SUPPRTIG IFRMATI For ACS Applied Materials & Interfaces S-1
Specific Fluorescence Probes for Lipid Droplets Based on Simple AIEgens Zhiming Wang,,,, # Chen Gui,,, Engui Zhao,, Jing Wang, # Xiaodong Li, Anjun Qin, Zujin Zhao,*, Zhenqiang Yu, Ben Zhong Tang*,,,,^ State Key Laboratory of Luminescent Materials and Devices, South China University of Technology (SCUT), Guangzhou 51060, China; KUST-Shenzhen Research Institute, o. 9 Yuexing 1st RD, South Area, i-tech Park, anshan, Shenzhen 518057, China; # School of Petrochemical Engineering, Shenyang University of Technology (SUT), Liaoyang 111003, China; Department of Chemistry, The ong Kong University of Science & Technology (KUST), Clear Water Bay, Kowloon, ong Kong, China; ^ong Kong Branch of Chinese ational Engineering Research Center (CERC) for Tissue Restoration and Reconstruction, ong Kong, China Corresponding Author Prof. Z. Zhao (e-mail: mszjzhao@scut.edu.cn). Prof. B. Z. Tang (e-mail: tangbenz@ust.hk). S-2
Table of Contents Experiment Details. S- Figure S1. ESIPT forms of FAS and DPAS, and the mechanism of ESIPT. S-7 Figure S2. The Uv-vis and PL spectra of FAS and DPAS. S-8 Table S1. The ratio of keto-emission to enol-emission in different solvents. S-8 Figure S3. The Uv-vis spectra of FAS and DPAS in mixtures. S-9 Figure S. The Uv-vis and PL spectra of FAS and DPAS in p buffers. S-9 Figure S5. MR spectra of FAS and its deprotonated form in CD 3 D. S-10 Figure S6. MR spectra of DPAS and its deprotonated form in CD 3 D. S-10 Figure S7. The fluorescent images of ela cells stained FAS, DPAS and S-11 BDIPY with different irradiation time. S-3
EXPERIMETAL DETAILS Materials and instrumentation All reagents were purchased from commercial suppliers and used as received without further purification. The absorption spectra were recorded using a UV-2600 spectrophotometer (Shimadzu) with quartz cuvettes of 1 cm pathlength. Fluorescence spectra were obtained using an LS-55 Fluorescence spectrophotometer (PerkinElmer) at room temperature. X-ray diffraction (XRD) intensity data were collected on a Bruker-onices Apex CCD diffractometer with graphite monochromated MoKα radiation. Processing of the intensity data was conducted using the SAT and SADABS routines, and the structure and refinement were caried out using the SELTL suits of X-ray progreams (version 6.10). igh-resolution mass spectra (RMS) were obtained on a GCT Premier CAB 08 mass spectrometer operated in MALDI-TF mode. Measurement of fluorescence quantum yield (Φ) The Φ values were measured by the optical dilute method with a standard of quinine sulfate (Φ r = 0.55, quinine in 0.05 mol L -1 sulfuric acid) calculated by: Φ s = Φ r (A r /A s )(n s /n r ) 2 (D s /D r ), where the subscripts s and r refer to the sample and reference standard solution respectively; n is the refractive index of the solvents; D is the integrated intensity. The value of A is absorption intensity at 365 nm. The refractive indices of the solvents at room temperature are taken from a standard source. Errors for Φ values (± 10 %) are estimated. S-
Measurement of AIE and p response For AIE property measurement, 0.05 ml stock solution was added to a 10.0 ml flask with different amount of water and TF. The solution was then allowed to stand at room temperature for 2 min. The p responses were measured in the mixed solvents of TF-aqueous (v : v = 0.5 : 99.5). B-R buffer solution (a mixture of 0.0 mol L -1 3 B 3, 3 P and C 3 C in water) follows the similar operation. The buffer solutions (p = 1 and 2) were prepared by a mixture of Cl and KCl. The p values of these solutions were detected by Sartourius PB-10 p meter, and standardized by standard buffer solutions (p =.01, 6.86 and 9.18). The concentration of FAS or DPAS was kept to ~1.0 10-5 mol L -1 in all of these buffer solutions. Cell culture and imaging Cell culture. ela cells were cultured in MEM containing 10% FBS and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin) in a 5% C 2 humidity incubator at 37 o C, and A59 cells were cultured in the DMEM in a 5% C 2 humidity incubator at 37 o C. Cell imaging. ela cells (or A59 cells) were grown overnight on a 35 mm petri dish with a cover slip or a plasma-treated 25 mm round cover slip mounted to the bottom of a 35 mm petri dish with an observation window. The cells were stained with 10 μm FAS (or DPAS) for 15 min, 30 min, 60 min (by adding 2μL of a 10-2 M stock solution of FAS (or DPAS) in DMS to a 2 ml culture medium) or 1μg/mL of BDIPY dyes for 15 min (by adding 2 μl of a 1mg/mL stock solution of BDIPY dyes in DMS to a 2 ml culture medium). The cells were imaged under an FL microscope (BX1 Microscope) using proper excitation for each dye: for BDIPY, excitation filter = 60-90 nm, S-5
