S1
Two-step FRET mediated metal ion induced signalling responses in a probe appended with
three fluorophores
Biswonath Biswal, Ajoy Pal and Bamaprasad Bag*
Colloids and Materials Chemistry Department, CSIR-Institute of Minerals and Materials Technology,
P.O.: R.R.L., Bhubaneswar-751 013, Odisha, India. Fax: (+) 91 674 258 1637; Tel: (+ 91) 674 237
9254, Email: [email protected]
Supplementary information
Absorption and fluorescence spectral data
Association constants and detection limit determination
Excited state life time data (TCSPC)
Characterization of the metal ion complexes
Characterization of the probes 1a-1f and 1
Characterization of the metal ion complexes, FT-IR and ESI-MS
Electronic Supplementary Material (ESI) for Dalton Transactions.This journal is © The Royal Society of Chemistry 2017
S2
300 400 500 6000.0
1.5
3.0
4.5A
bs.
Wavelength(nm)
CHCl3
DCM
THF
Ethyl acetate
EtOH
MeOH
MeCN
DMF
DMSO
1b
(a)
300 400 500 600 700 8000
1
2
3
4
Ab
s.
Wavelength(nm)
CHCl3
DCM
DMF
DMSO
EtOH
Ethyl acetate
MeCN
MeOH
n-PrOH
THF
1
(b)
Fig. S1: Absorption spectral pattern of (a) 1b and (b) 1 respectively in different solvents. [1b] =[1] =1
×10−4
M.
Table S1: The wavelength of absorption (λabs, nm) and corresponding molar extinction coefficients (ε,
dm3mol
−1cm
−1) of 1b and 1 in different solvents.
Solvents Probe λabs, nm
(ε, dm3mol
−1cm
−1)
Probe λabs, nm
(ε, dm3mol
−1cm
−1)
CHCl3 1b 320 (10359), 350 (11101),
368 (16022), 388 ( 14943)
1 323 (37865), 350 (25865), 369 (25865),
390 (24786), 478 (27955)
DCM 318 (29370), 350 (30449),
366 (45216), 386 (41670)
321 (33280), 481 (25528), 351 (23033),
368 (23370), 389 (21617)
THF 317 (15651), 348 (16359),
366 (19865), 386 (17910)
318 (21117), 351 (15056), 368 (15786),
389 (16887), 480 (15421)
Ethyl
acetate
317 (15115), 348 (15516),
364 (18853), 385 (17775)
320 (25528), 350 (17775), 368 (17775),
386 (16696), 474 (16696)
EtOH 317 (25865), 347 (26359),
365 (38202), 385 (35707)
274 (27955), 318 (16191), 348 (9348),
368 (9685), 388 (8943), 484 (10022)
MeOH 317 (27955), 347 (27955),
364 (41033), 383 (37153)
320 (22696), 350 (16359), 366 (17033),
386 (14606), 481 (1775)
MeCN 320 (9685), 347 (10022),
365 (14943), 383 (16528)
321 (23033), 351 (17438), 366 (17438), 388
(15685), 483 (18853)
DMF 320 (20022), 350 (21154),
366 (31853), 386 (30867)
320 (18488), 351 (14252), 389 (15719),
368 (15056), 487 (15056)
DMSO
320 (22146), 350 (23325),
369 (33629), 389 (32370)
321 (29707), 354 (22359), 369 (24115), 390
(20606), 493 (24115)
S3
400 450 500 550 600 650
λλλλex
= 350 nm
Flu
o. In
t. (
arb
. u
nits)
Wavelength(nm)
DCM
CHCl3
THF
Ethyl acetate
MeOH
EtOH
MeCN
DMF
DMSO
(a)
1
500 550 600 650 700 750 800
Flu
o.
Int.
(a
rb.
un
its)
Wavelength(nm)
DCM
CHCl3
THF
Ethyl acetate
MeOH
EtOH
MeCN
DMF
DMSO
(b)
1
λλλλex
= 465nm
400 450 500 550 600 650
Flu
o.
Int.
(arb
. units)
Wavelength(nm)
CHCl3
DCM
THF
Ethyl acetate
EtOH
MeOH
MeCN
DMF
DMSO
(c)
1b
400 450 500 550 600 650
Flu
o.
Int.
