Fluorogenic dansyl-ligated gold nanoparticles for the detection of sulfur mustard by displacement assay

Article abstract of DOI:10.1039/c3cc39105a

The dansyl fluorophore ligated to gold nanoparticles via imidazole and amine groups affords conjugates capable of detecting micromolar concentrations of the chemical warfare agent sulfur mustard by a fluorescence switching 'ON' displacement assay. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3cc39105a

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Fluorogenic dansyl-ligated gold nanoparticles for the
detection of sulfur mustard by displacement assay†
Richard C. Knighton,a Mark R. Sambrook,b Jack C. Vincent,b Simon A. Smith,b
Christopher J. Serpell,a James Cookson,c Matthew S. Vickersa and Paul D. Beer*a
Cite this: Chem. Commun., 2013,
49, 2293
Received 20th December 2012,
Accepted 6th February 2013
DOI: 10.1039/c3cc39105a
www.rsc.org/chemcomm
The dansyl fluorophore ligated to gold nanoparticles via imidazole
and amine groups affords conjugates capable of detecting micro-
molar concentrations of the chemical warfare agent sulfur mustard
by a fluorescence switching ‘ON’ displacement assay.
The colorimetric and fluorescent sensing of chemical warfare
agents (CWAs) has been largely dominated by the use of reaction
chemistries that generate new molecular species.1 This includes
cyclisation approaches for organophosphorus G-agent analogue
detection,2 and the use of amine-based PET sensors for the
detection of alkylating agents as simulants for bis-2-chloroethyl-
sulfide, or sulfur mustard (referred to hereafter as HD).3
Conversely, the sensing of CWAs through supramolecular
recognition has been reported only sporadically. As a necessity,
it often relies upon the use of low toxicity simulants rather than
the agents themselves for proof-of-concept. In the case of
organophosphorus nerve agents, sensing systems reported
include the recognition of pinacolyl methylphosphonate by
molecular imprinted polymers4 and ligand displacement via
PQOÁ Á Álanthanide coordination.5,6 More recently the binding
of the nerve agent GD, or Soman, by a series of hydrogen bond
donating receptors has been shown.7
Fig. 1 Schematic representation of sensing of sulfides by displacement assay.
The recognition of HD, by virtue of its limited functionality impregnated silica sol–gel matrix.9 A rare organic-based sensor was
for the formation of non-covalent bonds, is almost unknown. In reported by Hunt and Alder who demonstrated the quenching of
work which has implications for HD sensing, Tilley reported sodium fluorescein by HD,10 however, the mechanism for this
the stability of Ag(I) salts of 2-chloroethyl methyl sulfide quenching process is unclear.
(CEMS), HD and diethylsulfide in polar organic solvents.8
Herein we describe a proof-of-concept method for recognizing
Recently, the sensing of HD has been demonstrated through the and sensing sulfur mustard via a novel fluorescence switching ‘ON’
formation of R2SÁÁÁCu charge transfer complexes in a copper acetate displacement assay which exploits the hierarchy of binding energies
of different ligands with gold nanoparticle surfaces. The displace-
ment sensing assay concept is illustrated in Fig. 1. The weak donor
atom attachment of appropriately functionalised fluorophores in
a Chemistry Research Laboratory, Department of Chemistry, University of Oxford,
Mansfield Road, Oxford, OX1 3TA, UK. E-mail: paul.beer@chem.ox.ac.uk;
close proximity to a gold nanoparticle surface results in the fluoro-
phore being quenched through an energy transfer process.11
Exposure to a target sulfur-containing CWA interferes with the
relatively weak surface interaction between the metal nanoparticle
surface and functional group of the appended fluorophore,
triggering the displacement of the fluorophore from the surface
and a switching ‘ON’ of the fluorescence signal results.
Fax: +44 (0)1865 272960; Tel: +44 (0)1865 285182
b Detection Department, Dstl Porton Down, Salisbury, SP4 0JQ, UK.
