1

Supporting Information

Nitrogen Doped Thiol Functionalized Carbon Dots for

Ultrasensitive Hg (II) Detection

Abhishek Gupta, Abhishek Chaudhary, Pooja Mehta, Charu Dwivedi, Syamantak Khan, Navneet

Chandra Verma, Chayan Kanti Nandi*

School of Basic Sciences, Indian Institute of Technology, Mandi, Himachal Pradesh, 175001

India

Corresponding author

*Chayan K. Nandi

School of Basic Sciences,

Indian Institute of Technology Mandi,

Himachal Pradesh-175001, India

Email: chayan@iitmandi.ac.in

Table of Contents:

Figures (S1-S16)

Table (S1-S3)

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2015

2

Experimental Section:

Materials: All Glass wares were washed with aqua regia (3 HCl: 1 HNO3), followed by rinsing

several times with double distilled water. Chitosan, polyethylene glycol (PEG), dithiothreitol

(DTT), Glutathione and amino acids were purchased from Sigma Aldrich. All metal ions

including chloride of lead, mercury, chromium, cadmium, magnesium, copper, cobalt, nickel,

manganese, zinc and arsenic oxide with purity >99% and EDTA were purchased from Merck

India chemicals. Sodium Iodide (NaI, 99%) was purchased from Fisher Scientific. Double

distilled 18.3 mΩ deionized (DI) water (Elga Purelab Ultra) was used throughout for the

preparation of solutions.

UV-Vis Absorption spectroscopy

The UV-Vis absorption spectra were recorded using Shimadzu UV-Vis 2450 spectrophotometer.

The spectra were collected using a quartz cuvette of 10 mm path length and volume 1 ml. All the

measurements were repeated at least three times.

Transmission Electron Microscope (TEM)

The particle size and dispersity of the synthesized C-dots were checked using a TECNAI G2 200

kV TEM (FEI, Electron Optics) electron microscope with 200 kV input voltage. TEM grids were

prepared by placing 5 µL diluted and well sonicated sample solutions on a carbon coated copper

grid and evaporated the solution at room temperature completely. Precautions were taken to

avaoid contamination from various sources like dust particles and glasswares.

Fourier Transform Infrared Spectroscopy (FTIR)

3

FTIR spectra of C-dots and DTT modified C-dots were measured using a Perkin-Elmer FTIR

spectrophotometer equipped with a horizontal attenuated total reflectance (ATR) accessory

containing a zinc selenide crystal and operating at 4 cm−1

resolution. The use of the spectral

subtraction provided reliable and reproducible results.

Zeta Potential measurement

Zeta potential measurements were carried out on a Malvern Zetasizer Nano system equipped

with 633 nm He-Ne laser. 1 ml of the sample was used in a cuvette (Malvern-DTS1061) and

measured for 10 runs with 10 second time interval. The average of atleast five measurements

were reported in the data.

Atomic Force Microscopy (AFM)

AFM analysis of the synthesized C-dots for particle size determination was carried out using a

Digital Instruments Bruker AFM. Standard Veeco tapping mode silicon probes were used for

scanning the samples. Typically, aqueous suspensions of C-dots samples were dried on silicon

substrate for 3 hours. Once dried, samples were placed on the AFM stage and scanned. Pertinent

scanning parameters were as follows: Resonant frequency (probe): 60-80 kHz; Tip velocity for

all measurements are: (4 m/s for 2 m), (15 m/s for 5 m), (30 m/s for 10 m). Aspect ratio:

1:1; Resolution: 512 samples/line, 256 lines.

X-ray photoelectron spectroscopy (XPS)

X-ray Photo-Electron Spectroscopy (XPS) with Auger Electron Spectroscopy (AES) module PHI

5000 Versa Prob II, FEI Inc. and C60 sputter gun have been used for characterization and

scanning the spectra from C1s, N1s, O1s and S2p region. Al K X-ray radiation was used as the

4

source for excitation (1486.8 eV, 500 mm). Samples were loaded on copper strips, and surface

adherence done by double sided adhesive tape. C-dots samples were prepared by concentrating

the solution by centrifugation at 23000 rpm, followed by drying. The interference of DTT was

excluded by dialyzing the DTT modified C-dots in 3-5 kDa membrane (D-Tube™ Dialyzers,

Merck Millipore) for 24 hours. The resulting sample was concentrated by centrifugation at 23000

rpm and the pellet was dried to obtain DTT modified C-dots in powder form. Thus obtained C-

dots samples were used for recording XPS spectra.