(a
rb.
units)
Wavelength (nm)
χχχχ2 = 0.998
(d)
1
Fig. S2: Fluorescence spectral pattern of 1 upon excitation at (a) 350nm, (b) 465nm and that of 1b on
excitation at 350nm in different solvents. (d) Deconvulated fluorescence spectral pattern of 1 in MeCN
upon excitation at 350nm. [1b] = [1] = 1�M.
300 400 500 600 7000
1
2
3
4
Ab
s.
Wavelength(nm)
blank H+
Na+ K
+
Mn2+
Fe2+
Fe3+
Ni2+
Cu2+
Zn2+
Ag+ Pb
2+
Cd2+
Hg2+
Hg2+
(a)
Blank and
other metal ions
A557
Fig. S3: Absorption (a) spectra of 1b
Photographs depicting its metal ion induced
(below, on illumination of 350nm light
300 400 500 600 7000.00
0.05
0.10
0.15
0.20
Hg2+
Ab
s.
Wavelength (nm)
(a)
Blank and other
metal ions
Fig. S4: (a) absorption spectra of 1b
mixture, [1b] = 10�M. (b) Metal ion induced a
MeCN (9:1 v/v) as monitored at 557nm
Fig. S5: Photographs depicting absorption and fluorescence
solution in the absence and presence of various metal ions under normal condition (above) and
illuminated under 350nm (below).Solvent condition: MeCN (Left) and MeCH
S4
800
other metal ions
1b (1�M) in MeCN in absence and presence of various metal ions.
metal ion induced chromogenic (above, as observable) and
, on illumination of 350nm light) behaviour.
700 800
Blank
Mn2+
Fe2+
Co2+
Ni2+
Cu2+
Zn2+
Ag+
Pb2+
Cd2+
Hg2+
Na(
I)K(I)
Mn(II
)
Fe(II)
Fe(III
)
Co(II
)
Ni(I
I)
Cu(II
)
Zn(II)
Ag(I)
0
20
40
60
80
100
(εM
(I/I
I).1/ε
1) 5
57
Added Metal ions
MeCN
aq. MeCN
(b)
1b in presence of various metal ions in MeCN
Metal ion induced absorption enhancement factors of 1 in dry and aqueous
as monitored at 557nm.
Photographs depicting absorption and fluorescence of 1 as a function of colour change of the
solution in the absence and presence of various metal ions under normal condition (above) and
Solvent condition: MeCN (Left) and MeCH-H2O(9:1 v/v)
presence of various metal ions.
) and fluorogenic
Zn(II)
Ag(I)
Pb(II)
Cd(II
)
Hg(II
)
Added Metal ions
in presence of various metal ions in MeCN-H2O (9:1 v/v)
in dry and aqueous
as a function of colour change of the
solution in the absence and presence of various metal ions under normal condition (above) and
O(9:1 v/v).
S5
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
0.0
0.4
0.8
1.2
1.6
Abs.
[Pb2+
]/{[1]+ [Pb2+
]}
1
(a)
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Abs
[Hg2+
]/{[1b]+ [Hg2+
]}
(b)
Fig. S6: Plot of absorption of (a) 1 against mole fraction of Pb
2+ in MeCN and (b) 1b against mole
fraction of Hg2+
in MeCN-H2O (9: 1 v/v) (Job’s plot), λobs= 557 nm.
300 400 500 600 700 800
0.0
0.1
0.2
0.3
Abs.
Wavelength(nm)
0.0 eq.
20.0 eq.
[Hg2+
]
A557
(a)
1b
400 450 500 550 600 6500.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0 2 4 6 8 10 12 140.0
0.2
0.4
0.6
0.8
1.0
1.2
Flu
o.
Int. (
/E6,
arb
. units) 5
80
Eq. of Hg2+
added
(b) I420
Flu
o. in
t.(/
E6,
arb
. un
its)
Wavelength(nm)
I580
0.5 eq.
14.0 eq.
Hg2+
1b
Fig. S7: (a) Absorption and (b) fluorescence titration spectral pattern of 1b with Hg
2+ ion in MeCN-
H2O (9: 1 v/v).
300 400 500 6000.00
0.04
0.08
0.12
0.16
0.20
A517
A557
4.0 eq.
Abs.
Wavelength(nm)
Hg2+
0.0 eq.