E-mail: m.sambrook@dstl.gov.uk; Tel: +44 (0)1980 614301
c Johnson Matthey Technol Ctr, Reading RG4 9NH, Berks, England, UK
† Electronic supplementary information (ESI) available: GNP synthesis and
characterisation data, UV/vis spectral response to sulfides, additional fluores-
cence data. CCDC 916576. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c3cc39105a
c
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Chem. Commun., 2013, 49, 2293--2295 2293

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Fig. 2 1H NMR spectra of (top) AuÁ1 and (bottom) AuÁ1 in the presence of
ten equivalents of CEMS (CDCl3, 500 MHz, 298 K). 13C satellites marked with
an asterisk.
Studies of these materials by UV/Vis absorption spectro-
scopy provide further evidence of the occurrence of surface-
bound molecular events following exposure to both CEMS and
HD. The SPR bands of both AuÁ1 and AuÁ2 in CHCl3 were
observed to undergo significant bathochromic shifts from 528
to 536 nm and 520 to >600 nm,15 respectively, upon addition of
CEMS. Similarly, upon exposure to HD shifts of 528 to 540 nm
for AuÁ1 and 520 to 540 nm for AuÁ2 were observed (see ESI†).
This may indicate particle agglomeration following displace-
ment of the stabilising dansyl ligands. Indeed a significant
change in colour of the respective nanoparticle solution from
brown to clear was observed with the formation of a grey
precipitate upon addition of CEMS and HD (Fig. 3).
Scheme 1 Synthesis of ligands 1 and 2; (i) N-aminopropyl imidazole, Et3N,
CH2Cl2 (ii) N-Boc-ethylenediamine, Et3N, CH2Cl2 (iii) TFA/CH2Cl2.
We have recently demonstrated the capability of the imid-
azole ligand to stabilise gold nanoparticle materials.12 Due to
the ‘hardness’ of nitrogen donor ligands when compared to the
relatively ‘soft’ nature of sulfur donor compounds, we hypothesised
that upon treatment of dansyl fluorophore appended imidazole or
amine ligand stabilised gold nanoparticle materials with a bis alkyl
sulfide such as HD, the sulfur containing analyte will compete with
the respective nitrogen stabilising ligand for the gold nanoparticle
and displace the quenched ‘OFF’ dansyl fluorophore from the
surface leading to a switching ‘ON’ of the dansyl fluorescence.
The syntheses of the dansyl functionalised ligands were
accomplished using commercially available reagents. Ligand
1 was prepared by condensing dansyl chloride with N-amino-
propyl imidazole in dichloromethane in 77% yield. The amine
derivative was prepared by mono-BOC protection of ethylene-
diamine and subsequent reaction with the sulfonyl chloride to
yield the proligand. Deprotection with trifluoroacetic acid in
dichloromethane (1 : 4 v/v) afforded ligand 2 (see ESI† for X-ray-
single crystal structure) in an overall yield of 54% (Scheme 1).
Both ligands were ligated to gold nanoparticles using a
modified Brust method,13 using HAuCl4 and three equivalents of
the appropriate dansyl ligand. Reduction with NaBH4 in dichloro-
methane–toluene afforded the target dansyl-functionalised gold
nanoparticles (GNPs) AuÁ1 and AuÁ2 which were characterised by
TEM, elemental analysis, UV-vis and 1H NMR (see ESI†). The GNPs
Fig. 3 2.5 mL of a 0.04 mg mLÀ1 solution of AuÁ2 in CHCl3 (left), 30 minutes
after addition of 50 mL CEMS (right).
Both GNPs exhibited weak fluorescence from the ligated
exhibited characteristic UV-vis absorption bands assignable to the dansyl group with emission maxima around 500 nm. In order
ligand n-p* transition centred around 350 nm and broad maxima at to probe the fluorogenic properties of the GNPs and their
520–530 nm attributed to the Surface Plasmon Resonance (SPR) response to the addition of the sulfides, qualitative fluores-
band of the gold nanoparticles. Analysis of the TEM imaging data cence measurements were undertaken in chloroform. The
gave mean particle diameters of 3.1 Æ 0.7 nm and 3.2 Æ 0.7 nm for response of solutions of AuÁ1 and AuÁ2 upon addition of either
the imidazole and amine functionalised nanoparticles, respectively. CEMS or HD was monitored over five minute intervals (Fig. 4).
