values and effect of pH on absorbance of the fluorescent ... 28. Table 1. Absorption coefficients, fluorescence quantum yields and. pKa values of the probes. 29 ...
Electronic Supplementary Material (ESI) for Journal of Materials Chemistry B. This journal is © The Royal Society of Chemistry 2014
Supporting Information pH-Activatable Near-Infrared Fluorescent Probes for Detection of Lysosomal pH inside Living Cells Giri K. Vegesnaa, Jagadeesh Janjanam,a Jianheng Bia, Fen-Tair Luob*, Jingtuo Zhanga, Connor Oldsa, Ashutosh Tiwaria*, and Haiying Liua* aDepartment
of Chemistry, Michigan Technological University, Houghton, MI
49931 bInstitute
of Chemistry, Academia Sinica, Taipei, Taiwan 11529, Republic of
China
Table of Contents Figure S1. Figure S2. Figure S3. Figure S4. Figure S5. Figure S6. Figure S7. Figure S8. Figure S9. Figure S10. Figure S11. Figure S12. Figure S13. Figure S14. Figure S15. Figure S16. Figure S17. Figure S18 Figure S19. Figure S20. Figure S21
1H
NMR spectrum of compound 3 NMR spectrum of compound 3 1H NMR spectrum of compound 5 13C NMR spectrum of compound 5 1H NMR spectrum of fluorescent probe A 13C NMR spectrum of fluorescent probe A 1H NMR spectrum of fluorescent probe B 13C NMR spectrum of fluorescent probe B 1H NMR spectrum of fluorescent probe C 13C NMR spectrum of fluorescent probe C 1H NMR spectrum of fluorescent probe D 13C NMR spectrum of fluorescent probe D Normalized absorbance and fluorescence spectra of fluorescent probe A Normalized absorbance and fluorescence spectra of fluorescent probe B Normalized absorbance and fluorescence spectra of fluorescent probe C Normalized absorbance and fluorescence spectra of fluorescent probe D Fluorescence spectra of the fluorescent probe A in the presence of different concentrations of ethanol. Photostability of the fluorescent probe A Absorption spectra of fluorescent probe B at different pH values and effect of pH on absorbance of the fluorescent probe B Fluorescence spectra of fluorescent probe B at different pH values and effect of pH on fluorescence of the fluorescent probe B Fluorescence spectra of the fluorescent probe B in the 13C
1
3 4 5 6 7 8 9 10 11 12 13 14 15 15 16 16 17 17 18 18 19
Figure S22 Figure S23. Figure S24. Figure S25. Figure S26 Figure S27. Figure S28. Figure S29. Figure S30. Figure S31. Figure S32. Figure S33 Figure S34 Table 1.
presence of different concentrations of ethanol. Photostability of the fluorescent probe B Absorption spectra of fluorescent probe C at different pH values and effect of pH on absorbance of the fluorescent probe C Fluorescence spectra of fluorescent probe C at different pH values and effect of pH on fluorescence of the fluorescent probe C. Fluorescence spectra of the fluorescent probe C in the presence of different concentrations of ethanol. Photostability of the fluorescent probe C. Fluorescent responses of fluorescent probe B to different metal ions (200 µM) at pH 4.1 and 7.0. Fluorescent responses of 5µM fluorescent probe C to different metal ions (200 µM) at pH 4.1 and 7.0. Fluorescent responses of fluorescent probe D to different metal ions (200 µM) at pH 4.1 and 7.0. Absorbance spectra of compound 5 at pH 4.1 and 7.0 Fluorescence spectra of compound 5 at pH 4.1 and 7.0 Fluorescence images of MDA-MB-231 cells with 5 and 20 µM of dyes B, C, and D Fluorescence images of MDA-MB-231 cells incubated with increasing concentrations of Lysosensor-green dye Fluorescent images of MDA-MB-231 cells incubated with 5, 10, and 20 µM of probe 5. Absorption coefficients, fluorescence quantum yields and pKa values of the probes
2
20 20 21 22 22 23 23 24 24 25 26 27 28 29
Figure S1: 1H NMR spectrum of compound 3.
3
Figure S2:
13C
NMR spectrum of compound 3.