Steady state and Time Resolved Fluorescence Spectroscopy

Steady state fluorescence was measured using Horiba Fluorolog-3 Spectrofluorometer. All the

experiments including Hg2+

detection were performed at room temperature after equilibrating the

samples for 15 minutes. The fluorescence was measured in 1 ml quartz cuvette. For titration

experiment, to calculate the detection limit, different concentration of Hg2+

ions were added to a

fixed concentration of C-dots and the final volume is maintained as 1 ml. For determining the

selectivity for Hg2+

ion, 50 μM of each metal ion were added into a fixed concentration of

DTT/C-dots. The kinetics of the detection was investigated by recording the fluorescence spectra

of DTT/C-dots, immediately after the addition of Hg2+

ion upto 5 minutes (as most of the

quenching processes were over within 5 minutes of the contact time). The fluorescence lifetime

was measured using Horiba scientific Delta Flex TCSPC system with Pulsed LED Sources.

Ludox has been used as an IRF for de-convolution of the spectral value. 3 ml C-dots solution has

been used for all the lifetime measurements.

Calculation of quantum yield:

5

In the comparative method, the quantum yield (Φ) is calculated using the slope of the line

determined from the plot of the absorbance against the integrated fluorescence intensities. 2-

amino pyridine in 0.1 M H2SO4 is used as standard for determining the quantum yield. Equation

1 was used for calculating the quantum yield.

[

] [

] (Equation 1)

Where, m is the slope of the line obtained from the plot of the integrated fluorescence intensity

vs. absorbance, n is the refractive index of solvent (water n=1 in both cases) and the subscript R

refers to the reference (2-Amino pyridine).

Synthesis of C-dots and its Surface functionalization with DTT

Chitosan (CHS) gel (2%) was prepared by dissolving 2 gm CHS in 99 ml of water in the

presence of 1 ml of acetic acid, under vigorous stirring condition. 25 ml of PEG was dissolved in

75 ml water (25 % PEG solution). For synthesizing C-dots, in a beaker 4.5 ml of the prepared

CHS gel was mixed with 4.5 ml of the 25% of PEG. In the resulting solution 1 ml of 5 M NaOH

was added. The mixture was heated inside a domestic microwave at 600 W powers for 3

minutes. The resulting material was dissolved in 15 ml of distilled water. The solution was

filtered by Whatman filter paper. 1.5 ml of the filtered solution was centrifuged twice (Sorvall

Lynx 6000, thermo scientific) at 23000 rpm for 30 minutes each. The resulting pellets were

removed each time and the final supernatant was collected and used for experiments. For surface

functionalization with DTT, the above C-dots solutions were incubated for 2 hour with 10 M

DTT. Excess of DTT was removed as a supernatant by centrifugation at 25000 rpm for 5

minutes.

6

Figure S1. Schematic representation of DTT modified C-dots surface.

7

Figure S2. Characterization of synthesized C-dots (a) Multicolor fluorescence spectra recorded

from 280 to 600 nm with variable excitation with 20 nm increment (b) UV-Vis absorbance

spectrum (c) FTIR spectrum (d) AFM images and the height profile (e) TEM image and (f) TEM

size distribution of the C-dots.

a b

c d

e f

300 400 500 600 7000.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

nc

e

Wavelength (nm)

257

303

300 350 400 450 500 550 600 650 700

Co

un

ts

Wavelength (nm)

280

285

290

295

300

305

310

320

330

340

350

360

370

380

390

400

420

440

460

480

500

520

540

560

580

600

4000 3500 3000 2500 2000 1500 1000

Wavenumber (cm-1)

%T3331

29041574

1650

1071

1370

10nm

8

Figure S3. (a) XPS spectrum of the as synthesized C-dots shows three major peaks of C1s, O1s

and N1s respectively. (b), (c) and (d) showed the high resolution C1s, O1s and N1s spectra of the

as synthesized C-dots for characterizing the different functional groups.