emission filter = 515 nm and dichroic mirror = 505 nm; for FAS, excitation filter = 00-0 nm, emission filter = 65 nm and dichroic mirror = 55 nm; for DPAS, excitation filter = 330-385 nm, emission filter = 20 nm and dichroic mirror = 00 nm. For the photostability test, the cells were imaged by a confocal microscope (Zeiss Laser Scanning Confocal Microscope; LSM7 DU) using ZE 2009 software (Carl Zeiss). FAS (or DPAS) was excited at 05 nm (2% laser power) and BDIPY dye was excited at 88 nm (2% laser power). The scanning speed is 11.22 s/scan. ela cells with pretreatment of oleic acid were grown overnight on a 35 mm petri dish with a cover slip. The cells were incubated with different concentrations of oleic acid (50 μm) (by adding an appropriate volume of a 50 mm stock solution of oleic acid in DMS to a 2 ml culture medium) for 6 h. The oleic acid-treated cells were then stained with 10 μm FAS (or DPAS) for 15 min. Cell Viability evaluated by MTT assay Cells were seeded in 96-well plates at density of 5000 cells/well. After overnight culture, medium in each wells were replaced by fresh medium containing different concentrations of FAS (or DPAS). After 2 h of treatment, into each well, 10 μl MTT solution (5 mg/ml in phosphate buffer solution) was added. After h incubation at 37 o C, 100 μl SDS-Cl solution (10% SDS and 0.01 M Cl) was added to each well. After incubation at 37 o C for 6 h, the absorbance of each wells at 595 nm was recorded by the plate reader (Perkin-Elmer Victor 3TM). Each of the experiments was performed at least 5 times as parallel test. S-6
A B FAS DPAS Figure S1. ESIPT forms of FAS (A) and DPAS (B), and the mechanism of ESIPT (C). Abs intensity (a.u.) 0.50 0.25 A exane Toluene TF Acetonitrile Ethanol Isopropanol Abs intensity (a.u.) 0.75 0.50 0.25 B exane Toluene TF Acetonitrile Ethanol Isopropanol 0.00 300 00 500 600 0.00 300 00 500 600 S-7
PL intensity (a.u.) 1.00 C 0.75 0.50 0.25 exane Toluene TF Acetonitrile Ethanol Isopropanol PL intensity (a.u.) 2.0 1.5 1.0 0.5 D exane Toluene TF Acetonitrile Ethanol Isopropanol 0.00 00 500 600 0.0 00 75 550 625 Figure S2. The absorption and photoluminescence (PL) spectra of (A, C) FAS and (B, D) DPAS (10 μm) in different solvents, λ ex = 350 nm. Table S1 The ratio of keto-emission to enol-emission in different solvents. K / E exane Toluene TF Acetonitrile Ethanol Isopropanol FAS 0.028 0.087 0.039 0.117 0.121 0.250 DPAS 0.055 0.178 0.20 0.88 0.982 1.776 Abs intensity (a.u.) A f w (vol %) 99 95 90 80 60 0 20 0 Abs intensity (a.u.) B f w (vol %) 99 95 90 80 60 0 20 0 250 300 350 00 50 500 550 300 350 00 50 500 550 Figure S3. The absorption spectra of (A) FAS and (B) DPAS in TF-water mixtures with different water fractions (f w ). S-8
Abs intensity (a.u.) A FAS p1 p2 p3 p p5 p6 p7 p8 p9 p10 p11 p12 Abs intensity (a.u.) B DPAS p1 p2 p3 p p5 p6 p7 p8 p9 p10 p11 p12 250 350 50 550 650 250 350 50 550 650 PL intensity (a.u.) C FAS p1 p2 p3 p p5 p6 p7 p8 p9 p10 p11 p12 p13 PL intensity (a.u.) D DPAS p1 p2 p3 p p5 p6 p7 p8 p9 p10 p11 p12 p13 500 575 650 725 500 550 600 650 700 Figure S. (A, B) Absorption and (C, D) photoluminescence (PL) spectra of FAS and DPAS in different p buffers. 1 6 9,10 13 2 8,11,7,12 3 5,13 2 1 5 6 7 13 12 11 10 9 8 Int. (a.u.) 1 6 8,11 13 9,10 7,12 2 13 12 3 2 1 11 10 K 5 6 9 5 3 7 8 9 8 7 6 Chemical shift (ppm) Figure S5. 1 MR spectra of FAS and its deprotonated FAS (adding 5 eq of K in CD 3 D) in CD 3 D S-9
1 6,15 3 2 1 5 6 7 8 15 1 13 12 11 10 9 Int. (a.u.) 1 6,15 3 5 2 3 5 15 1 13 1 12 11 K 6 10 7 9 8 5 3 9 8 7 6 Chemical shift (ppm) Figure S6. 1 MR spectra of DPAS and its deprotonated form (adding 5 eq of K in CD 3 D) in CD 3 D. Figure S7. The fluorescent images of ela cells stained with FAS (10 μm, A-F, orange), DPAS (10 μm, G-L, yellow) and BDIPY (1 μg/ml, M-R, green) with different irradiation time. (FAS and DPAS: staining time: 15 min, excitation wavelength: 05 nm; BDIPY, staining time: 30 mins. Scale bar: 30 μm) S-10