Supporting Information. Animal by a Rationally Designed Molecular Switch

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1 Electronic Supplementary Material (ESI) for Analyst. This journal is The Royal Society of Chemistry 218 Supporting Information Dynamic Mapping of Spontaneous 2 S in Entire Cell Space and Live Animal by a Rationally Designed Molecular Switch Linlin Yang, a,b Jun Zhao, a Xinling Yu, a Ruilong Zhang, c Guangmei an, a Renyong Liu, a Tingting Zhao, a Ming-Yong an, a,d and Zhongping Zhang a,c,e* a CAS Center for Excellence in anoscience, Institute of Intelligent Machines, Chinese Academy of Sciences, efei, Anhui 2331, China. b University of Chinese Academy of Sciences, Beijing, 139, China. c School of Chemistry and Chemical Engineering, Anhui University, efei, Anhui 2361, China. d Institute of Material Research and Engineering, A-STAR, 3 Research Link, Singapore e State Key Laboratory of Transducer Technology, efei, Anhui 2331, China. * To whom all correspondence should be addressed: zpzhang@iim.ac.cn. S1

2 CEt Fig. S1 1 MR spectrum of compound C-. CEt f1 (ppm) Fig. S2 13 C MR spectrum of compound C-. [M-] - CEt Fig. S3 R-MS spectrum of compound C- (negative mode, calculated for C- [M-] - = ). S2

3 Fig. S4 1 MR spectrum of compound Cda-. f1 (ppm) Fig. S5 13 C MR spectrum of compound Cda-. [M-] - Fig. S6 R-MS spectrum of compound Cda- (negative mode, calculated for Cda- [M-] - = ). S3

4 2 2 f1 (ppm) Fig. S7 1 MR spectrum of probe Cda-DP. 2 2 Fig. S8 13 C MR spectrum of probe Cda-DP. [M+] Fig. S9 R-MS spectrum of probe Cda-DP (positive mode, calculated for Cda-DP [M+] + = ). S4

5 [M-] - S 2 2 DP-S [M-] - Cda- Fig. S1 R-MS spectrum of the products DP-S and Cda- resulting from the reaction of probe with 2 S (negative mode, calculated for DP-S [M-] - = and Cda- [M-] - = ). Fig. S11 (A) Absorption spectra of 2 M Cda-DP with the addition of S - (-5 M) at p 7.4 in 1 mm EPES/TF (7:3). (B) The plot of absorbances vs S - concentrations. Inset: the liner relationship between absorbances (at 45 nm) and S - concentrations. S5

6 FL Intensity (a.u.) (I-I )/I Time (min) 6 Time (min) Wavelength (nm) Fig. S12 Time-dependent fluorescence responses of Cda-DP (1 µm) to 5 µm S -. Inset is the plots of fluorescence enhancement vs the time of 5 µm S - response to Cda-DP. 8 S - FL Intensity (a.u.) Balnk, F -, Cl -, Br -, I -, C - 3, - 2, - 3, S - 4, S 2-3, S 2-4, S 2 2-3, S 2 2-8, SC -, 2 2, Cl -,, Cys, cy, GS Wavelength (nm) Fig. S13 Fluorescence spectra of Cda-DP (1 μm) at p 7.4 in 1 mm EPES/TF (7:3) in the presence of S - (5 μm) and other species (1 mm anions and, 2 μm Cys and cy, and 2 mm GS). S6

7 15 12 other species+s - 9 (I-I )/I 6 3 Blank F - Cl - Br - I - - C S 4 - S 4 2- S 2 3 SC - Cys cy GS S - Fig. S14 Interference tests of Cda-DP (1 M) in the presence of various species (Blank, 1 mm F -, Cl -, Br -, I -, C 3-, 2-, 3-, S 4 2-, S 4-, S , SC - ; 2 M Cys, cy; 2 mm GS; 5 M S - ) in 1 mm EPES/TF (7:3) Cell Viability (%) [Cda-DP]/ M Fig. S15 Viability of A549 cells treated with different concentrations of Cda-DP for 24 h by MTT assay. S7

8 A FL channel Merged µm Dose dependence 2 µm 4 µm 5 µm 6 µm B FL Intensity (a.u.) [MM]/ M Fig. S16 Screening of the appropriate dosage of 2 S scavenger (MM) to clean up endogenous 2 S in A549 cells. (A) Fluorescent intensity changes of cells with the addition of different concentrations of MM. The cells were separately incubated with, 2, 4, 5, 6 and 8 μm MM for 3 min, and then cultured with 5 μm Cda-DP for another 3 min after cells were washed with medium two times. (B) Dose-dependent evolutions of mean fluorescence intensities. Thirty cells were manually selected to collect the fluorescent intensities, and the mean fluorescent intensities at each concentration of MM were calculated from the 3 intensities. The error bars represent standard deviation (±SD). All these above quantification experiments were repeated with three batches of cells. A Dose dependence B FL channel Merged µm 5 µm 1 µm 15 µm 2 µm FL Intensity (a.u.) [S - ]/ M Fig. S17 Quantitative determination of exogenous 2 S in living cells. (A) The fluorescent intensity changes of A549 cells incubated with different concentrations of exogenous 2 S after removed endogenous 2 S. A549 cells were first pretreated with 6 μm MM, and then incubated separately with, 5, 1, 15 and 2 μm as, followed by the addition of 5 μm Cda-DP. For above tests, all incubation periods were 3 min. (B) Dosedependent evolutions of mean fluorescence intensities. Thirty cells were manually selected to collect the fluorescent intensities, and the mean fluorescent intensities at each concentration of 2 S were calculated from the 3 intensities. The error bars represent standard deviation (±SD). All these above quantification experiments were repeated with three batches of cells. S8

9 Fig. S18 Time-dependent fluorescence changes of A549 cells. The cells were incubated with 4 μl Reddot (a commercial nucleus tracker) for 3 min, and then the images were obtained from blue and red channel of A549 cells incubated with 5 μm probe Cda-DP for different times. Blue channel, λ ex = 45 nm, λ em = nm; Red channel, λ ex = 633 nm, λ em = nm. Fig. S19 Time-dependent fluorescence changes of A549 cells. The cells were incubated with 1 μl LysoTracker Deep Red (a commercial lysosome tracker) for 3 min, and then the images were obtained from blue and red channel of A549 cells incubated with 5 μm probe Cda-DP for different times. Blue channel, λ ex = 45 nm, λ em = nm; Red channel, λ ex = 633 nm, λ em = nm. S9

10 Fig. S2 Time-dependent fluorescence intensity changes of A549 cells. The cells were incubated with 1 μl MitoTracker ((a commercial mitochondria tracker)) for 3 min, and then the images were obtained from blue and red channel of A549 cells incubated with 5 μm probe Cda-DP for different times. Blue channel, λ ex = 45 nm, λ em = nm; Red channel, λ ex = 633 nm, λ em = nm. Fig. S21 Dose-dependent fluorescent images of probe for the detection of spontaneous 2 S in normal zebrafish. The seven-day old zebrafish were cultured with, 5, 1, 2, 3 and 4 μm Cda-DP for 6 min, respectively. S1