Ultrastable Amine, Sulfo Cofunctionalized Graphene Quantum Dots

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Ultra-stable amine, sulfo co-functionalized graphene quantum dots with high two-photon fluorescence for cellular imaging Liang Wang, Weitao Li, Ming Li, Qianqian Su, Zhen Li, Dengyu Pan, and Minghong Wu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03797 • Publication Date (Web): 15 Mar 2018 Downloaded from http://pubs.acs.org on March 16, 2018

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ACS Sustainable Chemistry & Engineering

Ultra-stable

amine,

sulfo

co-functionalized

graphene

quantum dots with high two-photon fluorescence for cellular imaging Liang Wang,†,*,¶ Weitao Li,†,¶ Ming Li,† Qianqian Su,† Zhen Li,‡ Dengyu Pan,*,§ Minghong Wu*,‡ †

Institute of Nanochemistry and Nanobiology, School of Environmental and Chemical

Engineering, Shanghai University, No.99 Shangda Road, BaoShan District, Shanghai 200444, P.R. China ‡

Shanghai Institute of Applied Radiation, Shanghai University, No.333 Nanchen Road,

BaoShan District, Shanghai 200444, P.R. China §

Department of Chemical Engineering, School of Environmental and Chemical

Engineering, Shanghai University, No.333 Nanchen Road, BaoShan District, Shanghai 200444, P.R. China To whom correspondence should be addressed: Tel: +86-21-66135276. *E-mail: [email protected] (L. Wang); *E-mail: [email protected] (D.Y. Pan); *E-mail: [email protected] (M.H. Wu). ¶ Liang Wang and Weitao Li contributed equally to this work. ABSTRACT: Fluorescent probes with superior two-photon fluorescence are highly attractive in the field of bioimaging. Herein, we report a one-pot hydrothermal route to synthesis amine, sulfo co-functionalized graphene quantum dots (GQDs), which is acted as efficient one-photon and two-photon fluorescent probes for cellular imaging. As synthesized GQDs exhibit ultra-stability due to the edge-site functionalization of amine and sulfo groups. In addition, the GQDs display attractive two-photon fluorescence properties. The two-photon absorption cross-section of GQDs reaches up to 31000 GM, which substantially exceeds that of the majority traditional fluorescent materials. Furthermore, the non-cytotoxicity GQDs exhibit negligible photothermal effect under 808 nm femtosecond laser irradiation, which is suitable for long-term 1

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two-photon imaging and observation. These findings open new possibilities for using two-photon fluorescent GQDs in various biological applications. Keywords: Two-photon fluorescence, bioimaging, graphene quantum dots, ultra-stability, functionalized INTRODUCTION Recent developments of fluorescent materials including CdSe/ZnS quantum dots,1,2 perovskite nanocrystals,3-5 and dyes6-9 have attracted tremendous attention for their potential applications in chemical sensors,10,11 optoelectronic devices1-5 and biological fields.6-9 Nevertheless, some problematic disadvantages of these fluorescent materials must be taken into account. (1) The commercial dyes possess good optical properties, but they could be easily photobleaching. Furthermore, complex chemical reactions consume a large amount of chemical reagents and numerous instruments.6-9 (2) Heavy metals (Pb, Cd) are embodied in perovskite nanocrystals and CdSe/ZnS quantum dots, restricting their biological applications owing to biosafety issues.1-5 There is consequently an urgent need, but it is still a significant challenge to rationally design fluorophores for deeply biological investigates. Shortly, zero-dimensional graphene material, graphene quantum dots (GQDs) as promising next-generation probes are extensive applied in biosensing12-15 and bioimaging,16-23 because of their eco-friendly, superior photostability, and wide range of fluorescence. Over the past decades, two-photon fluorescent (TPF) probes have been extensively developed9,24-33 due to the low background interference and weakly photobleaching. It is widely known that the most exciting discovery of GQDs30-33 is their admirable TPF property due to their large two-photon absorption (TPA) cross-section, giving them huge potential for TPF biosensing and bioimaging. However, exploration of GQDs for TPF imaging and sensing remains at an initial research stage. Herein, we report the design and synthesis of highly fluorescent amine, sulfo co-functionalized GQDs with controlled surface chemistry via a hydrothermal route. One-photon fluorescence (OPF) and TPF properties of GQDs were systematically investigated. Furthermore, GQDs were applied for efficient one-photon and two-photon 2