4
Figure S3: 1H NMR spectrum of compound 5.
5
Figure S4:
13C
NMR spectrum of compound 5.
6
Figure S5: 1H NMR spectrum of fluorescent probe A. 7
Figure S6:
13C
NMR spectrum of fluorescent probe A. 8
Figure S7: 1H NMR spectrum of fluorescent probe B.
9
Figure S8: 13C NMR spectrum of fluorescent probe B.
10
210
200
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
-10
-20
Figure S9: 1H NMR spectrum of fluorescent probe C.
11
Figure S10: 13C NMR spectrum of fluorescent probe C.
12
Figure S11: 1H NMR spectrum of fluorescent probe D 13
Figure S12: 13C NMR spectrum of fluorescent probe D 14
Normalized Absorbance
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
300
400
500
600
700
Normalized Fluorescence Intensity
Absorption Emission
1.0
800
Wavelength (nm)
Figure S13: Normalized absorbance and fluorescence spectra of fluorescent probe A in EtOH/pH8.0 HEPES Buffer (v/v = 2/3). Excitation wavelength: 350
Absorption Emission
Normalized Absorbance
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
300
400
500
600
700
Normalized Fluorescence Intensity
nm.
800
Wavelength (nm)
Figure S14: Normalized absorbance and fluorescence spectra of fluorescent probe B in EtOH/pH7.0 HEPES Buffer (v/v = 2/3). Excitation wavelength: 350 nm
15
Normalized Absorbance
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
300
400
500
600
700
Normalized Fluorescence Intensity
Absorption Emission
1.0
800
Wavelength (nm)
Figure S15: Normalized absorbance and fluorescence spectra of fluorescent probe C in EtOH/pH7.0 HEPES Buffer (v/v = 2/3). Excitation wavelength: 350
Absorption Emission
Normalized Absorbance
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
300
400
500
600
700
Normalized Fluorescence Intensity
nm.
800
Wavelength (nm)
Figure S16: Normalized absorbance and fluorescence spectra of fluorescent probe D in EtOH/pH8.0 HEPES Buffer (v/v = 2/3). Excitation wavelength: 350 nm
16
2.5x106 40% EtOH
Fluorescence Intensity
2.0x106
30% EtOH 20% EtOH 10% EtOH
6
1.5x10
5.0% EtOH
1.0x106 5.0x105 0.0 700
750
800
850
Wavelength (nm)
Figure S17. Fluorescent spectra of 5 µM fluorescent probe A in 40 mM citratephosphate buffer solution at pH 4 containing different concentrations of ethanol.
2.5x106
Fluorescence Intensity
2.0x106
1.5x106
1.0x106
5.0x105
0.0 0
10
20
30
40
50
60
Time (min)
Figure S18. Photostability of 5 µM fluorescent probe A at pH 4.1 in 40% ethanol solution. Sample was exposed under respective optimal excitation wavelength and fluorescence intensities were measured at 5-min intervals.
17
0.20 0.15 0.10 0.05
0.20
Absorbance
0.25
Absorbance
0.25
pH2.60 pH2.84 pH3.30 pH3.50 pH3.70 pH3.89 pH4.11 pH4.23 pH4.54 pH4.68 pH4.84 pH5.11 pH5.29 pH5.45 pH5.63 pH6.04 pH6.45 pH6.90 pH7.48
0.15 0.10 0.05 0.00
0.00 300
400
500
600
700
0
800
1
2
3
4
5
6
7
8
pH
Wavelength (nm)
Figure S19: Absorption spectra of 5 µM fluorescent probe B (with three repetitions/error bars) at different pH values (left) and effect of pH on absorbance of the fluorescent probe B at 718 nm (right). O
O
N NH2 N
pKcycl = 4.6 H
O
O
pKa = 1.6
NH NH2 N
NH NH2
H+
+
O
N
OH-
OH-
O
H
N
N
N
Fluorescent probe B
1600000
Fluorescence Intensity
1400000 1200000 1000000 800000 600000 400000 200000
1.5x106
Fluorescence Intensity
pH2.60 pH2.84 pH3.07 pH3.30 pH3.50 pH3.70 pH3.89 pH4.11 pH4.23 pH4.54 pH4.68 pH4.84 pH5.11 pH5.29 pH5.45 pH5.63 pH6.04 pH6.45 pH6.90 pH7.48
1800000
1.0x106
5.0x105
0.0
0 660
680
700
720
740
760
780
800
820
840
860
0
Wavelength(nm)
2
4
6
pH
18
8
10
Figure S20: Fluorescence spectra of 5 µM fluorescent probe B (with three repetitions/error bars) at different pH values (left) and effect of pH on fluorescence of the fluorescent probe B at 743 nm(right). Excitation wavelength: 670 nm
1.5x106
40% EtOH
Fluorescence Intensity
30% EtOH 20% EtOH 10% EtOH
1.0x106
5.0% EtOH
5.0x105
0.0 700
750
800
850
Wavelength (nm)
Figure S21. Fluorescent spectra of 5 µM fluorescent probe B in 40 mM citratephosphate buffer solution at pH 4 containing different concentrations of ethanol.
