Supporting Information of

Dicoumarol assisted synthesis of water dispersible gold nanoparticles for colorimetric sensing of

cysteine and lysozyme in biofluids

Betha Saineelima B. Kasibabu, Jigna R. Bhamore, Stephanie L. D’souza and Suresh Kumar

Kailasa*

Department of Applied Chemistry, S. V. National Institute of Technology, Surat-395 007, India*Corresponding author, Phone: +91-261-2201730; Fax: +91-261-2227334

E-mail: sureshkumarchem@gmail.com; skk@ashd.svnit.ac.in

O

O

O

O

O

O

O

OOH

O

OOH

O

O

OO

O

O

O O

HO

O

OHO

HAuCl4 +

DIC

DIC-Au NPs

Analytes

(Cys or lysozyme)

Electrostatic interaction

Hydrogen bonding and

π-π interactions

Aggregation of DIC-Au NPs induced by Cys or Lys

+

+

+

++

+

+

+

+

+

Scheme S1. A schematic representation of the mechanism for the colorimetric sensing Cys and Lys

using DIC-Au NPs as a probe.

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

Figure S1. UV-visible spectra of DIC-Au NPs at different concentrations of DIC (18.8 – 48.8 mM).

Inset: Photographic image of corresponding solutions.

400 500 600 700 800 900

0.0

0.3

0.6

0.9

1.2

1.5

18.8 mM

28.8 mM

38.8 mM

48.8 mM

Concentration of DIC (mM)

Ab

sorb

an

ce (

a.u

.)

Wavelength (nm)

1.5

400 500 600 700 800 900 1000

0.0

0.3

0.6

0.9

1.2

1.5

0°°°°C

25 °°°°C

50 °°°°C

75 °°°°C

100 °°°°C

Temperature (oC)

Figure S2. UV-visible spectra of DIC-Au NPs at different temperature ( 0 – 100oC). Inset:

Photographic image of DIC-Au NPs at different temperature.

Ab

sorb

an

ce (

a.u

.)

Wavelength (nm)

(a)

Tra

nsm

itta

nce

(%

)

C=O

C-H, aromatic (3087)

C-H aliphatic (2933)

C=C, aromatic

(1600, 1557)

-OH group stretching

(b)

Tra

nsm

itta

nce

(%

)

Wavenumber (cm-1)

C=O lactone

-OH group stretching was disappeared

Figure S3. FT-IR spectra of (a) pure DIC and (b) DIC-Au NPs.

(a)

2.0

AuNPs

2 pH

400 500 600 700 800 900 1000

0.0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

AuNPs

2 pH

3 pH

4 pH

5 pH

6 pH

7 pH

8 pH

9 pH

10 pH

11 pH

12 pH

(b)

Ab

sorb

an

ce (

a.u

.)

Wavelength (nm)

Figure S4. (a) UV-visible spectra of DIC-Au NPs in the presence of Cys (1 mM) at ammonium

acetate pHs from 2.0 to 12.0. Inset: Photographic image of corresponding solutions. (b) UV-

visible spectra of DIC-Au NPs in the presence of Cys (1 mM) at PBS pHs from 2.0 to 12.0.

Inset: Photographic image of corresponding solutions.

400 500 600 700 800 900 1000

0.0

0.5

1.0

1.5

2.0 2 pH

3 pH

4 pH

5 pH

6 pH

7 pH

8 pH

9 pH

10 pH

11 pH

12 pH

Ab

sorb

an

ce (

a.u

.)

Wavelength (nm)

400 500 600 700 800 900

0.00

0.25

0.50

0.75

1.00

DIC-Au NPs

pH 2

pH 3

pH 4

pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

pH 12

(b)

(a)

Ab

sorb

an

ce (

a.u

.)

Wavelength (nm)

Figure S5. (a) UV-visible spectra of DIC-Au NPs in the presence of Cys (1 mM) at Tris-HCl

pHs from 2.0- to 12.0. (b) Photographic image of corresponding solutions.

(b)

(b)

400 500 600 700 800 900 1000

0.3

0.6

0.9

1.2

1.5

1.8

2.1 AuNPs

2 pH

4 pH

6 pH

8 pH

10 pH

12 pH

(a)

1.2 AuNPs

2 pH

3 pH

Ab

sorb

an

ce (

a.u

.)

Wavelength (nm)

Figure S6. (a) UV-visible spectra of DIC-Au NPs in the presence of Lys (50 µM) at ammonium

acetate pHs from 2.0 to 12.0. Inset: Photographic image of corresponding solutions. (b) UV-

visible spectra of DIC-Au NPs in the presence of Lys 50 µM) at PBS pHs from 2.0 to 12.0. (c)

Photographic image of corresponding solutions.