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cellular imaging. The findings open the door to new possibilities of GQDs in bioimaging. RESULTS AND DISCUSSION

Figure 1. (a) AFM image and height distribution. (b) TEM image and lateral size distribution. (c) Large area HRTEM image. (d) Single HRTEM image and FFT pattern of the GQDs. The AFM image of GQDs (Figure 1a) that share similarities with the previously reports17-21 displays a high degree of monodispersity. In particular, the thickness of the GQDs was found to be 1.35 ± 0.3 nm, illustrating that the majority of GQDs consists of 4 graphene layers. Figure 1b shows that the GQDs are nearly monodisperse and have a narrow distribution in the average size of 1 nm to 4 nm, which were further confirmed to be good dispersion by High-resolution TEM (HRTEM) (Figure 1c). As shown in Figure 1d, the characteristic spacing of 0.21 nm corresponds to the (100) lattice planes of the graphite. As can be seen from the XRD spectrum of Figure S1a, we noted that the spacing between (002) layer of graphite is broad, which can be attributed to the co-functionalization of NH2 and SO3 groups. The Raman spectrum (Figure S1b) of GQDs includes a G band at 1584 cm-1, and a D band at 1352 cm-1, which is in agreement with the previous reports for GQDs obtained using GO as the precursor.34-36 The G band of GQDs is slightly intense compared to the D band, 3



indicating that the presence of edges groups in the GQDs is advantageous to fluorescence enhancement.17

c

C-OH

3500 2500 1500 Wavenumber (cm-1)

C-S SO3

500

N1s

200 400 600 Binding Energy (eV)

e

C1s

280

Intensity (a. u.)

C-OH

285 290 Binding Energy (eV)

295

390

SO3/2p1/2

800 160

f

N1s

C-C

C-N

Intensity (a. u.)

C-N N-H

C1s S2p

S2p SO3/2p3/2

O1s

N-H N-C

Intensity (a. u.)

C=C O-H

d

Intensity (a. u.)

b

Intensity (a. u.)

a

Intensity (a. u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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410 395 405 400 Binding Energy (eV)

165 170 Binding Energy (eV)

175

O1s O-H

O-C SO3

530 535 540 Binding Energy (eV)

Figure 2. (a) FT-IR spectrum of the GQDs. (b) XPS survey spectrum of the GQDs. (c) XPS S2p spectrum of the GQDs. (d) XPS C1s spectrum of the GQDs. (e) XPS N1s spectrum of the GQDs. (f) XPS O1s spectrum of the GQDs. Surface analysis of the GQDs was carried out using FT-IR and XPS, as displayed in Figure 2. As shown in Figure 2a, the peaks of FT-IR spectrum at 3420 and 1186 cm-1 could be ascribed to the bending vibration of OH and C-OH, while those at 3144 and 1350 cm-1 could be assigned to the stretching vibration of NH and C-N, respectively. Typical SO3 and C-S bands are appeared at 1036 and 617 cm-1, respectively. The bending vibration of the usual C=C group is obtained in 1589 cm-1. XPS analysis further verified the surface groups of GQDs in Figure 2b-2f. The four intense peaks at 167.1, 283.1, 400.7 and 530.3 eV in XPS survey spectrum of the GQDs correspond to the S2p, C1s, N1s, and O1s, respectively. The C, H, and N content of GQDs determined by elemental analyzer is 39%, 6.3%, and 19.3%, respectively. The SO3/2P3/2 and SO3/2P1/2 peak in the high-resolution S2p spectrum are observed at 166.8 and 167.9 eV, respectively. The peaks (283.3, 284.9, and 287.2 eV) for C1s spectrum indicated different carbon chemical environments, i.e., C-C, C-N and C-OH. The values of 400.2 eV (N-C) and 398 eV (N-H) are reported for 4


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GQDs in the N1s spectrum. In the O1s spectrum of the GQDs, the three O element signals, corresponding to O-H, O-C, and SO3, are also observed. This is consistent with the observation from FT-IR spectrum. On the basis of our findings, it can be concluded that the NH2, OH, and SO3 groups are co-functionalized at the site surface of GQDs.

a

b PL

ABS

300

400 500 Wavelength (nm)

390 nm 410 nm 430 nm 450 nm 470 nm

PL Intensity (a. u.)