19
Fluorescence Intensity
1.5x106
1.0x106
5.0x105
0.0 0
10
20
30
40
50
60
Time (min)
Figure S22. Photostability of 5 µM fluorescent probe B at pH 4.1 in 40% ethanol solution. Sample was exposed under respective optimal excitation wavelength and fluorescence intensities were measured at 5-min intervals.
0.30 pH 2.84 pH 3.50 pH 4.23 pH 4.54 pH 4.68 pH 4.84 pH 5.11 pH 5.29 pH 5.45 pH 5.63 pH 5.81 pH 6.04 pH 6.12 pH 6.45 pH 6.63 pH 6.90 pH 7.06 pH 7.48
0.25
Absorbance
0.20 0.15 0.10 0.05
0.25
Absorbance
0.30
400
500
600
700
0.15 0.10 0.05 0.00
0.00 300
0.20
0
800
1
2
3
4
5
6
7
8
pH
Wavelength (nm)
Figure S23: Absorption spectra of 5 µM fluorescent probe C (with three repetitions/error bars) at different pH values (left) and effect of pH on absorbance of the fluorescent probe C at 718 nm (right).
20
H O N N
H
O
pKcycl = 4.9 H
O
pKa = 1.8
NH
NH
H+
+
N
H
O
O
N
OH-
OH-
O
H
N
N
N
Fluorescent probe C
Fluorescence Intensity
2000000
1500000
1000000
500000
2.0x106
Fluorescence Intensity
pH2.84 pH3.07 pH3.30 pH3.50 pH3.70 pH3.89 pH4.11 pH4.23 pH4.54 pH4.68 pH4.84 pH5.11 pH5.29 pH5.45 pH5.63 pH5.81 pH6.04 pH6.12 pH6.45 pH6.63 pH6.90 pH7.06 pH7.48
1.5x106
1.0x106
5.0x105
0.0
0
700
750
800
0
850
2
4
6
8
10
pH
Wavelength(nm)
Figure S24: Fluorescence spectra of 5 µM fluorescent probe C with three repeated measurements at different pH values (left) and effect of pH on fluorescence of the fluorescent probe C at 743 nm(right). Excitation wavelength: 670 nm.
21
2.0x106
Fluorescence Intensity
40% EtOH 30% EtOH
6
1.5x10
20% EtOH 10% EtOH
1.0x106
5.0x105
0.0 700
750
800
850
Wavelength (nm)
Figure S25. Fluorescent spectra of 5 µM fluorescent probe C in 40 mM citratephosphate buffer solution at pH 4 containing different concentrations of ethanol.
Fluorescence Intensity
2.0x106
1.5x106
1.0x106
5.0x105
0.0 0
10
20
30
40
50
60
Time (min)
Figure S26. Photostability of 5 µM fluorescent probe C at pH 4.1 in 40% ethanol solution. Sample was exposed under respective optimal excitation wavelength and fluorescence intensities were measured at 5-min intervals. 22
pH 4.1 pH 7.0
Fluorescence Intensity
1.2x106 1.0x106 8.0x105 6.0x105 4.0x105 2.0x105 0.0
k Blan Cu Zn Mg Ca Fe Fe Co 2+
2+
2+
2+
3+
2+
2+
+
2+
2+
2+
Ag Ba Cd Pb
2+
2+
Ni Mn
Figure S27. Fluorescent responses of 5 µM fluorescent probe B to different metal ions (200 µM) at pH 4.1 and 7.0. Excitation wavelength: 670 nm.
