.04 mg/ml in artificia l cerebrospinal fluid (ACSF) bubbled with 95% O 2 and 5% CO 2 for 60 min at 37 °C. After washing with ACSF for three times , the slices were transferred to glass-bottomed dishes for TP F imaging.

Results and discussion NRCQDs were synthesized according to procedures outlined in scheme Figure 1a (for more details, see the experimental section). At the initial stage, the reaction of CA and GC in aqueous solution forms nanosheets through intermolecular H-bonding. NRCQDs were obtained after further dehydration and carbonization under violent hydrothermal condition. 34,3 5 NRCQDs in aqueous solution appear dark-brown in color (Figure 1b), and emit blue light even upon excitation by daylight (Figure S1). The emission of blue light significantly increased upon excitation by UV light (365 nm, Figure 1c). The yield of NRCQDs is 65%, which is suitable for industrial-scale production (Figure 1d).

Figure. 1 (a) Schematic illustration of the synthesis of NRCQDs by hydrothermal treatment of CA and GC. Photographs of NRCQDs aqueous solution under daylight (b) and fluorescence images under UV light (excited at 365 nm) (c). (d) Optical photograph of the NRCQDs powder synthesized in large quantities (about 5 g) under daylight.

Excitonic absorption and fluorescence of NRCQDs peak at ~335 and ~439 nm, respectively (Figure 2a, Figure S2). The photoluminescence (PL) of NRCQDs can be excited in a wide range of wavelengths. Regardless of the excitation wavelength, the maximum emission peak is well confined (Figure S3). The maximum excitation wavelength agrees well with the corresponding excitonic absorption peak (Figure 2a), suggesting


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Paper that the emission is characterized by band-edge exciton-state decay rather than defect-state decay.17-20 Time-resolved PL spectra analysis revealed that, unlike previously reported CQDs that have multi-exponential decay,17-20 NRCQDs exhibit monoexponentia l decay with a fluorescence lifetime of 14.21 ns (Figure S4). This result suggests that the excitons of NRCQDs are highly stable and the radiative decay is in high purity with minimum nonradiative components. Upon excitation at 800 nm by a femtosecond pulse laser, NRCQDs emit TPF, which peaks at ~ 480 nm (Figure 2b). The bright green TPF emission can be perceived with the naked eye (the inset of Figure 2b). We characterized the TPF property of NRCQDs by measuring their TPACSs at a series of wavelengths ranging from 720 to 880 nm with 20 nm increments in aqueous solution. Remarkably, we found that NRCQDs display maximum TPACS of 61,200 GM units at 780 nm with high QY of 63% (Figure 2c). We have selected the green fluorescent fluorescein as a second standard for the measurement of the TPACS of NRCQDs, which was determined to be about 60140 GM, slightly smaller than that of 61200 GM determined with Rhodamine B as standard. These results confirm that the as-prepared NRCQDs do show rather high TPACS (60000 GM at least). This degree of TPACS, which is greater than those which can be achieved by well-known heavy-metal containing semiconductor QDs (~60000 GM, QY>50%),27 -33,36,37 has not been previously reported for TPF carbon materials. We monitored the change of the green fluorescence intensity under a range of power supplies (Figure S5). Results in Figure 2d reveal a quadratic correlation between the integrated fluorescence intensity and the excited laser power, suggesting that the simultaneous absorption of two near-infrared photons is responsible for the bright green fluorescence in nature. 3

Figure. 2 (a) UV-vis absorption (black), PL excitation (green), and emission (blue) spectra of NRCQDs in aqueous solution. (b) TPF spectrum and image (inset) of NRCQDs in aqueous solution under 800 nm femtosecond pulse laser excitation. (c) The TPACS values of NRCQDs in aqueous solution under femtosecond pulse laser excitation with different wavelengths. (d) Quadratic relationship of the integrated TPF intensity of NRCQDs in aqueous solution with the different excitation laser powers at 800 nm.

We characterized the physical characteristics of NRCQDs. TEM analysis found that NRCQDs are uniform in size with a n

average diameter of 2.2 nm (Figure 3a-b). High-resolution TEM (HRTEM) image (insets of Figure 3a) revealed that NRCQDs exhibit uniform atomic arrangements and a high degree of crystallinity with lattice fringes (spacing: 0.21 nm) corresponding to that of graphene (100) planes. 12, 17 AF M images (Figure 3c-d) confirm the uniform distribution of nanoparticles with topographic height ranging from 0.4 to 1.2 nm, suggesting that most of NRCQDs consist of ca. 1-3 graphene layers.12,17

Figure. 3 TEM and HRTEM (inset) images (a) and the corresponding size distribution (b) of NRCQDs. AFM (c) and corresponding height (d) images of NRCQDs.

