(8)石墨烯量子点的红色,黄色和蓝色发光：合成,机理和细胞成像 001

Supporting InformationRed, Yellow, and Blue Luminescence by Graphene Quantum Dots: Syntheses, Mechanism, and Cellular ImagingTian Gao†, Xi Wang†, Li-Yun Yang†, Huan He†, Xiao-Xu Ba†, Jie Zhao†, Feng-Lei Jiang*,†, Yi Liu*,†,‡,§†State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry for Biology and Medicine, College of Chemistry and Molecular Sciences,Wuhan University, Wuhan 430072, P. R. China‡ School of Chemistry and Chemical Engineering, Wuhan University of Science and Technology, Wuhan 430081, P. R. China.§ College of Chemistry and Material Science, Guangxi Teachers Education University, Nanning 530001, P. R. China.*Corresponding author: yiliuchem@ (Y. Liu);fljiang@ (F. L. Jiang)1.Syntheses process of polyethyleneimine (PEI)-coated graphene quantum dots (GQDs)Simple and normal low temperature heating methods are adopted to synthesis PEI-coated GQDs. (Figure S1) At beginning, yellow photoluminescent (PL) GQDs are prepared from carbon black by acid assisted oxidation exfoliation with a serials of purification methods. To synthesis PEI-coated GQDs (PEI1800 GQDs and PEI600 GQDs), yellow GQDs and PEI were mixed together in ultrapure water, and the solution was heated to boiling temperature. After the gel formed, 10 mL ultrapure water was added in drop by drop to prevent it from drying and scorching. Then after adding about 3 times of ultrapure water, the PEI-coated GQDs were successfully prepared. By using PEI with different molecular weight, both blue and red PL PEI-coated GQDs can be obtained.Figure S1. Synthesis of PEI-coated GQDs with different PL emission colors.parison the powders of three different kinds of GQDs.Figure S2. Photos of GQDs and PEI-coated GQDs powder: (a) Uncoated GQDs; (b) PEI1800 GQDs; (c) PEI600 GQDs. All of them are pure and fluffy.3.Excitation dependent PL property of the Red light-emitting PEI600-coated GQDs and emission spectra of bare GQDs and PEI-coated GQDs under 365 nm After normalizing the PL intensity of the emission peaks under different excitation wavelength, it is clearly that the emission peaks red shifted (Figure S3a) with the increasing of excitation wavelength in a large scale. As shown in Figure S3b, the PL peaks under 365 nm of bare GQDs and PEI-coated GQDs located at different color region.Figure S3. (a) Excitation-dependent PL property of PEI600 GQDs (PL intensity normalized); (b) emission spectra of bare GQDs and PEI-coated GQDs under 360 nm (PL intensity normalized).4.XPS spectra of bare GQDs and PEI-coated GQDsAs shown in Figure S4a, b and c, apparently strong O 1s, N 1s and C 1s peaks can be seen both in the survey spectra of PEI1800 GQDs and PEI600 GQDs, while there is no obvious N 1s peak in that of bare GQDs. Figure S4d, e and f exhibit the C 1s high-resolution spectra, Figure S4g, h and I demonstrate the N 1s high-resolution XPS (HRXPS) spectra and Figure S4j, k and l show the O 1s high-resolution spectra of blue emitting PEI1800 GQDs, yellow emitting bare GQDs, and red emitting PEI600 GQDs, respectively. In the XPS data analyses, peak de-convolutions were performed by using a Gaussian-Lorentzian peak shape after a Shirley background subtraction. From C 1s spectra, it is evidently that there is a –COOH peak at 288.5 eV in the spectrum of bare GQDs. However, in the spectra of PEI1800 GQDs and PEI600 GQDs, there are no –COOH existed, but new –C-N peaks appear at 285.8 eV and –C=O peaks at 287.6 eV. Furthermore, it is easily to find that the N 1s spectrum of bare GQDs does not show apparent N 1s peaks, but which of both PEI1800 GQDs