Electronic Supplementary Material (ESI) for RSC Advances. This journal is The Royal Society of Chemistry 216 Electronic Supplementary Information A mitochondria-targeted near-infrared probe for colorimetric and ratiometric fluorescence detection of hypochlorite in living cells Junchao Xu, ac Houqun Yuan, b Caiqin Qin, a Lintao Zeng* ac and Guang-Ming Bao* b a Department of Chemistry and Materials Sciences, Hubei Engineering University, Xiaogan 4321, P.R. China. E-mail: zlt1981@126.com (L. Zeng). b College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 3345, P.R. China. Email: bycb25@gmail.com (G.-M. Bao). c School of Chemistry and Chemical Engineering, Tianjin University of Technology, Tianjin 3384, P.R. China. Contents 1. 1 H NMR, 13 C NMR and HR-MS spectra of CMBI 2. Summary of photophysical properties 3. Ratiometric response of CMBI toward ClO at low concentrations 4. Selectivity of CMBI toward ClO 5. Kinetics research for CMBI towards ClO, HSO3 and HS 6. Proposed mensing mechanism 7. Cell toxicity test of CMBI 8. Comparison of ratiometric fluorescent probes for ClO 9. 1 H NMR spectra of compounds 1 and 2
1. 1 H NMR, 13 C NMR and HR-MS spectra of CMBI Fig. S1 1 H NMR spectrum of CMBI in DMSO-d6 (4 MHz). Fig. S2 13 C NMR spectrum of CMBI in DMSO-d6 (1 MHz).
Fig. S3 HR-MS (ESI) spectrum of CMBI. 2. Summary of photophysical properties Table S1 Spectroscopic properties of CMBI (1 μm) and CMBI (1 μm) with ClO (1 μm) in EtOH/PBS solution (v/v = 1/9, ph 7.4, 1 mm). Compound λabs (nm) λem (nm) Φ Δν (cm 1 ) CMBI 592 658.12 1694 CMBI + ClO 4 475.141 3947 3. Ratiometric response of CMBI toward ClO at low concentrations Fluorescence Intensity 2 15 1 5 (a) 7.5 μm [ClO ] 7.5 μm 45 5 55 6 65 7 Wavelength (nm) F 475 /F 658.6.5.4.3 2 4 6 8 [ClO ] (μm).2.1. (b)..5 1. 1.5 2. 2.5 4 2 F 475 /F 658 y =.1399x +.713 R 2 =.9922 [ClO ] (μm) Fig. S4 (a) Fluorescence spectra changes of 1 µm CMBI in EtOH/PBS solution (v/v = 1/9, ph 7.4, 1 mm) upon addition of increasing amount of ClO ( 7.5 μm). (b) Linear relationship between fluorescence intensity of 1. µm CMBI versus concentrations of ClO in EtOH/PBS solution (v/v = 1/9, ph 7.4, 1 mm). Each spectrum was recorded after 3 min.
4. Selectivity of CMBI toward ClO Absorbance.8.6.4.2. (a) CMBI CMBI + ClO CMBI + Anion CMBI + HSO 3 CMBI + SH CMBI + ONOO 3 4 5 6 7 Wavelength (nm) Fluorescence Intensity 25 2 15 1 5 (b) CMBI CMBI + ClO CMBI + Anion CMBI + HSO 3 CMBI + SH CMBI + ONOO 4 45 5 55 6 65 7 Wavelength (nm) 6 (c) 4 A 4 /A 592 2 Blank ClO F Cl Br N 3 NO 2 NO 3 AcO HCO 3 2 HPO 4 H 2 PO 4 SH 2 SO 3 2 SO 4 2 S 2 O 3 Cys Hcy GSH H 2 O 2 OH TBHP TBO O 2 1 O2 NO ONOO 1 Fig. S5 (a) UV-vis absorption, (b) fluorescence spectra changes and (c) UV-vis absorption ratios (A4/A592) of 1 μm CMBI in EtOH/PBS solution (v/v = 1/9, ph 7.4, 1 mm) in the presence of various small molecular species (5 μm) and ROS/RNS (1 μm). Each spectrum was recorded after 3 min.
5. Kinetics research for CMBI towards ClO, HSO3 and HS F 475 ln(1-f t /F max ) 25 2 15 1 5.2. -.2 -.4 -.6 -.8-1. -1.2-1.4 (a) 3 6 9 12 15 18 (c) CMBI + ClO CMBI + HSO 3 CMBI + SH Response Time (s) y =.139x.571 R 2 =.9916 CMBI + HSO 3 k' =.139 s 1 15 3 45 6 75 9 Response Time (s) ln(1-f t /F max ) ln(1-f t /F max ).5. -.5-1. -1.5-2. -2.5-3. -3.5.5. -.5-1. -1.5-2. -2.5-3. (b) y =.322x.186 R 2 =.9947 15 3 45 6 75 9 (d) CMBI + ClO k' =.322 s 1 Response Time (s) y =.29x.36 R 2 =.9971 CMBI + SH k' =.29 s 1 15 3 45 6 75 9 Response Time (s) Fig. S6 Time-resolved fluorescence responses (a) and Pseudo-first-order kinetic plot of 1 μm CMBI towards 1 µm ClO (b), HSO3 (c) and HS (d) in EtOH/PBS solution (v/v = 1/9, ph 7.4, 1 mm).