XRD patterns of NRCQDs show a narrow (002) peak centered at ~23°(Figure 4a), which confirms the high crystallinity structure of NRCQDs. The high degree of graphitization of NRCQDs is reflected in the Raman spectra (Figure 4b), where the crystalline G band at 1610 cm-1 is much stronger than the disordered D band at 1380 cm-1.12, 17 A large G to D intensity ratio (IG/ID) is 1.7. The chemical composition and detailed structure of NRCQDs were comprehensively studied by FTIR and XPS. As shown in Figure 4c, the strong stretching vibration bands for O-H, N-H C=C, C=O, C=N and C-N were observed at 3420, 3190, 1655, 1720, 1580 and 1290 cm-1 , respectively. This indicates the presence of amino (NH 2) and imino (NH), as well as carboxyl (COOH) and hydroxyl (OH) groups at the edge sites of NRCQDs. 17-19 The zeta potential of NRCQDs was determined to be about 8.14 mV, als o suggesting the surface functionalization of NRCQDs wit h numerous nitrogen containing groups. The full scan XPS spectrum revealed a high nitrogen content in NRCQDs with an atomic percentage of 17.67% (Figure 4d), which is significantly greater than that for previously reported CQDs (< 6%). 17 -19 The high resolution C 1s and N 1s spectra ( Figure 4e-f) further confirmed the functionalization of amino, imino, carboxyl and hydroxyl groups at the edge sites of NRCQDs. The presence of C=C (284.5 eV), C-N (285.4 eV) and C=N (287.8) bonds , and several oxygen-bearing functional groups, including C -O (286.1 eV), C=O (287.8 eV) and O-C=O (288.8 eV), were observed (Figure 4e). Furthermore, the N 1s spectrum



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Paper (Figure 4f) identified the nitrogen form of C=N -C, C-NH2 , C=NH and C3-N, which are centered at 398.9, 399.4, 400. 2 and 401.1 eV, respectively. 1 7-19

test this, we first evaluated NRCQDs for cell imaging using HeLa cells. After 6 h incubation with NRCQDs, HeLa cells were extensively washed and imaged using a microscope wit h excitation at 800 nm. Results in Figure 5a-c showed that NRCQDs efficiently penetrated HeLa cells and distribute d within the cytoplasm without penetration into the nucleus (Figure 5b), which was confirmed by Z-axis confoca l imaging (Figure S6). NRCQDs exhibited limited toxicity t o HeLa cells. Treatment of NRCQDs at concentrations up t o 500 μg/mL for 24 h did not significantly reduce cell viabilit y (Figure 5d).

Figure. 4 XRD pattern (a) and Raman spectrum (b) of NRCQDs. (c) FTIR spectra of CA, GC and NRCQDs. XPS full spectrum (d), high-resolution C 1s (e) and N 1s (f) spectra of NRCQDs.

Two-photon active organic probes typically contain three essential components, including π -electron donor, π-electron acceptor, and rigid π-bridge. The TP ACS of probes depends on the number of each component, the electron donating/accepting ability, and the degree of conjugation in the π-conjugated system. 3 Our data suggest that numerous strong electron-donating groups (amino, imino and hydroxyl) and electron-accepting groups (carboxyl) are functionalized at the edge sites of NRCQDs, which could act as π-electron donors and acceptors, respectively. The high crystallinity rigid graphitic nitrogen (C3 -N) doped aromatic π-conjugate d structure which could act as a elaborate rigid π-bridge couple d with the π -electron donors and acceptors make the whole of NRCQDs a plane rigid high crystallinity nitrogen rich D-π-A structure. The highly enriched strong electron-donating groups in the D-π-A structure greatly increase the electron cloud density and facilitate the radiative recombination of confine d electrons and holes, leading to significant improvement in QY. There are also numerous strong electron-donating groups wit h lots of lone pair electrons. These groups are capable of forming p-π conjugation with the aromatic rings. Consequently, the π-conjugated system can be further expanded, leading to further improvement of TP F performance. In addition, the nitrogen elements in graphitic nitrogen form within NRCQDs are capable of markedly improving the mobility and intramolecular charge transfer efficiency, and, thus, exponentially enhancing the TP ACS. 3 Moreover, the TP ACS and QY of different kinds of NRCQDs prepared with different mass ratios of CA (carbon source) t o GC (nitrogen source) from 1:0 to 1:1.5 are in the range of 900-61200 GM and 8-81%, respectively, as shown in Table S1, which indicates that the appropriate high nitrogen content in NRCQDs leads to the best TP F performance by forming well defined D-π-A structure with the elaborate balance of the strong electron-donating and electron-withdrawing groups. Bearing such a tremendous TP F capacity, NRCQDs might have great potential for living cell and deep-tissue imaging. T o

