· 557 fig. s3: absorption (a) spectra of 1b photographs depicting its metal ion induced ... [ln...

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


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


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+

)


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+

)


Adj. R-Squ 0.99021


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+


)] 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+

)


Adj. R-Sq 0.99434


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+


)] 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


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


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.

S13

Fig. S23: 1H-NMR spectrum of 1(CDCl3).

Fig. S24: 13

C-NMR spectrum of 1(CDCl3).

S14

Fig. S25: ESI-MS of 1

Fig. S26:

1H-NMR spectrum of 1a(CDCl3).

Fig. S27: 13

C-NMR spectrum of 1a(CDCl

Fig. S28: ESI-MS spectrum of 1a.

S15

(CDCl3).

S16

Fig. S29:

1H-NMR spectrum of 1b(CDCl3).

Fig. S30:

13C-NMR of 1b(CDCl3).

S17

Fig. S31: ESI-MS spectrum of 1b.

Fig. S32: 1H-NMR spectrum of 1c (CDCl3).

Fig. S33: 13

C-NMR spectrum of 1c (CDCl

Fig. S34: ESI-MS spectrum of 1c.

S18

(CDCl3).

S19

Fig. S35:

1H-NMR spectrum of 1d (CDCl3)..

Fig. S36: 13

C NMR spectrum of 1d(CDCl3).

S20

Fig. S37: ESI-MS spectrum of 1d.

Fig. S38: ESI-MS spectrum of 1e.

S21

Fig. S39: 1H NMR spectrum of 1e(CDCl3)..

Fig. S40: 13

C NMR spectrum of 1e(CDCl3).

S22

Fig. S41: 1H NMR spectrum of 1f (CDCl3)..

Fig. S42: 13

C NMR spectrum of 1f (CDCl3).

Fig. S43: ESI-MS spectrum of 1f.

Fig. S44: ESI-MS spectrum of 1b-Hg

S23

Hg2+

complex.

Fig. S45: ESI-MS spectrum of 1-Hg2+

Fig. S46: ESI-MS spectrum of 1-Pb2+

S24

2+ complex.

2+ complex.

· 557 fig. s3: absorption (a) spectra of 1b photographs depicting its metal ion induced ... [ln...

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