Supporting Information German Edition: DOI: Red, Green, and Blue Luminescence by Carbon Dots: Full-Color Emission Tuning and Multicolor Cellular Imaging** Kai Jiang, Shan Sun, Ling Zhang, Yue Lu, Aiguo Wu, Congzhong Cai, and Hengwei Lin* ange_2151193_sm_miscellaneous_information.pdf
Experimental Materials Reagent grades of o-, m-, p-phenylenediamines were purchased from Aldrich. Ethanol, methylene chloride, polymethyl methacrylate (PMMA) and poly(vinyl alcohol) (PVA) (Mw=175±5) were provided by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All chemicals were used as received without further purification unless otherwise specified. Deionized (DI) water was used throughout this study. Characterizations Transmission electron microscopy (TEM) observations were performed on a Tecnai F2 microscope. Atomic force microscope (AFM) measurements were carried out with Veeco Dimension 31V. Fourier transform infrared (FT-IR) spectra were obtained on a Nicolet 67 FT-IR spectrometer. XPS were carried out with ESCALAB 25Xi (Thermo Scientific). UV-vis absorption spectra were recorded on a PERSEE T1CS UV-Vis spectrophotometer. PL emission and excitation spectra were measured on a Hitachi F-46 spectrophotometer at ambient conditions. PL lifetime was measured using Fluorolog 3-11 (HORIBA Jobin Yvon). UCPL was carried out using a femtosecond pulsed laser excitation at 8 nm (Coherent Inc.) connecting an optical-multichannel analyzer (OMA) system (Spectrapro-3i, ARC). Photographs of PL and UCPL were taken using a Canon camera (EOS 55) under excitation by a hand-hold UV lamp (365 nm) or femtosecond pulsed laser (8 nm) with bandpass filter 4-7 nm. Cellular imaging was performed with a confocal laser fluorescence microscopy (TCS SP5II, Leica, Germany). Synthesis of, m-cds and opd (mpd or ppd) (.9 g) was firstly dissolved in 9 ml ethanol, and then the solutions was transferred into poly(tetrafluoroethylene)-lined autoclaves. After heating at 18 ºC in oven for 12 h and cooling down to room temperature naturally, orange, gray and dark-red suspensions were obtained from opd, mpd and ppd, respectively. The crude products were then purified with a silica column chromatography using mixtures of methylene chloride and methanol as eluents. After removing solvents and further drying under vacuum, the three purified, m-cds and could be finally obtained in 1-2 wt% yields. Preparation of multicolor emissive films A typical formulation used for emission films: 2 µl of (m-cds or ) solution (1. mg/ml in ethanol) were firstly diluted with 3 µl DI water, and then mixed with 1.5 ml of PVA (Mw=175±5) solution (1. g in 15 ml water). PVA composite emission films. PVA emission films were fabricated referring to a previously described method. [S1] Typically, the above obtained formulations (single or mixtures with appropriate ratios)
were dropped casting on cleaned glass substrates and allowed drying for overnight under ambient circumstances. The resulting freestanding films were then peeled off from the glass substrates. Quantum yields (QYs) measurements. QYs of the obtained three CDs were determined by a relative method. [S2] Specially, quinine sulfate (QY=55% in.1 M H 2 SO 4 ) was selected as the reference for the emission range of 4-48 nm (for m-cds herein), rhodamine 6G (QY=95% in ethanol) for the emission range of 48-56 nm (for herein), and rhodamine B (QY=56% in ethanol) for the emission range of 58-61 nm (for herein). The QY of a sample was then calculated according to the following equation: A I n I A n ' 2 ' ' '2 where ϕ is the QY of the testing sample, I is the testing sample s integrated emission intensity, n is the refractive index (1.33 for water and 1.36 for ethanol), and A is the optical density. The superscript