andPEI600 GQDs possess apparent N 1s peaks for –CH2NH2 (around 398.3 eV) and –CONHR (around 399.6 eV). Thus, take both C 1s and N 1s spectra into account, it can prove that carboxyl function groups (-COOH) changed to amide function groups (-CONH-) during the coating reactions. The results are consistent with the observation from FTIR spectra of the bare GQDs and PEI-coated GQDs.Figure S4. XPS spectra and C 1s, N 1s, and O 1s high-resolution XPS (HRXPS) spectra of GQDs and PEI-coated GQDs. (a), (b), and (c) are survey XPS spectra of PEI1800 GQDs, bare GQDs, and PEI600 GQDs, respectively. C 1s HRXPS spectra of PEI1800 GQDs (d), bare GQDs (e), and PEI600 GQDs (f) reveal that the carboxyl function group changed to amide bond during the coating reaction. From N 1s HRXPS spectra of PEI1800 GQDs (g), bare GQDs (h), and PEI600 GQDs (i), it is foundthat –NH2 and C-N bond only exist in PEI-coated GQDs. In additon, O 1s HRXPS of PEI1800 GQDs (j), bare GQDs (k), and PEI600 GQDs (l) are presented.5.Elemental content analyses of GQDs and PEI-coated GQDs by elemental atomic ratio from XPSTable S1 exhibits the (N 1s)/(C 1s) and (O 1s)/(C 1s) atomic ratios of GQDs and PEI-coated GQDs. For yellow-emitting bare GQDs, there were little nitrogen element and rich of oxygen function groups. However, after coating reaction, both of blue-emitting PEI1800 GQDs and red-emitting PEI600 GQDs possess a high nitrogen/carbon ratio and lower oxygen/carbon ration because of the carboxyl function group transformed into amide function groups during the reaction. For PEI, the (N 1s)/(C 1s) ratio is about 2, while in bare GQDs, the ratio is 0.038, so when the PEI linked to the surface of GQDs, the (N 1s)/(C 1s) would increase a lot. At the same time, the (O1s)/(C 1s) atomic ratio decreases, because there is no oxygen in PEI, and during the reaction, -COOH changed to –CONHR, which lost an oxygen atom in every amidation. Comparing the ratios between blue-emitting PEI1800 GQDs and red-emitting PEI600 GQDs, we can find that the red-emitting GQDs have a higher nitrogen/carbon ratio but lower oxygen/carbon ratio, which means that more short-chain PEI600 molecules were coated on the same amount bare GQDs than that of long-chain PEI1800 molecules. Moreover, it can further confirm that the structure of PEI600 GQDs is like a big cage that contains more PEI units.Table S1. (N 1s)/(C 1s) and (O 1s)/(C 1s) atomic ratios determined from the full-range XPS spectra of bare PEI1800 GQDs, GQDs, and PEI600 GQDsN 1s / C 1s ratio O 1s / C 1s ratio PEI1800 GQDs 19.6% 24.7%Bare GQDs 3.8% 59.5%PEI600 GQDs 21.1% 16.1%6.XRD spectra of GQDsAs exhibited in Figure S5, It is apparently to find the XRD peaks for both bare GQDs and PEI-coated GQDs. For bare GQDs, there is a comparable narrow peak around 23.1° in the spectrum, which coincided with weak broad (002) facet of graphite structure.1-2 However, this peak moved to 20° when PEI coated on the surface of GQDs in PEI1800 GQDs and PEI600 GQDs systems. The decrease of the 2-θ degree indicated that the lattice parameter increased, resulted from the adding of disordered carbon and nitrogen atoms from PEI chain. That further manifested that stable core-shell structures formed after the coating reaction.Figure S5. XRD spectrum of GQDs. An obviously peak shift from 23.1° to 20° occurred after the coating reaction.7.Zeta potential of GQDsIt is clearly that the GQDs are electronegtive with -57.27 mV and the PEI-coatedGQDs are almost electroneutral.Figure S6. Zeta potential of GQDs and PEI-coated GQDs.8.Schematic diagram of the key reaction that