(a)
A465
0 1 2 3 4 5
0.0
0.1
Ab
s [(A
-A0) 5
57]
Eq. of Hg2+
added
(b)
Fig. S8: (a) Absorption spectral pattern of 1 with Hg
2+ ion in MeCN-H2O (9: 1 v/v).
S6
300 400 500 600
0.0
0.1
0.2
0.3
0.4
0.5
0 1 2 3 40.00
0.05
0.10
0.15
0.20
0.25
0.30
Ab
s.(
A55
7)
[Pb2+
]x (10-5) M
Ab
s.
Wavelength(nm)
[Pb2+
]
0.2 eq.
4.0 eq.
A557
Fig. S9: (a) absorption titration spectra of probe 1 in MeCN on addition of Pb2+
ion, (inset):
corresponding plot of absorption (A557) as a function of concentration of added Pb2+
ion.
1x105
2x105
3x105
4x105
5x105
0
20
40
60
80
100
120
140
1/(
A-A
0) 5
57
1/[Pb2+] M-1
Y= -4.893651+ 2.553E-4 X
( r2 = 0.998 )
Fig. S10: Double reciprocal plot of change in absorption of 1 as a function of added Pb2+
ion in
MeCN. The reciprocal of slope estimates the association constant through Benesi-Hildebrand
equation. [1] = 10�M.
S7
0 5 10 15 20
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Flu
o.
Int.
(/E
6,
arb
. un
its)
Eq. of Pb2+
added
(a)
-16 -15 -14 -13 -12 -11
0.0
0.5
1.0
1.5
2.0
2.5
3.0
[Flu
o.
Int.
(F
-F0) 5
80,
/E6
, arb
. un
its]
ln C(Pb2+
)
Equation y = A2 + (A1-A2)/(1 + exp((x-x0)/dx))
Adj. R-Squar 0.99678
Value Standard Erro
A1 70679.5867 13419.10744
A2 3.18037E6 62876.02611
x0 -11.69335 0.02033
dx 0.35396 0.01521
(b)
Fig. S11: (a) Fluorescence titration profile of 1 as a function of equivalents of added Pb2+
ion in
MeCN and (b) corresponding plot of change in fluorescence intensities (I-I0)580 as a function of
concentration of Pb2+
ion added [ln C(Pb2+
)] for determination of association constant. Conditions: [1]
= 1�M, λex =350nm, em. and ex. b. p. =5nm, RT.
0 2 4 6 8 10 12 14 16
0.1
0.2
0.3
0.4
0.5
Flu
o.
Int.
(/E
6,
arb
. units)
Eq. of Hg2+
added
(a)
-16 -15 -14 -13 -12 -11
0.0
0.1
0.2
0.3
0.4
0.5
Flu
o.
Int.
[(F
-F0) 5
80,
/E6
, arb
. un
its]
ln C(Hg2+
)
Equation y = A2 + (A1-A2)/(1 + exp((x-x0)/dx))
Adj. R-Squ 0.99765
Value Standard Error
(F-F0)580 A1 4844.24771 2942.6206
(F-F0)580 A2 585381.75938 15718.03209
(F-F0)580 x0 -11.79149 0.02921
(F-F0)580 dx 0.40741 0.01916
(b)
Fig. S12: (a) Fluorescence titration profile of 1 as a function of equivalents of added Hg2+
ion in
MeCN-H2O (9: 1 v/v) and (b) corresponding plot of change in fluorescence intensities (I-I0)580 as a
function of concentration of Hg2+
ion added [ln C(Hg2+
)] for determination of association constant.
Conditions: [1] = 1�M, λex =350nm, em. and ex. b. p. =5nm, RT.