In order to test the displacement assay concept (Fig. 1) a qualitative Addition of either sulfide species resulted in a significant
1H NMR analysis was conducted initially using AuÁ1 and the mustard increase in the fluorescence intensity of the sample. This
simulant CEMS in CDCl3 solution. As expected, the 1H NMR spectrum increase in fluorescence is explained by the highly distant-
of AuÁ1 reveals very broad resonances (see ESI†), consistent with dependant nature of the non-radiative processes responsible
chemical shift anisotropy of the bound species as a result of being for the quenching of the fluorescent dansyl group. Proximity of
chemisorbed to the gold surface.14 Upon addition of 10 equivalents of the fluorophore to the metal surface in the initial GNP con-
CEMS new sharp resonances associated with the dansyl moiety were jugate results in weak fluorescence due to efficient quenching
seen (Fig. 2). This observation is evidence of the liberation of the free processes between the dansyl group and the gold surface,
ligand species 1 by the aurophilic mustard simulant.
attributed to through-space relaxation by Nanosurface Energy
c
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the ligand–nanoparticle conjugates are shown to be highly
sensitive to the presence of the mustard derivative, displaying
a response to micromolar concentrations of sulfur mustard.
Assuming the displacement is a 1 : 1 ligand exchange
process with one guest sulfide molecule displacing one surface
stabilised ligand, and ignoring any cooperativity effects between
surface ligand exchange sites, SPECFIT analysis17 of the
fluorescence titration data gave association constants log K =
4.65 Æ 0.06 and log K = 4.19 Æ 0.05 for CEMS and HD,
respectively, for AuÁ2.
As expected, the relative surface binding affinities of the two
sulfide analytes in the surface displacement of the amine
stabilised dansyl ligand of AuÁ2 are of similar magnitude.
In summary, we have demonstrated a proof-of-concept
capability of imidazole and amine dansyl-ligated gold nano-
Fig. 4 Fluorescence spectra of a 0.04 mg mLÀ1 solutions of AuÁ1 (left) and AuÁ2
(right) upon addition of CEMS (50 mL) in CHCl3 at 298 K (lex = 345 nm).
Transfer (NSET).11 Substitution of the dansyl ligand by the particles to detect the chemical warfare agent sulfur mustard.
sulfide analyte at the gold surface releases the dansyl group The mechanism of detection occurs via the displacement of a
into solution and results in the loss of the NSET through-space nanoparticle surface energy transfer quenched fluorophore,
quenching process. Displacement for both analytes with both resulting in a ‘switching-on’ fluorescence sensing response.
nanoparticles occurred almost instantaneously, with the max-
We thank the ESPRC and the Helmore Foundation for DPhil
imum increase in fluorescence occurring within five minutes studentship (RK) and the Centre for Defence Enterprise and the
for both samples. Importantly analogous titration experiments Defence Science and Technology Laboratory (Dstl) for funding.
of AuÁ1 and AuÁ2 with octanol and di-n-butyl ether gave no CJS thanks Johnson Matthey and the EPSRC for a CASE
significant fluorescence increase. The selective sulfide fluores- studentship. We also thank Oxford University Chemical Crystal-
cence response together with the rapid kinetics of displacement lography for instrument use.
suggests that conjugates AuÁ1 and AuÁ2 are promising prototype
sensory materials for mustard detection.16
Notes and references
In order to probe the lower detection limit of the GNPs
towards HD the surface coverage of AuÁ2 was calculated using
sulfur elemental analysis data, with surface coverage deter-
mined to be 13.5% ligand by weight. Titrations were conducted
using a 0.04 mg mLÀ1 solution of GNPs and the fluorescence
changes monitored upon addition of 0–10 molar equivalents
of either CEMS or HD (Fig. 5). As can be seen from the spectra,
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Fig. 5 Fluorescence titration of a 0.04 mg mLÀ1 solution of AuÁ2 with HD in
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c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 2293--2295 2295