refers to the referenced fluorescence dyes of known QYs. To obtain more reliable results, a series of solutions of CDs and referenced fluorescence dyes were prepared with concentrations adjusted such that the optical absorbance values were between.1 at 365 nm (or other desired). The PL spectra were measured and the PL intensity was integrated. QYs were determined by comparison of the integrated PL intensity vs absorbance curves (refractive index, n, had also to be considered) (see Figures S4-S6). Cytotoxicity assay In vitro cytotoxicity of the CDs (i.e., m-cds and ) against MCF-7 cells was assessed by the standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Typically, 1 μl of cells were seeded in a 96-well plate with a density of 1 1 5 cells per ml and allowed to adhere overnight. After incubation for 24 h at 37 ºC, the culture medium was discarded and then cells were treated with Dulbecco's Modified Eagle's Medium (DMEM), containing various concentrations of CDs (1-5 μg /ml) for another 24 h. At the end of the incubation, the culture medium was removed, and 1 μl of MTT (5. mg/ml in PBS) was added into each well. After additional 4 h incubation, the growth medium was removed and 1 μl of DMSO was added into each well to dissolve MTT. Finally, the optical density of each sample was recorded using a microplate reader (Imark 168-113, Biorad, USA) at a wavelength of 55 nm. Cellular imaging The potential for cellular imaging of the three as obtained CDs was tested by the use of MCF-7 cancer cells. 2. ml of MCF-7 cells in DMEM medium at an initial density of 4 1 4 cell per ml were seeded in each dish and cultured at 37 for 24 h under a humidified atmosphere containing 5% CO 2. The
dispersion of the CDs was prepared in DMEM medium with a concentration of 4 μg/ml. Cells were cultured with the dispersion of CDs for 6 h and then washed three times with PBS to remove the free CDs. Finally, the samples were observed with confocal laser fluorescence microscopy (TCS SP5II, Leica, Germany). References: [S1] W. Kwon, S. Do, J. Lee, S. Hwang, J. K. Kim, S.-W. Rhee, Chem. Mater. 213, 25, 1893. [S2] a) D. F. Eaton, Pure Appl. Chem. 1988, 6, 117; b) M. Grabolle, M. Spieles, V. Lesnyak, N. Gaponik, A. Eychmüller, U. Resch-Genger, Anal. Chem. 29, 81, 6285; c) T. Karstens, K. Kobs, J. Phys. Chem. 198, 84, 1871.
a) 1. 435 nm.8.6.4.2. 25 3 35 4 45 5 b) 1. 535 nm.8.6.4.2. 25 3 35 4 45 5 55 c) 1. 68nm.8.6.4.2. 3 35 4 45 5 55 6 Figure S1. PL excitation spectra of m-cds (a), (b), and (c) at their maxima emission wavelengths in ethanol solution.
Integrated PL 13 11 9 7 5 m-cds λ ex =365 nm in EtOH Quinine Sulfate λ ex =365 nm in.1m H 2 SO 4 m-cds.3.4.5.6.7 Integrated PL 11 9 7 5 Quinine Sulfate.2.3.4.5.6 m-cds Quinine Sulfate Abs.32.41.53.69.25.37.44.56 Integrated PL 6157 7629 9579 11841 51715 73384 85474 1936 Slope 1.54 1 5 1.85 1 6 QY 4.8% 55% Figure S2. Plots of integrated PL intensity of m-cds and quinolone sulfate (referenced dye) as a function of optical absorbance at 365 nm and relevant data.
Integrated PL 15 12 9 6 3 λ ex =365 nm in EtOH.2.3.4.5.6 Integrated PL 12 1 8 6 4 Rhodamine 6G λ ex =365 nm in EtOH Rhodamine 6G 2.2.3.4.5.6.7 Rhodamine 6G Abs.2.35.48.59.26.44.56.64 Integrated PL 4444 796 1528 13674 39643 6256 98143 11944 Slope 2.32 1 5 2.12 1 6 QY 1.4% 95% λ ex =42 nm in EtOH Rhodamine 6G λ ex =42 nm in EtOH Integrated PL 28 24 2 16 12 8.3.4.5.6.7.8 Integrated PL 16 14 12 1 8 6 Rhodamine 6G.2.3.4.5.6.7.8 Rhodamine 6G Abs.3.43.53.74.29.53.65.8 Integrated PL 168 16823 19741 2716 58447 1641 1312 158458 Slope 3.66 1 5 1.97 1 6 QY 17.6% 95% Figure S3. Plots of integrated PL intensity of and rhodamine 6G (referenced dye) as a function of optical absorbance at 365 nm and 42 nm and relevant data.