S8
-14 -13 -12 -11 -10
0.00
0.02
0.04
0.06
0.08
0.10
0.12A
bs (
A-A
0) 5
57
ln C(Hg2+
)
Equation y = A2 + (A1-A2)/(1 + exp((x-x0)/dx))
Adj. R-Squ 0.99021
Value Standard Error
A-A0 A1 -0.00234 0.00257
A-A0 A2 0.11585 0.00386
A-A0 x0 -11.32867 0.04113
A-A0 dx 0.40941 0.03904
(a)
0 2 4 6 8 10 12 14 16 180
500
1000
1500
2000
2500
3000
3500
4000
[(A
-A0)/
A0] 5
57
1/[Hg2+
] (x 107) M
-1
Y= 5.79683+2.053E-5 X
r2 = 0.998
(b)
1
Fig. S13: Graphs for determination of association constants from absorption titration of 1 (10µM) with
added Hg2+
ion in MeCN-H2O (9: 1 v/v) solvent. (a) Plot of change in absorption intensities (A-A0)557
as a function of concentration of Hg2+
ion added [ln C(Hg2+
)] and (b) double reciprocal plot of change
in absorption intensities [(A-A0)/A0]557 as a function of concentration of Hg2+
ion added (1/[Hg2+
])
through Benesi-Hildebrand formalism.
0 1x107
2x107
3x107
4x107
5x107
0.0000
0.0002
0.0004
0.0006
0.0008
1/(
I F-I
0) 5
80
1/[Hg2+ + + +
] M-1
Y= -7.45761E-6 + 1.59762E-11 X
r2 = 0.997
Ka = [intercept] / [slope]
(a)
1
0 1 2 3 4 50.000
0.004
0.008
0.012
0.016
0.020
1/(
I F-I
0)
1/[Pb2+
] (x107)M
-1
Y= 4.84E-4 + 3.60E-10 X
(r2 = 0.995)
(b)
Fig. S14: Double reciprocal plots of change in fluorescence intensities [1/(I-I0)]580 of 1 as a function of
concentration of metal ion added (1/[M2+
]) through Benesi-Hildebrand formalism, (a) Hg2+
ion in
MeCN-H2O (9: 1 v/v) and (b) Pb2+
ion in MeCN medium. Conditions: [1] = 1�M, λex =350nm, em.
and ex. b. p. =5nm, RT.
The association constants determined through Benesi-Hildebrand equation are comparable with those
determined through the equation (equation 6, mentioned in the manuscript).
S9
0 2 4 6 8 10 12 140.0
0.2
0.4
0.6
0.8
1.0
1.2F
luo
. In
t. (
/E6,
arb
. u
nits)
Eq. of Hg2+
added
(a)
λobs
= 580nm
-16 -15 -14 -13 -12 -11
0.0
0.2
0.4
0.6
0.8
1.0
Flu
o.
Int.
[(I
-I0) 5
80,
/E6
, a
rb.
units)
lnC(Hg2+
)
Equation y = A2 + (A1-A2)/(1 + exp((x-x0)/dx))
Adj. R-Sq 0.99434
Value Standard Error
I-I0 A1 14159.04308 10190.46775
I-I0 A2 1.32416E6 60166.25735
I-I0 x0 -11.97194 0.04222
I-I0 dx 0.34075 0.0301
(b)
Fig. S15: (a) Fluorescence titration profile of 1b as a function of equivalents of added Hg2+
ion in
MeCN-H2O (9: 1 v/v) and (b) corresponding plot of change in fluorescence intensities (I-I0)580 as a
function of concentration of Hg2+
ion added [ln C(Hg2+
)] for determination of association constant.
Conditions: [1b] = 1�M, λex =350nm, em. and ex. b. p. =5nm, RT.
0.0 2.0x10-7
4.0x10-7
6.0x10-7
8.0x10-7
1.0x10-6
1.2x10-6
0.065
0.070
0.075
0.080
0.085
0.090
Fl. I
nt.
ratio
(I 5
80/
I 42
0)
[Hg2+
]
Equation y = a + b*x
Adj. R-Square 0.99061
Value Standard Error
I580/I420 Intercept 0.06261 4.54511E-4
I580/I420 Slope 24969.70998 768.26446
(a)
0.0 1.0x10-7
2.0x10-7
3.0x10-7
4.0x10-7
0.164
0.166
0.168
0.170
0.172
0.174
0.176
0.178
0.180
Flu
o.
Int.
ratio (
I 58
0/I
420)
[Pb2+
]
Equation y = a + b*x
Adj. R-S 0.9705
Value Standard Error
Intercept 0.16479 7.61974E-4
Slope 35819.03064 3110.74392
(b)
Fig. S16: Linear regressions of the plot of fluorescence spectral intensity ratio (I580/I420) of 1 as a
function of concentrations of (a) Hg2+
ion in MeCN-H2O(9:1 v/v) medium (b) Pb2+
ion in MeCN for
determination of sensitivity of detection (S/N = 5).
