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Electronic Supplementary Information (ESI)

Characterization of Mixed-Ligand Shells on Gold Nanoparticles by Transition Metal and Supramolecular Surface Probes

Mohamed Nilam,a Mostafa Ahmed,a,b Mohammad A. Alnajjar,a Andreas Hennig*,a

a Department of Life Sciences and Chemistry, Jacobs University Bremen, Campus Ring 1, 28759

Bremen, Germany

b Department of Chemistry, Faculty of Science (New Valley), Assuit University, Egypt

* Corresponding author: a.hennig@jacobs-university.de

Table of Contents

Abbreviations....................................................................................................................................S-2

Molecular Structure of Ligands ........................................................................................................S-2

DTNB Assay.....................................................................................................................................S-3

Analysis of Particle Size Distribution by TEM ................................................................................S-4

Thermogravimetric analysis (TGA)..................................................................................................S-5

Other Supporting Figures and Tables ...............................................................................................S-5

Electronic Supplementary Material (ESI) for Analyst.This journal is © The Royal Society of Chemistry 2018

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Abbreviations

AMADA-Put: N-adamantylmethylbutane-1,4-diamine; AO: acridine orange; AuNPs: gold

nanoparticles; CB7: cucurbit[7]uril; DTNB: 5,5´-dithio-bis-(2-nitrobenzoic acid) (Ellman’s

reagent); EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride salt; Hepes: 2-[4-

(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; MES: 2-(N-morpholino)ethanesulfonic acid;

MPA: sodium 3-mercaptopropionate; MPS: sodium 3-mercapto-1-propanesulfonate; MUA: sodium

11-mercaptoundecanoate; PV: pyrocatechol violet; TEG: triethylene glycol mono-11-

mercaptoundecyl ether.

Molecular Structure of Ligands

O

O-Na+HS Na+S

O

O O-HS

Sodium 3-mercaptopropanoate (MPA) Sodium 3-mercapto-1-propanesulfonate (MPS)

O

O-Na+HS

Sodium 11-mercaptoundecanoate (MUA)

OO

OOH

HS

Triethylene glycol mono-11-mercaptoundecyl ether (TEG)

Chart S1. Molecular structures of ligands.

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DTNB Assay

Stock solutions of DTNB and MPA were prepared in 0.1 M NaH2PO4, 1 mM EDTA, pH 8.0.

Varying concentrations of MPA (5-25 µM) and 40 µM DTNB were added to each solution.

Solutions were mixed and incubated at room temperature for 5 minutes, and then absorption spectra

were recorded. The prepared stock solutions of the AuNPs (ca. 1.5 µM AuNPs) were centrifuged

for 25 min at 16400 rcf, and then, 40 µM DTNB was added to the supernatant and absorption

spectra were recorded.

5 10 15 20 25

0.1

0.2

Abso

rban

cce

[MPA] M)

b)

300 400 500

0.00

0.15

0.30

Abs

orba

nce

Wavelength (nm)

Supernatant of MPA/MPS-AuNPs

a)

300 400 5000.00

0.25

0.50

0.75

Abs

orba

nce

Wavelength (nm)

Au/MPA-MPS 1:5 Au/MUA-TEG 1:1 Au/MUA-TEG 1:100 Au/MPA-MPS 1:83

c)

Fig. S1 a) Absorption spectra of 40 µM DTNB with varying concentration of MPA (5-25 µM) and

MPA/MPS-AuNPs nanoparticles in 0.1 M NaH2PO4, 1 mM EDTA, pH 8.0. b) Corresponding plot

of the absorbance at 412 nm against the concentration of MPA. The limit of detection (LOD) of

DTNB assay was calculated using the equation; LOD = 3sa/b where, sa is the standard deviation of

the blank and b is the slope of the calibration line. c) Absorption spectra of 80 µM DTNB with

different molar ratio of the ligand capped nanoparticles (after five months storage at 4 ˚C) in 0.1 M

NaH2PO4, 1 mM EDTA, pH 8.0.

S-4

Analysis of Particle Size Distribution by TEM

The synthesized mixed-ligand nanoparticles (ca. 1.5 µM) were fivefold diluted with NANOpure

water and then deposited onto a carbon film-coated copper net TEM grid (PLANO GmbH).

Samples were allowed to air dry and then dried under high vacuum before characterization by TEM

at 80 kV. To obtain the size of distribution of the nanoparticles from TEM images, ImageJ 1.47d

(National Institute of Health, USA) was used and at least 200 AuNPs from various areas of the grid

were considered.