Integrated PL 36 28 2 λ ex =365 nm in EtOH 12.2.3.4.5.6.7 Integrated PL 9 8 7 6 5 Rhodamine B λ ex =365 nm in EtOH Rhodamine B 4.3.4.5.6.7 Rhodamine B Abs.24.37.52.67.35.41.53.67 Integrated PL 14562 288 28185 34481 444 565 69257 86454 Slope 4.66 1 5 1.27 1 6 QY 2.6% 56% Integrated PL 7 6 5 4 3 λ ex =51 nm in EtOH 2.2.3.4.5.6.7 Integrated PL 16 14 12 1 8 6 Rhodamine B λ ex =51 nm in EtOH Rhodamine B.3.4.5.6.7.8.9 Rhodamine B Abs.26.3.43.65.35.42.59.82 Integrated PL 27143 34576 44322 62986 62432 7959 119119 154512 Slope 9.13 1 5 1.96 1 6 QY 26.1% 56% Figure S4. Plots of integrated PL intensity of and rhodamine B (referenced dye) as a function of optical absorbance at 365 nm and 51 nm and relevant data.
a) 1 1 1 1 IRF m-cds b) 1 1 1 1 1 2 4 6 8 1 Time (ns) IRF c) 1 1 1 1 1 2 4 6 8 1 Time (ns) IRF 1 2 4 6 8 1 Time (ns) Figure S5. PL decay spectra and fitting curves of m-cds (a), (b), and (c). Table S1. Photoluminescent lifetimes of m-cds, and in ethanol solutions. λ ex [nm] λ em [nm] B 1 [%] τ 1 [ns] B 2 [%] τ 2 [ns] τ avg [ns] χ 2 m-cds 319 435 92.41.93 7.86 6.9.99 1.26 457 544 1 4.44 - - 4.44 1.13 457 61 1 9.39 - - 9.39 1.25
Normalized PL Intensity 1..5 苯二胺产物荧光稳定性 a) b) c) m-cds in ethanol Normalized PL Intensity 1..5 in ethanol Normalized PL Intensity 1..5 in ethanol. 1 2 3 Time (s). 1 2 3 Time (s). 1 2 3 Time (s) d) e) f) Normalized PL Intensity m-cds in PVA film 1..5 Normalized PL Intensity in PVA film 1..5 Normalized PL Intensity 1..5 in PVA film. 1 2 3 Time (s). 1 2 3 Time (s). 1 2 3 Time (s) Figure S6. Photostability of m-cds, and in ethanol solutions (a-c), and in PVA films (d-f) under continuous excitation at 365 nm for one hour using a spectrofluorometer equipped with an xenon lamp (15 W). UCPL 2 1 a 1.19 W 1.7 W 2.7 W 2.53 W 2.83 W 3.23 W UCPL 2 1 b 1.19 W 1.87 W 2.32 W 3.2 W 3.23 W UCPL 6 4 2 c 1.19 W 1.87 W 2.32 W 3.2 W 3.23 W 4 45 5 55 6 65 45 5 55 6 65 7 5 55 6 65 7 75 UCPL 2 1 3 6 d e f 2 4 6 8 1 (Laser Power) 2 (W 2 ) 2 1 2 4 6 8 1 (Laswer Power) 2 (W 2 ) UCPL 4 2 2 4 6 8 1 (Laser Power) 2 (W 2 ) Figure S7. (a-c) UCPL spectra of m-cds, and, respectively, with variable excitation laser powers; (d-f) relationships between UCPL intensities of m-cds, and, respectively, and square of laser powers (8 nm femtosecond pulsed laser was throughout applied).