6. Proposed mensing mechanism [CMBI] + [1 + H] + Fig. S7 HR-MS (ESI) spectrum of CMBI with ClO. 7. Cell toxicity test of CMBI 1 Survival Rate (%) 8 6 4 2 1 2.5 5 1 15 2 [CMBI] (μm) Fig. S8 Cell toxicity effect of CMBI. HeLa cells were incubated with CMBI ( 2 μm) for 24 h. Results are mean ± SD, n = 3.
8. Comparison of ratiometric fluorescent probes for ClO Table S2 Comparison of ratiometric fluorescent probes for ClO Probes Phenanthroimidazole Probe 1 [1] 1b [2] λex /nm 394 54 464 Cou-Rho-HOCl [3] 41 BODIPY-DCDHF 465 Probe 1 [4] Cy7-NR1 [5] 54 Z2 [6] 46 CDH [7] 316 41 HRS1 [8] 554 DPNO [9] 35 DPH [1] 344 CMCY [11] 46 Coumarin-Pyridinium 42 Probe 1 [12] 1,8-diaminonaphthalene [13] 325 Coumarin-Rhodamine 41 Probe 2 [14] λem /nm 439 59 585 55 473 594 629 52 78 566 59 6 376 456 51 578 354 43 495 375 631 48 631 488 44 518 58 47 Reagents Detection Response limit/nm time/s Application ph 9. PBS Within (8% DMF) MCF-7 cells (Ex) 2 < 6 (2% DMF) Ex = exogenous Bel 772 cells (Ex) (5% DMF) 52 < 6 En = endogenous (1% EtOH,.1 % 5 ~ 4 RAW 264.7 cells (Ex, En) Triton X-1) (.5% DMF) 19.5 ~ 36 HeLa cells (Ex) 63 < 12 Test paper ph 7.4 HEPES 17 < 6 (4% CH 3CN) TLC plate strips ph 8.5 PBS (4% DMF) 24 < 6 ph 7.4 HEPES 56 < 6 (6% CH 3CN) TLC plate strips ph 7.4 HEPES (5% EtOH) 35 8 < 12 HeLa cells (Ex) 93 ~ 2 (75% THF) ph 7. HEPES 2 < 2 HepG2 cells (Ex) ph 5. PBS (4% DMF) < 5
BTHT [15] 365 562 476 ph 7.4 HEPES (5% CH 3CN) 14 < 48 TLC plate strips NIR-1 thiospirolactone Probe 1 [16] 43 65 486 77 (3% CH 3CN) 4 Within HeLa cells (Ex) TAM [17] 43 63 485 ph 7.1 H 2O (4% THF) 7 ~ 1 TLC plate strips PBMCs (Ex) Dansyl-Rhodamine Probe 1 [18] 4 494 578 ph 8. PBS (1% CH 3CN) 113 < 12 Test paper PMN-TPP [19] 41 522 64 ph 7.3 PBS (.5% DMSO, 1 mm Triton X-1) 43 Few Nude mouse (En) Naph-Rh [2] 35 44 585 ph 6. PBS (3% EtOH) 1 < 5 RMClO-2 [21] 412 54 476 57 (.1% DMSO).84 Within Normal and cancer cells HPQ-Cy2 [22] 365 575 435 (.5% EtOH) 28 < 5 A375 cells (En) CRSH [23] 35 47 58 ph 5. PBS (5% EtOH) 21 ~ 6 STBR [24] 37 514 595 ph 7.5 PBS (2% CH 3CN) 22 TLC plate strips Baker s yeast (Ex) AETU-HOCl [25] 38 533 473 (5% EtOH) 3.9 ~ 6 QC1 [26] 4 482 381 ph 7.4 HEPES (1% DMSO) 41 Naphthalimide-Indolium Probe 1 [27] 43 62 515 ph 7. HEPES (5% MeOH) 53 ~ 5 HepG2 cells (Ex) HES-BODIPY [28] 48 562 532 ph 7.1 PBS (1% EtOH) 43 Within BRT [29] 525 54 58 ph 5. PBS (6% EtOH) 38 ~ 15 CARSH [3] 35 49 58 ph 5. PBS (5% EtOH) 2 ~ 6 Lyso-HA [31] 42 45 586 ph 5. PBS (3% DMSO) 11 Within HeLa cells (Ex)
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9. 1 H NMR spectra of compounds 1 and 2 Fig. S9 1 H NMR spectrum of compound 1 in CDCl3 (4 MHz). Fig. S1 1 H NMR spectrum of compound 2 in DMSO-d6 (4 MHz).