Intensity (a. u.)

PLE

600

400


700

d 20 PL Intensity (a. u.)

Zeta potential (mV)

c

0 -20 -40 -60

3

4

5

6 7 8 pH value

9

10 11

e

2

4

6 8 pH value

10

12

TPF OPF

400


700

TPF Intensity (a. u.)

f PL Intensity (a. u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

1 2 3 PEXC2 (mW2*105)

4

Figure 3. (a) UV-visible absorption, PL, and PLE spectra of GQDs. (b) PL spectra of GQDs excited at different wavelengths. Dependence of PL intensity (c) and Zeta potential (d) of GQDs on pH values. (e) OPF and TPF spectra of GQDs. (f) Quadratic relationship between the GQD fluorescence intensity and the different excitation laser powers at 800 nm (PEXC, as measured at the focal plane). The optical behaviors of GQDs are then characterized and discussed as follows. As 5



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presented in Figure 3a, direct absorption peaks appear at 288 nm due to π−π* transition of aromatic C=C bond and 376 nm assigned to an n-π* transition of the aromatic sp2 domains containing C-O and C-N bonds in UV-vis absorption spectra of GQDs,37 with an optical absorption edge at about 480 nm. Photoluminescence (PL) and PL excitation (PLE) spectra of GQDs are also shown in Figure 3a. The maximum emission wavelength located at 520 nm with a full width at half maximum 105 nm was observed. The PLE spectrum reveals two peaks at 310 and 440 nm. The quantum yield of cyan PL was calculated to be 33% (Figure S2), which is considerably higher than the most reported values.38,39 The PL maximum of the GQDs shifted only 5 nm when the excitation wavelength changed from 390 to 470 nm (Figure 3b). The phenomenon exhibited excitation wavelength-independence behavior of GQDs due to the monodisperse size of GQDs40. Moreover, a monoexponential decay feature of GQDs was observed with lifetime of 4.6 ns (Figure S3). The alkali and acid resistance property of GQDs in aqueous solution enables new opportunities of using GQDs in different application environment. As shown in Figure 3c, the PL intensity of GQDs was relative stable in alkaline and weak acid condition. It is helpful to note that the GQDs with acid resistance has a promising prospect of application in vitro and in vivo imaging for the reason that the pH value of physiological conditions ranging from 6.5 to 7.4.41 Besides, the Zeta potentials of GQDs in water at different pH values were all negative charged (Figure 3d), due to the existence of a large amount of negatively charged OH and SO3 groups on the edge sites of GQDs, which has illustrated by the FT-IR and XPS survey spectrum of GQDs. A set of experiments were carried out to investigate the aggregation behavior of GQDs by controlling temperature, illumination, storage time and solid re-dissolution conditions.

As shown in Figure

S2 and S4-S7. Compared with

other

group-functionalized GQDs23 (Figure S4), the synthesized GQDs powder re-dispersed in solution remained high PL QYs up to 30% due to the SO3 group protection, despite the fact that the GQDs were annealed at the temperature as high as 250 oC (Figure S2). Besides, the GQDs show high optical stability without being photo-bleached under lasting UV irradiation for several hours (Figure S5). Furthermore, The GQDs exhibit 6