400 500 600 700 800 900 1000

0.2

0.4

0.6

0.8

1.0

3 pH

4 pH

5 pH

6 pH

7 pH

8 pH

9 pH

10 pH

11 pH

12 pH

Ab

sorb

an

ce (

a.u

.)

Wavelength (nm)

Figure S7. UV-visible spectra of DIC-Au NPs in the presence of Lys (50 µM) at Tris-HCl pH

400 500 600 700 800 900 1000

0.0

0.3

0.6

0.9

1.2

1.5

AuNPs

2 pH

4 pH

6 pH

8 pH

10 pH

12 pH

Ab

sorb

an

ce (

a.u

.)

Wavelength (nm)

Figure S7. UV-visible spectra of DIC-Au NPs in the presence of Lys (50 µM) at Tris-HCl pH

from 2.0 to 12.0. Inset: Photographic image of corresponding solutions.

1.2

AuNPs

2 pH(b)

400 500 600 700 800 900 1000

0.0

0.5

1.0

1.5

2.0

2.5

AuNPs

2 pH

4 pH

6 pH

8 pH

10 pH

12 pH

Ab

sorb

an

ce (

a.u

.)

Wavelength (nm)

(a)

400 500 600 700 800 900 1000

0.0

0.3

0.6

0.9

2 pH

4 pH

6 pH

8 pH

10 pH

12 pH

Figure S8. (a) UV-visible spectra of DIC-Au NPs in the presence of ammonium acetate buffer in pH range

from 2.0 to 12.0 without anlytes. Inset: Photograph of corresponding solutions. (b) UV-visible spectra of

DIC-Au NPs in the presence of PBS in pH range from 2.0 to 12.0. Inset: Photograph of corresponding

solutions.

(b)

Ab

sorb

an

ce (

a.u

.)

Wavelength (nm)

0.0

0.5

1.0

1.5 AuNPs

2 pH

4 pH

6 pH

8 pH

10 pH

12 pH

Ab

sorb

an

ce (

a.u

.)

Figure S9. UV-visible spectra of DIC-Au NPs in the presence of Tris-HCl buffer in pH range from 2.0 to

12.0 without anlytes. Inset: Photograph of corresponding solutions.

400 500 600 700 800 900 1000

0.0

Wavelength (nm)

0.0

0.3

0.6

0.9

1.2

1.5

AuNPs

0.05 M

0.1 M

0.2 M

0.3 M

0.4 M

0.5 M

0.6 M

0.7 M

0.8 M

0.9 M

1.0 M

Concentration of NaCl (0.05-1.0 M)

Ab

sorb

an

ce (

a.u

.)

400 500 600 700 800 900 1000 1100

0.0

Wavelength (nm)

Figure S10. UV-visible spectra of DIC-Au NPs by the addition of NaCl from 0.05 to 1.0 M

without target analytes. Inset images are the corresponding photographs of Au NPs color.

A666/A

525

y = 0.013x - 0.015

R² = 0.994

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 20 40 60 80 100

LOD 2.2 µM

Figure S11. Calibration graph for the detection of Cys in the concentration range of 10-90 µM

using DIC-Au NPs as a probe at absorption ratio at A666/A525 versus Cys concentration.

Concentration (µM)

A570/A

525

y = 0.441x + 0.485

R² = 0.996

0.4

0.5

0.6

0.7

0.8

0.9

0 0.2 0.4 0.6 0.8 1

LOD 8.7 nM

Figure S12. Calibration graph for the detection of Lys in the concentration range of 100-800 nM

using DIC-Au NPs as a probe at absorption ratio at A570/A525 versus Lys concentration.

Concentration (µM)

0 0.2 0.4 0.6 0.8 1

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

DIC-Au NPs

DIC-Au NPs+Mixture of A.A

DIC-Au NPs+Mixture of A.A+Cysteine

DIC-Au NPs+Mixture of A.A+Lysozyme

DIC-Au NPs+Cysteine

DIC-Au NPs+Lysozyme

Ab

sorb

an

ce (

a.u

.)

Figure S13. UV-visible absorbance spectra of DIC-Au NPs in the presence of various

aminoacids (phenyl alanine, histidine, 4-hydroxyproline, proline, trypophan, tyrosine, alanine,

isoleucine, aspargine, aspartic acid, methonine, lysine, serine and leucine, 1 mM) for detection

of Cys and Lys. Inset: the corresponding photographic image.

