1. Calculation of the detection limit: Figure S1: Fl. Intensity ratio (I462 nm/ I359 nm) Vs. .... 450. 500. 550. 600. 0.0. 5.0x105. 1.0x106. 1.5x106. 2.0x106. 2.5x106.
Electronic Supplementary Material (ESI) for RSC Advances. This journal is © The Royal Society of Chemistry 2014
Supporting Information Cascade reaction based rapid and ratiometric detection of H2S/S2over bio-thiols with live cell-imaging: demasking of ESIPT approach Shyamaprosad Goswami, Abhishek Manna, Monalisa Mondal and Debasish Sarkar
CONTENTS 1. Calculation of the detection limit……………………………………………….....2 2. Calculation of rate constant………………...……..……………………………….3 3. Fluorescence responses of FBBP + interfering species + S2- …………………….3 4. 1H-NMR, 13C-NMR and Mass spectra…………………………………………..4-7 5. Fluorescence spectra of FBBP with different guest spices ………………….....8-10 6. Fluorescence emission spectra of FBBP with Na2S at λex=405 nm…………….11 7. MTT assay of the probe FBBP…………………………………………………...12 8. References…………………………………………………………………….…...12
1. Calculation of the detection limit: 1.1 1.0
y=85402x+0.1096 2 R =0.99
0.9
I462/I359
0.8 0.7 0.6 0.5 0.4 0.3 -6
4.0x10
-6
6.0x10
-6
8.0x10 2-
-5
1.0x10
-5
1.2x10
[S ]
Figure S1: Fl. Intensity ratio (I462 nm/ I359 nm) Vs. Conc. of S2- plot. The detection limit DL of FBBP for S2- was determined from the following equation. [S1] DL = K* Sb1/S Where K = 2 or 3 (we take 2 in this case); Sb1 is the standard deviation and S is the slope of the calibration curve. From the graph we get slope = 85402, and Sb1 value is 0.021958. Thus using the formula we get the Detection Limit = 0.51 µM i.e. FBBP can detect S2- in this minimum level.
2
2. Calculation of rate constant: From Fl. Intensity ratio (I462/I359) vs. time (sec.) plot using first order rate equation (Figure S2), we get rate constant K = slope × 2.303 = 0.0086 × 2.303 = 1.9 × 10-2 sec-1
+ 0.177 1.4 y=0.0086x 2 R = 0.98 1.6
1.2 1.4 1.2 1.0
I462/I359
I462/I359
1.0 0.8 0.6
0.8 0.6 0.4 0.2
0.4
0.0 0
0.2 0
20
40
60
80
100
120
140
20
40
160
60
80 100 120 140 160 180 200
Time (sec.)
Time (sec.) Figure S2: The time vs. Fl. Intensity ratio (I462 nm/ I359 nm) plot between 20 to 160 secend and the full plot is in inset. 3. Fluorescence responses of FBBP + various species + S2-:
1.4 1.2
I 462nm /I 359nm
1.0 0.8 0.6 0.4
3
P + + S 2 S 2- + +C S 2- y + s G + S S 2- H + + S 2- F + + CN + S 2- S 2- + + N + SC 3 S 2- N + +H S 2- O 2 + +O 2 S 2- C + +O l S 2- H . + S 2 O 2
F
B
B
0.0
-
0.2
Figure S3: Ratiometric fluorescence responses (I462nm/I359nm) of FBBP (c = 1.0 x 10–5 M) to S2- (1 equiv) containing 10 equiv of various metal species.
3
4. 1H NMR, 13C NMR and ESI MS spectra: 1 H NMR spectrum and its expansion of Receptor i.e. FBBP in CDCl3:
Expansion of the NMR:
4
1
H NMR spectrum and its expansion of Receptor i.e. FBBP in d6-DMSO:
13
C NMR spectrum of Receptor i.e. FBBP:
5
ESI MS Mass Spectrum of FBBP:
1
H NMR spectrum and its expansion of FBBP + Na2S in d6-DMSO:
6
ESI MS Spectrum of FBBP + Na2S:
7
5. Fluorescence emission spectra of FBBP with different species such as Cysteine, Glutathione, F-, CN-, N3-, SCN-, SO32-, S2O32-, H2O2, HSO3-, Hydroxyl radical, Hypochlorite, Ascorbic Acid, NH4+, Co2+, Al3+, Hg2+ and Cu2+ in CH3CN : H2O (2:8, v/v) (The solutions of anions and oxidants were prepared from Cysteine, Glutathione, TBAF, KCN, NaN3, KSCN, KSO3, K2S2O3, NaHSO3, H2O2, Femton’s reagent, NaOCl, Ascorbic Acid, NH4Cl, CoCl2, Al(NO3)3, 9H2O, HgCl2 and CuCl2 respectively in H2O). (a)
(b) 6
3.5x10 6
6
Cysteine
6
2.5x10
3.0x10
Fl. Intensity (a.u.)
Fl. Intensity (a.u.)