Figure. 5 TPF imag ing of Hela cells under bright fie ld (a) and 800 n m femtosecond pulse laser excitation (b ). (c) The merged image o f a and b. (d) Cell v iability after incubation with different concentrations of NRCQDs for 24 h.

Figure. 6 TPF imag ing of a fresh live rat liver t issue slice stained with NRCQDs taken at different penetration depths (0-440 μm) with 800 n m femtosecond pulse laser excitation.

We next evaluated NRCQDs for deep-tissue imaging using rat liver tissues. Liver tissues were selected as they have strong scattering and refraction of visible excitation light and are challenging for light imaging. Results in Figure 6 suggested that NRCQDs allow TPF imaging of liver tissues at


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Paper depths of up to 440 μm. The strongest fluorescence signals could be observed at the depths of approximately 200 μm, which may be ascribed to a large concentration of NRCQDs due to aggregation effect. More further deep understanding is currently studied underway. By contrast, one-photon fuorescence (OPF) imaging penetrated liver tissues by no more than 30 μm (Figure S7). The TPF imaging depth of 440 μm achieved by NRCQDs exceeds the depths achieved by organic dyes (400 μm).38-42 Finally, we investigated the photostability of NRCQDs. Photostability is an important parameter for TPF imaging, especially for long-term dynamic bioimaging. 3 To determine the photostability, we monitored the changes of fluorescence intensity after NRCQDs were excited by a femtosecond pulse laser at 800 nm for multiple times or exposed to long-term UV irradiation. Results in Figure 7a-c showed there were no significant changes in the fluorescence intensity after 100 times continuous excitations/scans or after 3 h continuous UV irradiation. The NRCQDs maintained high photostability across a wide pH range, with emission intensity just slightly decreased in the strong acidic or alkaline conditions, and emission peaks unchanged (Figure S8), which demonstrated the great potential of NRCQDs for a wide pH range biological applications.

Supporting Information The Supporting Information is available free of charge on the ACS Publications websites. The photographs of NRCQDs under daylight; UV-vis absorption spectra of CA and GC in aqueous solution; PL spectra of NRCQDs excited at different wavelengths; time-resolved PL spectra of NRCQDs; TPF spectra of NRCQDs under 800 nm femtosecond laser excitation with different laser powers; TPACS and QY of different kinds of NRCQDs prepared with different mass ratios of CA to GC from 1:0 to 1:1.5; TPF images of Hela cells incubated with NRCQDs at different Z positions; OPF imaging of fresh rat liver slice stained with NRCQDs taken at different penetration depths; fluorescence spectra and pH-dependent fluorescence emission intensity of NRCQDs at different pH values; fluorescence spectra and photostability of NRCQDs in water, PBS and serum.

Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by NSFC grant 21573019, NSFC Major Research P lan grant 21233003, Central Universities Fundamental Research Funds, and NIH grant NS095817.

References

Figure. 7 (a, b) Photostability of NRCQDs under 800 nm femtosecond pulse laser excitation with different scans. (c) Time-dependent fluorescence intensity behavior of NRCQDs under UV irradiation for 3 h.

Conclusions We have developed procedures for fabrication of nove l NRCQDs in a industrial scale based on a D-π-A design strategy. The resulting NRCQDs demonstrated a tremendous TP F capacity with TP ACS and QY up to 61200 GM units and 63%, respectively, which has not been previously achieved by carbon mate rials. Structural and optical characterizations revealed that the nitrogen rich D -π-A structure and the high crystallinity, large plane, rigid, graphitic nitrogen dope d π-conjugated system are responsible for the observed TP F property. We demonstrated that NRCQDs have limite d cytotoxicity and allow imaging of live cells as well as live rat liver tissue at depths of up to 440 μm. In summary, due t o their great TP F property, deep tissue penetrability, and minimal toxicity, NRCQDs have the potential to be widely used as a robust TP F probe for a variety of applications in biological systems. The research of NRCQDs for TP F dee p tumor tissue imaging and a combined two-photon excitation photodynamic and photothermal therapy in living mice is currently underway in our lab, which will be reported in due course.

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