400 500 600 700 800 900

0.0

Wavelength (nm)

400 500 600 700 800 900 1000

0.0

0.3

0.6

0.9

1.2

1.5

Ab

sorb

an

ce (

a.u

.)

Wavelength (n.m)

AuNPs

AuNPs+ Mixture of metal ions

AuNPs+ Mixture of metal ions+ Cys

AuNPs+ Mixture of metal ions+ Lys

Figure S14. UV-visible absorbance spectra of DIC-Au NPs in the presence of Cys and Lys upon addition

of various metal ions (Na+, K+, Br2+, Ca2+,Cu2+, Zn2+, Ni2+, Fe2+, Fe3+ and Al3+, 1.0 mM). Inset: the

corresponding photographic image.

Wavelength (nm)

400 500 600 700 800 900 1000

0.0

0.3

0.6

0.9

1.2

1.5

Abso

rbance (

a.u

.)

Wavelength (n.m)

AuNPs

AuNPs+ Mixture of anions

AuNPs+ Mixture of anions+Cys

AuNPs+ Mixture of anions+ Lys

Figure S15. UV-visible absorbance spectra of DIC-Au NPs in the presence of Cys and Lys upon addition

of various anions (Cl-, Br-, I- , F- , SO42- ,S2- , PO4

3- and Cr2O72- , 1.0 mM). Inset: the corresponding

photographic image.

Wavelength (nm)

0.3

0.6

0.9

1.2

1.5

Ab

sorb

an

ce

(a

.u.)

AuNPs

AuNPs+ Mixture of pesticides

AuNPs+ Mixture of pesticides+ Cys

AuNPs+ Mixture of pesticides+ Lys

400 500 600 700 800 900 1000

0.0

Figure S16. UV-visible absorbance spectra of DIC-Au NPs in the presence of Cys and Lys upon

addition of various pesticides (chlorpyrifos, quinalphos, hexaconazole, tricyclazole, acephate,

metsulfuron, isoproturon, and chlopropham, 1.0 mM). Inset: the corresponding photographic image.

Wavelength (nm)

0.3

0.6

0.9

1.2

1.5

Ab

sorb

an

ce (

a.u

.)

AuNPs

Egg without spiking

Egg spiking with 0.2 µM

Standard solution of 0.2 µM

Ab

sorb

an

ce (

a.u

.)

400 500 600 700 800 900 1000

0.0

0.3

Wavelength (n.m)Wavelength (nm)

Figure S17. UV-visible spectral variations of DIC-Au NPs in egg white (without spiking of

Lys), and with spiking 0.2 µM of Lys and the spectrum of DIC-Au NPs in the presence of

standard Lys at PBS pH 7.

Sample Cys Lys

Addeda

FoundaRecovery

(%)b

RSD Addeda

FoundaRecovery

(%)b

RSD

Urine 1 40 39.8 99.5 0.28 0.2 0.21 104.8 2.2

60 59.9 99.8 0.18 0.5 0.52 104.0 2.9

80 78.0 97.5 1.82 0.8 0.81 102.2 2.2

Urine 2 40 39.1 97.8 0.12 0.2 0.19 98.6 2.7

60 57.6 96.0 0.38 0.5 0.51 101.0 2.1

80 77.8 97.3 0.98 0.8 0.81 100.8 1.6

Urine 3 40 41.2 103.2 0.23 0.2 0.20 101.3 0.7

60 63.7 106.2 0.18 0.5 0.49 99.4 1.1

80 85.2 106.5 0.42 0.8 0.81 101.3 1.3

Plasma 1 40 42.2 105.7 0.45 0.2 0.19 97.2 0.3

60 65.0 108.3 0.14 0.5 0.47 95.1 1.8

80 85.8 107.2 1.79 0.8 0.75 94.3 1.8

Plasma 2 40 0.2

Table S1. Analysis of Cys and Lys in biological samples using DIC-Au NPs as a probe.

Plasma 2 40 39.1 97.8 0.10 0.2 0.19 95.3 2.6

60 58.9 98.3 0.11 0.5 0.49 98.9 1.3

80 78.1 97.6 1.80 0.8 0.77 96.0 1.8

Plasma 3 40 39.1 97.7 0.44 0.2 0.21 102.0 0.9

60 57.5 95.9 0.14 0.5 0.50 100.2 1.5

80 77.3 96.6 0.93 0.8 0.79 99.15 2.3

Egg white - - - - 0.2 0.22 108.4 2.5

- - - - 0.5 0.55 110.6 2.0

- - - - 0.8 0.86 108.5 2.6

aµM, bn=3