10 15 20 25 300

20

40

60

80

Cou

nts

Diameter (nm)

10 15 20 25 300

20

40

60

Cou

nts

Diameter (nm)

Fig. S2 TEM images of MPA/MPS-AuNPs (top) and MUA/TEG-AuNPs (bottom) and respective

size distribution histograms.

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Thermogravimetric analysis (TGA)

Thermogravimetric analysis (TGA) was performed with a SDT Q600 (TA Instruments).

MUA/TEG-AuNP samples (2 - 5 mg) were prepared by drying and the temperature range between

20 ˚C and 700 ˚C was scanned at a rate of 5 ˚C/min under a nitrogen flow of 100 mL/min.

Other Supporting Figures and Tables

400 500 600 700 800

0.0

0.5

1.0

Abs

orba

nce

Wavelength (nm)

MPA:MPS 1:2 MPA:MPS 1:5 MPA:MPS 1:10 MPA:MPS 1:15 MPA:MPS 1:20 MPA:MPS 1:50

a)

400 500 600 700 8000.0

0.5

1.0

Abs

orba

nce

Wavelength (nm)

MUA:SPEG 1:1 MUA:SPEG 1:2 MUA:SPEG 1:5 MUA:SPEG 1:10 MUA:SPEG 1:20 MUA:SPEG 1:100

b)

Fig. S3 Normalized absorption spectra of functionalized particles: a) MPA/MPS-AuNPs,

b) MUA/TEG-AuNPs.

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Fig. S4 1H NMR spectra of MUA/TEG-AuNPs with varying molar ratio of MUA and TEG after

digestion with aqua regia and subsequent dilution with D2O (pD 1.6). a) MUA only and MUA/TEG

molar ratio b) 1:1, c) 2:1, d) 5:1, e) 10:1, f) 20:1, g) 100:1.

100 200 300 400 500 600 70050

75

100

Wei

ght L

oss

(%)

Temperature (C)

MUA/TEG(10:1)-AuNPs: 1.46 ligands/nm2 MUA/TEG(5:1)-AuNPs: 1.30 ligands/nm2

Fig. S5 TGA plots of MUA/TEG-AuNPs coated with different molar ratios of MUA and TEG.

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Fig. S6 Reproducibility measurements of different batches of MPA/MPS(1:5)-AuNPs by 1H NMR

spectroscopy of after digestion with aqua regia and subsequent dilution with D2O (pD 1.6).

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0 20 40 60 80 1000.15

0.20

0.25

0.30

Abs

orba

nce

Volume of AuNP stock solution (L)

a)

0 50 100

0.1

0.2

0.3

400 600 8000.0

0.2

0.4

Abs

orba

nce

Wavelength (nm)

0 20 40 60 80

0.15

0.20

0.25

0.30

0.35

Abs

orba

nce

Volume of AuNP stock solution (L)

b)

400 600 8000.0

0.2

0.4

Abs

orba

nce

Wavelength (nm)

0 20 40 60 800.15

0.20

0.25

0.30

Abs

orba

nce

Volume of AuNP stock solution (L)

c)

400 600 8000.0

0.2

0.4

Abs

orba

nce

Wavelength (nm)

0 20 40 60 80 100

0.15

0.20

0.25

0.30

Abs

orba

nce

Volume of AuNP stock solution (L)

d)

400 600 8000.0

0.2

0.4

Abs

orba

nce

Wavelength (nm)

0 50 100 150 200

0.15

0.20

0.25

0.30

Abs

orba

nce

Volume of AuNP stock solution (L)

400 600 8000.0

0.2

0.4

Abs

orba

nce

Wavelength (nm)

e)

0 20 40 60 800.15

0.20

0.25

0.30

Abs

orba

nce

Volume of AuNP stock solution (L)

400 600 8000.0

0.2

0.4

Abs

orba

nce

Wavelength (nm)

f)

Fig. S7 Results of NiPV assay for quantification of negatively charged surface functional groups on

AuNPs. Dependence of the absorbance at 650 nm versus the volume of particles a) MPA only and

MPA/MPS molar ratio b) 1:5, c) 1:10, d) 1:30, e) 1:50, f) 1: 83.