Fluorescence Intensity
pH 4.1 pH 7.0
1.5x106
1.0x106
5.0x105
0.0 +
2 2 2 nk u2 Zn Mg Ca Bla C +
+
+
3+
Fe
2+
2+
Fe Co
+
2+
2+
2+
Ag Ba Cd Pb
2+
2+
Ni Mn
Figure S28. Fluorescent responses of 5µM fluorescent probe C to different metal ions (200 µM) at pH 4.1 and 7.0. Excitation wavelength: 670 nm.
23
pH 4.1 pH 7.0
Fluorescence Intensity
1.5x106
1.0x106
5.0x105
0.0
k Blan Cu Zn Mg Ca Fe Fe Co 2+
2+
2+
2+
3+
2+
2+
+
2+
2+
2+
Ag Ba Cd Pb
2+
2+
Ni Mn
Figure S29. Fluorescent responses of 5 µM fluorescent probe D to different metal ions (200 µM) at pH 4.1 and 7.0. Excitation wavelength: 670 nm.
pH 7.0 pH 4.1
0.35 0.30
Absorbance
0.25 0.20 0.15 0.10 0.05 0.00 300
400
500
600
700
800
Wavelength(nm)
Figure S30. Absorbance spectra of compound 5 at pH 4.1 and 7.0.
24
350000
Fluorescence Intensity
300000
pH 7.0 pH 4.1
250000 200000 150000 100000 50000 0
680 700 720 740 760 780 800 820 840 860
Wavelength (nm)
Figure S31. Fluorescence spectra of compound 5 at pH 4.1 and 7.0.
25
A
B
Figure S32. Fluorescence images of MDA-MB-231 cells incubated with fluorescent probes B, C, and D. Cells were incubated with 5 µM (A) or 20 µM (B) of fluorescent probes B, C, and D for 2 h and imaged for co-localization in presence of 5 µM LysoSensor Green, a lysosomal stain and Hoechst, a nuclear stain. The images were acquired using inverted fluorescence microscope at 60X magnification.
26
Figure S33. Fluorescence images of MDA-MB-231 cells incubated with 0.3, 1.0, and 3.0 µM of Lysosensor-green dye and 1.0 µg/ml of Hoechst33342 dye for 2 h. The images were acquired using inverted fluorescence microscope at 60X magnification.
27
Figure S34. Fluorescence images of MDA-MB-231 cells incubated for 2 h with 5, 10, and 20 µM concentrations of probe 5 and 1 µM of Hoechst33342 dye. The images were acquired using inverted fluorescence microscope at 60X magnification.
28
Table 1: Absorption coefficients, fluorescent quantum yields, and pKa values of the probes Compound
Absorption coefficient (EtOH)
pKa, pKcycl (Citratephosphate buffer with 40%EtOH) 1.56, 5.80
Quantum Yield at pH 4.1 (Citratephosphate buffer with 40%EtOH) 0.48
Quantum Yield at pH 7.4 (Citratephosphate buffer with 40%EtOH) 0
A
9.8 x 104
B
5.2 x 104
1.55, 4.60
0.57
0
C
6.2 x 104
1.81, 4.86
0.37
0
D
1.1 x 105
1.56, 5.38
0.46
0
5
8.8 x 104
Determination of the fluorescence quantum yield : Fluorescence quantum yields for A, B, C, D were determined using Rodamin 6G (Φf = 0.95 in ethanol) as fluorescence standard. The equation used for calculating is as follows: Φf(x) = Φf(s) ( AsFx / AxFs ) ( nx / ns )2 Where Φf is the fluorescence quantum yield, A is the absorbance at the excitation wavelength (670nm), F is the integration of the emission curve (area under the curve), and n is the refractive index of the solvents used. Subscripts s and x refer to the standard and unknown respectively.
29