800 600 400 200 0

0

5000

10000

15000

20000

25000

30000

C/s

Binding Energy (eV)

529.8 (O1s

)

283.2 (C1s

)

401(N1s

)

C-C

C-N

C=O

C-O-C

C=O

C-N-C

N-H

a b

c d

9

Figure S4. (a) XPS spectrum of DTT/C-dots shows a new peak at 162.2 eV corresponding to

S2p. (b) the high resolution scan of S2p confirms the presence of bound and free thiol group in

DTT functionalized C-dots (DTT/C-dots).1,2

(c) FTIR spectra of DTT/C-dots: a new absorbance

bands at 655 and 2426 cm-1

appear in DTT/C-dots corresponding to C-S and free –SH stretch3

and (d) change in the zeta-potential value of C-dots after DTT functionalization.

a b

1000 800 600 400 200 0

0

5000

10000

15000

20000

25000

227 S2s

C/s

Binding Energy (eV)

529.8, O1s

399.1, N1s

284.2 C1s

162.2 S2p

DTT C-dots C-dots-DTT

-15

-10

-5

0

5

10

15

-7.2Z

eta

Po

ten

tia

l (m

V)

-12.4+2.1

d c

4000 3500 3000 2500 2000 1500 1000 500

2426

% T

Wavenumber (cm-1)

3339

2874 1597

1377

1014

655

C-S

Free -SH

162.6 (bound -SH)

161.8

164 (free -SH)

10

Figure S5. (a) Comparison of UV-Vis absorbance spectra of only C-dot and DTT/C-dots shows

no significant change in the spectra after the modification with DTT and (b) multicolor

fluorescence emission spectra of DTT/C-dots recorded from 280 to 640 nm excitation with 20

nm increment.

300 400 500 600 700

Co

un

ts

Wavelength (nm)

280

285

290

295

300

305

310

320

330

340

350

360

370

380

390

420

440

460

480

500

520

540

560

580

600

620

640

a b

300 400 500 600 700 800

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rban

ce

Wavelength (nm)

C-dots

DTT modified C-dots

11

Figure S6. Effect of (a) pH and (b) NaCl concentration (0, 10, 50, 100, 200, 300, 400 and

500mM) on the fluorescence of C-dots. These results suggest that C-dots shows maximum

fluorescence intensity at physiological pH (pH 7.4) and also its fluorescence is not affected even

at high salt concentration.

2 4 6 8 10

Co

un

ts (

Arb

. u

nit

)

pH

Effect of pH on Fluorescence

a b

0 100 200 300 400 500

C

ou

nts

(A

rb.

un

it)

NaCl Salt (mM)

Effect of NaCl Salt Conc. on Fluorescence

12

Figure S7. (a) UV-Vis absorbance spectra of DTT/C-dots and conjugated with Hg2+

(50 M) and

(b) life time data of only DTT/C-dots and with Hg2+

(50 M).No significant changes were

observed in the UV-Vis spectrum. However, a decrease in excited state lifetime after the Hg2+

complex formation confirms that the quenching process is dynamic in nature.

a b

300 400 500 600 700 800

0.0

0.5

1.0

1.5

2.0

2.5

3.0

DTT modified C-dots

DTT modified C-dots with Hg2+

Ab

so

rban

ce

Wavelegth (nm)

Average τ DTT C-

dots = 1.69ns

Average τ DTT C-

dots + Hg2+ = 1.23ns

10

100

1000

10000

40 50301C

ou

nts

Time (ns)

Prompt

DTT/C-dots

DTT/C-dots with Hg2+

10 20

13

Figure S8. (a) The fluorescence spectra of DTT/C-dots in the presence of different halide ions

(50 M). No interference of halide ions (chloride, bromide and iodide) was observed on the

fluorescence intensity of DTT/C-dots (b) Fluorescence quenching of DTT/C-dots with increasing

concentration of Hg2+

in presence of 1 mM NaCl (c) the DL is determined to be as 19 pM in

presence of 1 mM NaCl when the data of (b) is fitted using Stern-Volmer equation (d)

fluorescence quenching of DTT/C-dots with increasing concentration of Hg2+

in presence of 1

mM KI (e) the DL is determined to be 23 pM when the data of (d) fitted using Stern-Volmer

equation. Each data point is presented as standard deviation from three replicate assays.