3 3 2 1 2 15 1 5 5 6 7 8 9 1 7 8 9 1 2 nm 1 7 8 9 1 11 12 13 14 15 Size (nm) 2 nm 1 e) 2 5 f) Height (nm) Height (nm) 1 3 12 2 nm 2 nm 4 nm 1 μm Height (nm) Height (nm) 11 Size (nm) Size (nm) d) 6 4 Height (nm) 4 5 Height (nm) c) 25 Percentage (%) b) 4 Percentage (%) 5 Percentage (%) a) Figure S8. a-c) TEM images of m-cds, and (from left to right), inset: histograms and Gauss fittings of particles size distribution of these CDs; d-f) AFM images of m-cds, and (from left to right), bottom: height-profiles analysis along the corresponding lines in Figures S1d-f.
a) 341 3184 3293 2918 2848 1645 152 1367 1226 1459 1279 165 848 Transmittance b) 3434 3363 132 1641 1336 317 2852 1225 3298 1261 292 761 1493 3311 3169 2918 2854 m-cds 1645 1384 1234 1281 1518 139 4 35 3 25 2 15 1 5 Wavenumber (cm -1 ) 337 33 32 ppd 163 152 127 829 833 Transmittance 339 329 319 opd mpd 164 127 1495 748 321 34 333 132 16 149 688 4 35 3 25 2 15 1 5 Wavenumber (cm -1 ) Figure S9. FT-IR spectra of, m-cds and (a), and their corresponding strating materials, i.e. opd, mpd and ppd (b).
a) b) C1s N1sO1s m-cds Sample C (%) N (%) O (%) m-cds 84.56 3.69 11.76 76.74 7.32 15.94 P-CDs 77.39 15.57 7.4 2 4 6 8 Figure S1. a) XPS survey of, m-cds and ; b) the relative contents of C, N and O atoms for, m-cds and (determined by XPS). a) m-cds C1s C-C/C=C (284.7 ev) C-N (285.5 ev) C-O (286.3 ev) Raw data m-cds N1s pyridinic N (398.4 ev) amino N (399. ev) pyrrolic N (4.2 ev) Raw data m-cds C1s C-OH/C-O-C (532.2 ev) 282 284 286 288 29 396 4 44 528 532 536 54 b) C1s C-C/C=C (284.7 ev) C-N (285.4 ev) C-O (286.3 ev) Raw data N1s pyridinic N (398.6 ev) amino N (399.2 ev) pyrrolic N (4.4 ev) Raw data C1s C-OH/C-O-C (532.2 ev) 282 284 286 288 29 396 4 44 528 532 536 54 c) C1s C-C/C=C (284.6 ev) C-N (285.4 ev) C-O (286.3 ev) Raw data N1s pyridinic N (398.4 ev) amino N (399.1 ev) pyrrolic N (4.2 ev) Raw data C1s C-OH/C-O-C (532.1 ev) 282 284 286 288 29 396 4 44 528 532 536 54 Figure S11. Deconvoluted high-resolution XPS spectra of m-cds (a), (b), and (c) for C1s and N1s.
a 365 nm UV lamp b Normalized PL Intensity 1..5 426 in PVA (365 nm) m-cds in PVA (365 nm) in PVA (365 nm) 529 63. 4 5 6 7 Figure 12. PVA films doped with, m-cds and under daylight and 365 nm UV lamp (a), and PL spectra of these PVA films under 365 nm UV excitation (b). I II III IV i ii iii iv v vi vii viii ix x xi xii Figure S13. Photographs of the three CDs doped PVA composite films under daylight: (I) m-cds, (II), (III), (IV) : m-cds : = 2:4:1 (w/w/w); (i-iv) : m-cds = 1:8, 1:4, 1:2 and 1:1; (v-viii) : = 4:1, 2:1,1:1 and 1:2; (ix-xii) m-cds : = 1:4, 1:2, 1:1 and 4:1 (all ratios are w/w). All of these films are uniform and transparent.
UCPL 6 4 2 a UCPL 3 2 1 b 4 45 5 55 6 65 45 5 55 6 65 UCPL 4 3 2 1 c 5 55 6 65 7 75 Figure S14. UCPL spectra and images (inset) of PVA films doped with m-cds (a), (b) and p- CDs (c), respectively, under excitation with a femtosecond pulsed laser (8 nm).
a 1 Cell viability (%) 8 6 4 2 b 1 1 2 3 4 5 m-cds ( g/ml) Cell viability (%) 8 6 4 2 1 2 3 4 5 ( g/ml) c 1 Cell viability (%) 8 6 4 2 1 2 3 4 5 ( g/ml) Figure S15. Cellular cytotoxicity assessment of m-cds (a), (b), and (c).