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high water solubility in water and buffer solution without obvious fluorescence quenching phenomenon, even after storing at room temperature for seven days (Figure S6) or even three weeks in water (Figure S7). Meanwhile, the size of GQDs in water and buffer solution tested at seventh day are agree with that at first day (Figure S8), which is illustrated that the GQDs in water and buffer solution are qualified to be stored for long times at room temperature. In addition, the GQDs solution displayed high solid re-dispersion stability dried at 80 oC (Figure S9). Our ﬁndings suggested that the sulfonic functionalization of GQDs might render them a strong protection for improving their stability. Furthermore, TPF property of GQDs was systematically investigated on a self-established optical platform using a femtosecond (fs) pulse laser (800 nm). As shown in Figure 3e, similarly to the OPF, TPF spectra with λmax=520 nm was observed under excitation at 800 nm using the fs pulse laser. As demonstrated in the Figure 3f, the apparent linearly quadratic relationship between the fluorescence intensity of GQDs and the excited laser power density was observed, indicating that the TPF process is accurately responsible for the emission of GQD in nature. To further evaluate the optical performance of the GQDs for TPF, the TPA cross section of GQDs was measured using equation S1 published in previous reports.26,32,42 The TPA cross section of GQDs was recorded to be as high as 31000 GM with excitation at 800 nm. The observed large TPA cross section can be ascribed to the highly efficient intramolecular charge transfer between large π-conjugated systems of GQDs and the strong electron denoting OH and NH2 groups. This charge transfer process could enhance the TPA cross section significantly.32 For comparison, TPA cross section of the as-prepared GQDs, traditional fluorescent materials26,43-45 and other GQDs32,40 reported by the previous works are listed in Table S1, the TPA cross section of as-prepared GQDs in this study substantially exceeded that of the majority of traditional fluorescent materials, which is high enough to be qualified for two-photon

bioimaging

and

biosensing.

Furthermore,

the

GQDs

display

non-cytotoxicity even at high concentrations and long incubation time (Figure S10), which is suitable for long-term observations in living cancer cells. 7



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Figure 4. Merged image (a) and image (b) of Hela cells treated with GQDs under 405 nm excitation. Two-photon cell imaging under 800 nm excitation (c, d). Due to the astonishing optical properties with one-photon and two-photon excitation, the non-cytotoxicity GQDs were utilized in cellular imaging. Figure 4a,b demonstrated that the GQDs were internalized by the cytoplasm of Hela cells at 405 nm excitation. The localization of GQDs were determined by means of co-stain with a nucleus-tracker dye (Figure S11) and z-axis scanning cell imaging (Figure S12). As shown in Figure 4c,d, the GQDs distributed the cytoplasm position with excitation at 800 nm as proved by high contrast fluorescence image, suggesting that the GQDs can stain the cytoplasm of Hela cells under two-photon excitation. For safety considerations of two-photon excitation, the photothermal experiments were measured (Figure S13). The temperature of the GQDs solution (10 mg/L) after 808 nm irradiation for 10 min changes slightly, indicating a negligible photothermal effect as the same as that of the pure water. Therefore, the GQDs is suitable for long-term TPF imaging and observation.

8


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1.5

a

b -6 Eneerg (eV)

1.0 Current (mA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.5 -0.0 -0.5 -1.0 -1.5

-1.46 V -2.0

1.0 -1.0 0.0 Potential (V)

LUMO

-5 2.9 eV -4

π

-3

σ

4.13 eV HOMO δE = 1.23 eV

2.0

Figure 5. (a) The CV of the GQDs. (b) Fluorescence mechanism of the GQDs. The origin of common PL is directly attributed to a carbene-like triplet ground state described as σ1π1, which have been demonstrated in our previous work39. The reduction potentials of the GQDs determined by CV were −1.46 eV (Figure 5a). Therefore, the ELUMO of the GQDs was calculated to be −2.94 eV according to the equation S2. The two electronic transitions were observed in the PLE spectra of the GQDs (Figure 3a), which denoted the transitions from the ground state to σ (300 nm, 4.13 eV) and π orbitals (427 nm, 2.9 eV), respectively. The corresponding EHOMO was calculated to be −5.84 eV using the equation S3. The emission mechanism is demonstrated in Figure 5b. For the triplet carbene structure, it is worth noting that energy difference (δE) between the σ and π orbitals should be below 1.5 eV46. The δE was thus calculated to be 1.23 eV, falling within the expected range (

Ultrastable Amine, Sulfo Cofunctionalized Graphene Quantum Dots

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