3.0x10
6
2.0x10
6
1.5x10
6
1.0x10
Glutathione
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
5
5.0x10
5
5.0x10
0.0
0.0 350
400
450
500
550
350
600
(c)
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
(d) 6
3.5x10
6
3.5x10
-
Fl. Intensity (a.u.)
Fl. Intensity (a.u.)
6
6
-
3.0x10
F
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
CN
3.0x10
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
5
5
5.0x10
5.0x10
0.0
0.0 350
400
450
500
550
600
350
N3
-
SCN
6
3x10
Fl. Intensity (a.u.)
Fl. Intensity (a.u.)
3.0x10
500
550
600
(f)
6
3.5x10
6
450
Wavelength (nm)
Wavelength (nm)
(e)
400
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
-
6
2x10
6
1x10
5
5.0x10
0.0
0 350
400
450
500
Wavelength (nm)
550
600
350
400
450
500
550
600
650
Wavelength (nm)
8
(g)
(h) 6
3.5x10
6
3.5x10
Fl. Intensity (a.u.)
6
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
S 2 O3
3.0x10
Fl. Intensity (a.u.)
SO3
6
3.0x10
2-
2-
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
5
5
5.0x10
5.0x10
0.0 350
400
450
500
550
600
0.0 350
400
Wavelength (nm)
450
(i)
550
600
(j) 6
6
3.5x10
3.5x10
H2O2
-
3.0x10
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
HSO3
6
3.0x10
Fl. Intensity (a.u.)
Fl. Intensity (a.u.)
6
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
5
5
5.0x10
5.0x10
0.0
0.0 350
400
450
500
550
600
350
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
(k)
(l)
6
3.5x10
6
3.5x10
.
OH
6
Fl. Intensity (a.u.)
3.0x10
Fl. Intensity (a.u.)
500
Wavelength (nm)
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
OCl
6
3.0x10
-
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
5
5
5.0x10
5.0x10
0.0
0.0 350
400
450
500
Wavelength (nm)
550
600
350
400
450
500
550
600
Wavelength (nm)
9
(m)
(n) 6
6
4.0x10
Ascorbic Acid
6
3.0x10
NH4
6
3.5x10
+
6
3.0x10
Fl. Intensity (a.u.)
Fl. Intensity (a.u.)
3.5x10
6
2.5x10
6
2.0x10
6
1.5x10
6
2.5x10
6
2.0x10
6
1.5x10
6
6
1.0x10
1.0x10
5
5.0x10
5
5.0x10
0.0
0.0
350
350
400
450
500
550
400
600
450
500
550
600
Wavelength (nm)
Wavelength (nm) (o)
(p) 6
4.0x10
6
4.0x10
3.5x10
Co
3.5x10
Al
6
2+
6
3+
6
3.0x10
Fl. Intensity (a.u.)
Fl Intensity (a.u.)
6
3.0x10
6
2.5x10
6
2.0x10
6
1.5x10
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
6
1.0x10
5
5.0x10
5
5.0x10
0.0 0.0
350 350
400
450
500
550
400
600
450
500
550
600
Wavelength (nm)
Wavelength (nm)
(q)
(r) 6
6
4.0x10
4.0x10
Hg
6
2+
6
3.5x10
6
6
3.0x10
Fl. Intensity (a.u.)
Fl. Intensity (a.u.)
3.5x10
Cu
2+
6
2.5x10
6
2.0x10
6
1.5x10
6
3.0x10
6
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
1.0x10
5
5
5.0x10
5.0x10
0.0
0.0
350
400
450
500
Wavelength (nm)
550
600
350
400
450
500
550
600
Wavelength (nm)
10
Figure S4-S22: Fluorescence emission spectra of FBBP with different species such as Cysteine, Glutathione, F-, CN-, N3-, SCN-, SO32-, S2O32-, H2O2, HSO3-, Hydroxyl radical, Hypochlorite, Ascorbic Acid, NH4+, Co2+, Al3+, Hg2+ and Cu2+ in CH3CN : H2O (2:8, v/v) respectively.
6. Fluorescence emission spectra of FBBP with Na2S (excitation at 405 nm): 6
3.5x10
6
Na2S
3.0x10
6
Fl. Intensity (a.u.)
2.5x10
6
2.0x10
6
1.5x10
6
1.0x10
5
5.0x10
0.0 450
500
550
600
Wavelength (nm)
Figure S23: Fluorescence emission spectra of FBBP (c = 1.0 x 10–5 M) with Na2S (c = 1.0 x 10–5 M) at pH 7.5 in CH3CN: H2O (2:8, v/v) (excitation at 405 nm).
11
7. MTT assay of the probe FBBP: 100
Absorbance (%)
80
60
40
20
0 10
20
30
40
50
60
70
80
90 100
[FBBP] in µM
Figure S24: MTT assay with different concentration of probe (FBBP) after 24h.
8. References: [S1] M. Zhu, M. Yuan, X. Liu, J. Xu, J. Lv, C. Huang, H. Liu, Y. Li, S. Wang, D. Zhu, Org. Lett. 2008, 10, 1481-1484.
12