350 400 450 500 550 600

Co

un

ts (

Arb

. u

nit

)

Wavelength (nm)

0

5 X 104 nM

Hg2+

0 50 100

0.00

0.07

0.14

F0 /

F -

1

Conc. Hg2+ (nM)

350 400 450 500 550 600

(Co

un

ts)

Arb

.un

it

Wavelength (nm)

0

5 X 104 nM

Hg2+

0 50 1000.00

0.03

0.06

0.09

F0

/ F

- 1

Conc. Hg2+

(nM)

b c

d e

350 400 450 500 550C

ou

nts

(A

rb. u

nit

)

Wavelength (nm)

DTT/C-dots

Cl- (NaCl)

Cl- (KCl)

Br- (NaBr)

Br- (KBr)

I- (NaI)

I- (KI)

a

14

Figure S9. (a) Fluorescence quenching of the as synthesized C-dots in the presence of Hg2+

(b)

the detection limit of 6.8 nM was observed when the data of (a) fitted using Stern-Volmer

equation. Each data point is presented as standard deviation from three replicate assays.

0 25 50 75 100

0.00

0.02

0.04

0.06

0.08

0.10

F0

/ F

- 1

Conc. Hg2+ (nM)

350 400 450 500

Co

un

ts (

Arb

. u

nit

)

Wavelength (nm)

0

2X104 nM

a b

15

0.0

0.2

0.4

0.6

0.8

1.0

DTT C

-dots

Cd (2+)

Co (2+)

Mg (2+)

Mn (2+)

Ni (2+)

Pb (2+)

Zn (2+)

Ca (2+)

Na (+)

K (+)

AsO2(-)

Cr (3+)

Cu (2+)

Hg (2+)

Co

un

ts D

ec

rem

en

t

(

No

rma

lize

d)

Metal ions

Figure S10. Normalized fluorescence intensity changes in DTT/C-dots in presence of various

other metal ions (50 M).

16

Figure S11. Selectivity of different metal ions (50 M) in presence of C-dots surface

functionalized with (a) GSH and (b) Cysteine

350 400 450 500 550

Cys/C-dots

Cd2+

Co2+

Cr3+

Cu2+

Hg2+

Mg2+

Mn2+

Ni2+

Pb2+

Zn2+

AsO2

_

Co

un

ts (

Arb

. u

nit

)

Wavelength (nm)

350 400 450 500 550

Co

un

ts (

Arb

. u

nit

)

Wavelength (nm)

GSH/C-dots

Cd2+

Co2+

Cr3+

Cu2+

Hg2+

Mg2+

Mn2+

Ni2+

Pb2+

Zn2+

AsO2

_

a b

17

Figure S12. The data showed the change in the fluorescence intensity of DTT/C-dots in presence

of the mixture of various metal ions with and without adding Hg2+

. No change in the

fluorescence intensity of the DTT/C-dots was observed when all the metal ions were added

excluding Hg2+

, however, extensive quenching was observed when Hg2+

was added to the

system.

350 400 450 500 550

C

ou

nts

(A

rb.

un

it) DTT/C-dots + Other metals

except Hg2+

DTT/C-dots + Hg2+

Wavelength (nm)

DTT/C-dots

DTT/C-dots +

Other metals with Hg2+

18

Figure S13. Fluorescence quenching pattern of DTT/C-dots in presence of Hg2+

ion spiked in

tap water. Inset is showing the Stern-Volmer fitting to calculate the detection limit as 50 pM.

Each data point is presented as standard deviation from three replicate assays.