affect the PL property most. Before coating reaction, there were numerous carboxyl function groups on the surface of GQDs, and the C=O part could conjugate with the inner graphene core. Thus, the electron can transfer between them freely. While, after amidation, the atoms in amide bond were in co-plane and blocked the electron transfer between inner core and the outside chain. Thus, the amidation reaction changed the conjugation modes of PEI-coated GQDs.Figure S7. Schematic diagram of the influence of function group change.9.Theoretical calculation of a serials of GQDsIt is quite difficult to study a single nanoparticle by traditional methods, so the theoretical calculation is an excellent method to understand the PL mechanism. As shown in Figure S8, the conjugation mode between the two functional groups and inner graphene core was calculated, respectively. By theoretical calculation with density functional theory (DFT) B3LYP/6-31G*, the molecular orbitals of amide-GQD G1 (C50H21NO) (Figure S8a), carboxyl-GQD G2 (C49H18O2) (Figure S8b) and G1-N (C53H28N2O) (Figure S8c) were obtained. G1 and G2 are different in the linking groups and G1-N simulates a real PEI branch chain. It is evident that the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital(LUMO) play important roles in the chemical and PL properties of a compound. Compared with carboxyl-GQDs, amide-GQDs can induce a bigger energy gap. In reality, although there are always many functional groups linked to the grapheme core, in the theoretical calculation, only one functional group was taken into account for convenience. Therefore, the influence caused by amidation of carboxyl groups may be much more significant in real system. As a result, the changes of the energy gap of PEI-coated GQDs would be influenced cumulatively. In addition, the width of band gap is positively correlated with that of energy gap. Therefore, when the band gap was enlarged, the PL emission peak would be blue-shifted.Figure S8.Molecular orbitals of GQDs: (a) G1, amide-GQD; (b) G2, carboxyl-GQD; (c) G1-N, PEI chain-GQDs; (d) Comparison of the energy gaps of GQDs with different substituents.10.Energy gap information of GQDsTable S2. Energy gaps of a series of GQDs.GQDs G1 G1-2C G1-3C G1-4C G1-5C G1-N G2ΔE (eV) 1.21798 1.21825 1.21798 1.21825 1.21825 1.21771 1.21254 11.PL lifetime, chemical stability and optical stability of GQDsFigure S9. (a) PL lifetime of three kinds of GQDs. (b) Dependence of PL intensity on excitation times at maximum excitation wavelength for GQDs by fluorescence spectrophotometer. (c) Effect of ionic strengths on the PL intensity of three GQDs (ionic strengths were controlled by various concentrations of KCl). (d) Dependence ofPL intensity on excitation time for all these three GQDs under 365 nm UV lamp (24W).12.Biological stability and toxicity test of GQDs and PEI-coated GQDsTwo kinds of cell lines, human embryonic kidney cell line 293 (HEK-293) and human primary glioblastoma cell line 87 (U-87), are used to test the toxicity of GQDs. After 24 hours co-incubation, all of the GQDs showed quite low cytotoxicity when the cell lines exposed to GQDs with concentration from 0 to 200μg mL-1.Figure S10. Cell viability of the HEK-293 (a, c, e) and SGC-7901 (b, d, f) cells after 24 h incubation with the three kinds of GQDs: PEI1800 GQDs (a) and (b); uncoated GQDs (c) and (d); PEI600 GQDs (e) and (f). Mean ± SD, n=6.13.Quantum Yields (QYs) measurement of GQDs and PEI-coated GQDsIn order to valuate the luminescent properties of GQDs and PEI-coated GQDs, the QYs were measured by