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0 20 40 60 80

0.15

0.20

0.25

0.30

0.35

0.40

Abs

orba

nce

Volume of AuNP stock solution (L)

400 600 8000.0

0.2

0.4

Abs

orba

nce

Wavelength (nm)

a)

0 50 100 150 200 2500.15

0.20

0.25

0.30

Abs

orba

nce

Volume of AuNP stock solution (L)

400 600 8000.0

0.2

0.4

Abs

orba

nce

Wavelength (nm)

b)

0 50 100 1500.15

0.20

0.25

0.30

Abs

orba

nce

Volume of AuNP stock solution (L)

400 600 8000.0

0.2

0.4

Abs

orba

nce

Wavelength (nm)

c)

0 50 100 150 2000.15

0.20

0.25

0.30

Abs

orba

nce

Volume of AuNP stock solution (L)

400 600 8000.0

0.2

0.4

Abs

orba

nce

Wavelength (nm)

d)

0 50 100 150 200 2500.15

0.20

0.25

0.30

Abs

orba

nce

Volume of AuNP stock solution (L)

400 600 8000.0

0.2

0.4

Abs

orba

nce

Wavelength (nm)

e)

0 100 200 300 4000.20

0.25

0.30

Abs

orba

nce

Volume of AuNP stock solution (L)

400 600 8000.0

0.2

0.4A

bsor

banc

e

Wavelength (nm)

f)

Fig. S8 Results of NiPV assay for quantification of negatively charged surface functional groups on

AuNPs. Dependence of the absorbance at 650 nm versus the volume of particles a) MUA only and

MUA/TEG molar ratio b) 100:1, c) 20:1, d) 10:1, e) 5:1, f) 2:1.

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Table S1. Reproducibility measurements of MPA/MPS(1:5)-AuNPs by the NiPV assay.

Au-MPA/MPS Ligand density (ligands/nm2)Sample 1 5.449Sample 2 5.374Sample 3 5.426Sample 4 5.415Sample 5 5.578

Sample 6 (different batch) 5.454Average 5.449 ± 0.070Coefficient of Variation (%) 1.3

Table S2. Reproducibility measurements of MPA/MPS(1:5)-AuNPs by the CB7 assay.

Au-MPA/MPS Ligand density (ligands/nm2)Sample 1 0.07798Sample 2 0.07559Sample 3 0.07902Sample 4 0.09128Sample 5 0.08533Sample 6 0.09279Average 0.0837 ± 0.0073Coefficient of Variation (%) 8.6

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0 20 40 60 80

5

10

Fluo

resc

ence

Volume (L)

500 600 7000

5

10

Fluo

resc

ence

Wavelength (nm)

b)

0 20 40 60 80

5

10

Fluo

resc

ence

Volume (L)

500 600 7000

5

10

Fluo

resc

ence

Wavelength (nm)

c)

0 30 60 90 120 150

5

10

Fluo

resc

ence

Volume (L)

500 600 7000

5

10

Fluo

resc

ence

Wavelength (nm)

d)

0 50 100 150 200

5

10

Fluo

resc

ence

Volume (L)

500 600 7000

5

10

Fluo

resc

ence

Wavelength (nm)

f)

Fig. S9 Results of CB7 assay for quantification of accessible surface functional groups on

MPA/MPS-AuNPs a) MPA only and MPA/MPS molar ratio b) 1:5, c) 1:10, d) 1:15, e) 1:30,

f) 1:83. Shown is the variation of fluorescence spectra with increasing volume of AuNPs stock

solution (see main text for details) (λem = 510 nm, λexe = 450 nm).

0 10 20 30 40

5

10

Fluo

resc

ence

Volume (L)

a)

500 600 7000

5

10

Fluo

resc

ence

Wavelength (nm)

0 30 60 90 120 150

5

10

Fluo

resc

ence

Volume (L)

500 600 7000

5

10

Fluo

resc

ence

Wavelength (nm)

e)

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0 10 20 30 40

5

10

Fluo

resc

ence

Volume (L)

500 600 7000

5

10

Fluo

resc

ence

Wavelength (nm)

a)

0 40 80 120 160

5

10

Fluo

resc

ence

Volume (L)

500 600 7000

5

10

Fluo

resc

ence

Wavelength (nm)

b)

0 30 60 90 120 150

5

10

Fluo

resc

ence

Volume (L)

500 600 7000

5

10

Fluo

resc

ence

Wavelength (nm)

c)

0 40 80 120 160

5

10

Fluo

resc

ence

Volume (L)

500 600 7000

5

10

Fluo

resc

ence

Wavelength (nm)

d)

0 50 100 150

5

10

Fluo

resc

ence

Volume (L)

500 600 7000

5

10

Fluo

resc

ence

Wavelength (nm)

e)

0 50 100 150 200 250

5

10

Fluo

resc

ence

Volume (L)

f)

500 600 7000

5

10

Fluo

resc

ence

Wavelength (nm)

Fig. S10 Results of CB7 assay for quantification of accessible surface functional groups on

MUA/TEG-AuNPs a) MUA only and MUA/TEG molar ratio b) 100:1, c) 20:1, d) 10:1, e) 5:1,

f) 2:1. Shown is the variation of fluorescence spectra with increasing volume of AuNPs stock

solution (λem = 510 nm, λexe = 450 nm).