350 400 450 500 550

C

ou

nts

(A

rb.

un

it)

Wavelength (nm)

Spiked Hg2+

in Tap water

0

2X104 nM

0 8 16

0.00

0.02

0.04

F0

/ F

-1

Conc. Hg2+ (nM)

19

Figure S14. Fluorescence recovery of quenched C-dots/Hg2+

assay after the addition of

Cysteine, The Stern-Volmer fitting (inset) showed detection limit as 72 nM. Each data point is

presented as standard deviation from three replicate assays.

350 400 450 500 550

C

ou

nts

(A

rb.

un

it)

Wavelength (nm)

50M Cys

0C-dots

C-dots + Hg2+

0 100 200 300 400 5000.02

0.03

0.04

0.05

0.06

F0

/ F

- 1

Conc. Cys (nM)

20

350 400 450 500 550

C

ou

nts

(A

rb.

un

it)

Wavelength (nm)

C-dots

C-dots + 100M GSH

C-dots + 100M Cysteine

Figure S15. Control experiment (without addition of Hg2+

) revealed that the fluorescence

intensity of the as synthesized C-dots (without DTT modification) had a negligible change in

presence of biothiols (GSH and Cys).

21

Gly Asn Ala Leu Ile Asp Pro Lys EDTA GSH Cys

0.00

0.45

0.90

1.35

F/F

0 -

1

Figure S16. Relative change in the fluorescence recovery of quenched C-dots/Hg2+

after addition

of EDTA, GSH, cysteine and several other amino acids (50M). Apart from GSH and Cys, no

other amino acids showed any significant effect on fluorescence recovery of quenched C-

dots/Hg2+

.

22

Table S1: Comparison of the detection limit using different nanoparticle-based probes for Hg2+

detection.

S.

No.

Methods Detection limit Supporting

References

1 Surface-modified CdTe quantum dots 4 nM 4

2 Fluorescent gold nanoparticle 5 nM 5

3 Au@Ag core-shell Nanoparticles 9 nM 6

4 DNA-functionalized gold nanoparticles 25 nM 7

5 Mononucleotides-stabilized gold nanoparticles 50 nM 8

6 Cysteine functionalized Ag nanoparticles 65 nM 9

7 Carbon dots 226 and 845 nM 10

8 Carbon quantum dots 60 nM 11

9 Fluorescent carbon dots 5.9 nM 12

10 Carbon nano dots 4.2 nM 13

11 Carbon dots-labeled oligodeoxyribonucleotide 2.6 nM 14

12 Fluorescent carbon nanoparticles 0.23 nM 15

13 Carbon-dot 0.05 nM 16

14 Disposable strip biosensor 1 pM 17

15 Gold nanorod 2.4 pM 18

16 Lysozyme type VI stabilized gold nanoclusters 3.0 pM 19

17 DTT/ C-dots and C-dots 18 pM and 6.8 nM (our method)

23

Table S2: Physicochemical properties of the Uhl River water, Kamand Village of Mandi,

Himachal Pradesh 175001, India

Physicochemical property Experimental value

Turbidity (NTU) 4.9 NTU

Conductivity (μS) 95.6μS

pH 7.56

Alkalinity 40.00 mg/L

Total Hardness as CaCO3 80.00 mg/L

Ca2+

16.00 mg/L

Mg2+

9.76 mg/L

Na+ 2.70 mg/L

K+ 1.80 mg/L

Cl- 25.56 mg/L

SO42-

2.90 mg/L

HCO3- 40.00 mg/L

F- 0.00 mg/L

24

Table S3: Physicochemical properties of the tap water of Mandi, Himachal Pradesh 175001,

India.

Physicochemical property Experimental value

Turbidity (NTU) 0.00 NTU

Conductivity (μS) 34.8 0μS

pH 7.79

Alkalinity 64.00 mg/L

Total Hardness as CaCO3 240.00 mg/L

Ca2+

64.00 mg/L

Mg2+

19.50 mg/L

Na+ 11.30 mg/L

K+ 5.30 mg/L

Cl- 19.88 mg/L

SO42-

2.80 mg/L

HCO3- 64.00 mg/L

F- 0.35 mg/L

25

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