FL emission spectroscopy and UV-vis absorption spectroscopy by using reference method. Quinine sulfate (QS) in 0.1 M H2SO4was used as the reference for yellow-emitting bare GQDs and blue-emitting PEI1800GQDs, and Rhodamine B (RhB) was used as the reference for red-emitting PEI600 GQDs. All the QYs of GQDs and PEI-coated GQDs can be calculated according to the equation below:Q QIn the equation, Q represents the quantum yield, I stands for the measured integrated emission intensity, A is the UV-vis absorbance at PL excitation wavelength, and n is the refractive index, R means the reference.Because the solutions of QS, bare GQDs, and PEI1800 GQDs were prepared by 0.1 M H2SO4, the n1 were always 1.34 (400 - 550 nm at 20℃). And the solution of RhB and PEI600 GQDs were prepared by ultrapure water, the n2 were always 1.33. Hence, the (n2/n R2) ratio always equals to 1.In a typical measuring processs, more than 5 groups of samples with different intensity of UV absorbance (A), which should be less than 0.1, were prepared. (The wavelength of absorbance were 350 nm for QS, bare GQDs, and PEI1800 GQDs, and 495 nm forRhB and PEI600 GQDs.) And then these sampls were measured by FL specstropy and their intensity graphs were saved for integral operation, integrating all the effective peak area, to get the integrated emission intensity (I). Then, the I/A ratio could be abtained from the slope value which calculated by linear fitting to the data. At last, according to the known QYs of QS and RhB, the QYs of bare GQDs and PEI-coated GQDs could be calculated by the equation above.Figure S11. Integrated emission intensity (I) vs. absorbance (A) graph and I/A linear fitting of quinine sulfate (a), bare GQDs and PEI1800 GQDs (b), Rhodamine B (c), and PEI600 GQDs (d).Table S3. Calculation results of QYs of bare GQDs and PEI-coated GQDsSample Integrated emissionIntensity (I)Absorbance (A) Refractive index of solvent(n)Quantum yield(Q)Quinine Sulfate 835789 1.34 55.7% Bare GQDs 52349 1.34 3.5%PEI1800 GQDs 42545 1.34 2.8% Rhodamine B 1795200 1.33 31%PEI600 GQDs 97668 1.33 1.7%As shown in Figure S11, more than five groups of data were used to do the linear fitting for every sample. The slope of the linear line correlated to I/A ratio. Table S3 exhibited the data in details, according to former researches, the QY of QS is 55.7%,3 and the QY of RhB is 31% in water,4 so other QYs could be calculated. The QYs of bare GQDs, PEI1800 GQDs, and PEI600 GQDs are 3.5%, 2.8%, and 1.7%, respectively. The slightly decrease of QY of monocoated PEI1800 GQDs was of highly possibilities caused by the energy dissipation from the vibration of PEI chain. The QY decrease of PEI600 GQDs was from the self-absorption from their linking and stacking multicoated capsule-like structure. These QYs are comparable to the GQDs synthesized before by other groups and sufficient for applications, such as bioimaging in this paper.References:(1) Li, Y.; Zhao, Y.; Cheng, H.; Hu, Y.; Shi, G.; Dai, L.; Qu, L. Nitrogen-Doped Graphene Quantum Dots with Oxygen-Rich Functional Groups. J. Am. Chem. Soc. 2012,134, 15-8.(2) Tang, L.; Ji, R.; Li, X.; Teng, K. S.; Lau, S. P. Size-Dependent Structural and Optical Characteristics of Glucose-Derived Graphene Quantum Dots. Part. Part. Syst. Charact. 2013,30, 523-531.(3) Eastman, J. W. Quantitative Spectrofluorimetry-the Fluorescence Quantum Yield of Quinine Sulfate. Photochem. Photobiol. 1967,6, 55-72.(4) Magde, D.; Rojas, G. E.; Seybold, P. G. Solvent Dependence of the Fluorescence Lifetimes of Xanthene Dyes. Photochem. Photobiol. 1999,70, 737-744.。