S10
450 500 550 600 650 700 750 800
Flu
o.
Int.
(a
. u
.)
Wavelength(nm)
1c + Hg2+
1f + Hg2+
1 + Hg2+
(a)
400 450 500 550 600 650
Flu
o.
Int.
(a
. u
.)
Wavelength (nm)
1e + Hg2+
1 + Hg2+
1b + Hg2+
1d + Hg2+
1a + Hg2+
(b)
Fig. S17: Fluorescence spectra of the probes (1, 1f and 1c), λex = 420nm (a) and (1, 1a, 1b, 1d, 1e) (b)
in presence of Hg2+
in MeCN-H2O (9: 1 v/v) mixture, λex = 350nm, RT, [probes] = 1µM.
90:10 80:20 70:30 60:40 50:50 40:60 30:70 10:900
1
2
3
4
5
6
7
8
Flu
o. In
t. (
I 580/I
420)
MeCN:H2O Composition (v/v)
(a)
400 450 500 550 600 650
Flu
o.
Int.
(a.
u.)
Wavelength(nm)
MeCN: H2O (v/v)
9:1
8:2
7:3
6:4
5:5
4:6
3:7
1:9
(b)
Fig. S18: (a) Fluorescence intensity ratio (F580/ F420) and (b) corresponding spectra of 1-Hg
2+ complex
in various composition of MeCN-H2O (9:1 v/v) mixture, HEPES buffer, pH = 7.02, λex= 350nm, em
and ex bp = 5nm, RT, [1] =1�M, [Hg2+
] = 5�M.
S11
5 10 15 200
2000
4000
6000
8000
10000
λem
= 420nm
Ph
oto
n C
oun
ts
Time (ns)
1d + Hg2+
1 + Hg2+
(a)
0 5 10 15 200
2000
4000
6000
8000
10000
Pho
ton
Co
unts
Time (ns)
1f + Hg2+
1 + Hg2+
λem
= 525nm
(b)
0 5 10 15 200
2000
4000
6000
8000
10000 1
1+Hg2+
Photo
n C
ounts
Time (ns)
λem
= 580nm
(c)
Fig. S19: TCSPC decay profile of in-situ Hg
2+ complexes of (a) 1 in comparison to 1d(monitored at
420nm), (b) 1 in comparison to 1f (monitored at 525nm) and that of (c) 1 alone and in presence of
Hg2+
in MeCN-H2O(9:1 v/v) medium, λex = 350nm, RT.
400 450 500 550 600 650
Flu
o.
Int.
(a
rb.
un
its.)
Wavelength(nm)
X X+Cl-
X+NO3
- X+SO
4
2-
X+I- X+PO
4
3-
X+Cr2O
7
- X+HCO
3
-
X+AcO- X+En
X+EDTA X+SCN-
X= = = = [1 + Hg2+
](a)
Fig. S20: Fluorescence spectra of 1 in presence of Hg
2+ in MeCN-H2O (9: 1 v/v) in presence of
various anions and chelating agents such as EDTA / ethylene diamine (En). [1]= 1µM. λex = 350nm.
S12
400 450 500 550 600 650
Flu
o.
Int.
(arb
. un
its)
Wavelength(nm)
pH 3 pH 4
pH 5 pH 6
pH 7 pH 8
pH 9 pH 10
pH 11 pH 12
(a)
3 4 5 6 7 8 9 10 11 120.0
0.2
0.4
0.6
0.8
1.0
F5
80/F
42
0
pH
(b)
Fig. S21: (a) Fluorescence spectra and (b) corresponding intensity ratio(F580/F420) of 1 in various pH
showing its stability over a wide pH range. Conditions: MeCN-H2O (9:1 v/v) mixture, HEPES buffer,
λex= 350nm, em and ex bp = 5nm, RT, [1] =1�M
1000 1200 1400 1600 1800 2000
0.6
0.7
0.8
0.9
1.0
% T
ran
sm
itta
nce
W avenumber (cm -1)
1
1+Pb2+
1682cm-1
1638cm-1
Fig. S22: FT-IR spectra of 1 and 1-Pb
2+ complex.