Ratio-Au: A FRET-based fluorescent probe for ratiometric determination of gold ions and nanoparticles

Article abstract of DOI:10.1002/chem.201003586

Digging for gold: Ratio-Au the first ratiometric fluorescent probe for Au³⁺ based on fluorescence (or Foerster) resonance energy transfer has been judiciously designed and synthesized on the basis of the newly discovered Au³⁺-mediate

Full text of DOI:10.1002/chem.201003586

DOI: 10.1002/chem.201003586
Ratio-Au: A FRET-based Fluorescent Probe for Ratiometric Determination
of Gold Ions and Nanoparticles**
Xiaowei Cao, Weiying Lin,* and Yundi Ding[a]
Recently, gold nanoparticles (AuNPs) have attracted in-
tense attention due to their advantageous chemical and pho-
tophysical properties, such as small size (1–100 nm), large
surface-to-volume ratio, and tunable physicochemical pro-
files. AuNPs have been widely employed in the fields of ma-
terial sciences, chemistry, biology, and medicine. For in-
stance, AuNPs have been used as catalysts,[1] biosensors,[2]
bioimagining agents,[3] drug-delivery carriers,[4] gene-trans-
fection carriers,[5] and additives in cosmetics, hair tonic, and
soap.[6] On the other hand, despite of their advantages,
AuNPs may elicit undesirable interactions with biological
systems and the environment, which generates toxicity.
AuNPs may cause intracellular damage in human epithelial
cells and oxidative stress in Mytilus edulis.[7] Thus, it is of
high importance to detect AuNPs.
Inductively coupled plasma optical emission spectroscopy
(ICP-OES), inductively coupled plasma atomic emission
spectroscopy (ICP-AES), inductively coupled plasma mass
spectroscopy (ICP-MS), and anodic stripping voltammetry[8]
have been used for detection of gold ions and AuNPs. The
fluorescence-analysis method is advantageous due to the
simplicity in operation and the high sensitivity. A few fluo-
rescent probes for gold ions have been recently reported.[9]
However, all of these have not been applied for the detec-
tion of AuNPs. Furthermore, most of the known fluorescent
probes for gold ions are fluorescent-intensity based and sus-
ceptible to environmental effects. By contrast, ratiometric
fluorescent probes have a built-in correction for environ-
mental effects as ratiometric probes allow the measurement
of fluorescence intensities at two wavelengths. This renders
ratiometric fluorescent probes favorable for quantitative
measurements. Thus, the ratiometric fluorescent probes for
quantitative detection of AuNPs are highly sought. Fluores-
cence (or Fçrster) resonance energy transfer (FRET) is an
effective signaling mechanism for ratiometric-fluorescent-
probe development.[10] Herein, we describe the rational
design, synthesis, and spectral properties of the first FRET-
based ratiometric fluorescent gold probe that is capable of
quantitative determination of AuNPs.
To judiciously design a new fluorescent probe for gold
ions and AuNPs, a chemical reaction selectively promoted
by gold ions is desirable. Inspired by our previous report,
that Hg2+ could mediate the cyclization of thioamide-termi-
nal alkynes on a rhodamine platform in phosphate buffer so-
lution (25 mm, pH 7.2, containing 20% DMF),[11] we envi-
sioned that by tuning the chemical structure as well as the
solvent system, a selective, new, gold-ion-mediated reaction
may be obtained. Indeed, we were able to find that thio-
AHCTUNGTREGaNNNU mide-phenyl-substituted alkyne on a rhodamine platform,
compound 1 (for the synthesis see Scheme S1 in the Sup-
porting Information), may be selectively promoted by Au3+
over Hg2+ ions in aqueous ethanol to afford 5-ketothiazole
2 (Scheme 1A and Figure S1 in the Supporting Informa-
Scheme 1. A) The newly discovered Au3+-promoted transformation of
thioamide-phenyl-substituted alkynes to 5-ketothiazole in aqueous etha-
nol. B) Synthesis of the new, FRET-based, ratiometric probe Ratio-Au.
(DIPEA=diisopropylethylamine)
[a] X. Cao, Prof. W. Lin, Y. Ding
State Key Laboratory of Chemo/Biosensing and Chemometrics
College of Chemistry and Chemical Engineering
Hunan University, Changsha, Hunan 410082 (P.R. China)
Fax : (+86)731-88821464
tion). The structure of compound 2 has been well estab-
1
lished by H NMR and 13C NMR spectroscopy as well as by
E-mail: weiyinglin@hnu.cn
HRMS (Figure S2–4 in the Supporting Information). To the
best of our knowledge, the Au3+-mediated transformation
of thioamide-phenyl-substituted alkynes to 5-ketothiazoles
[**] FRET=fluorescence (or Fçrster) resonance energy transfer
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201003586.
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Chem. Eur. J. 2011, 17, 9066 – 9069

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is unprecedented. Although the mechanism of this new reac-
tion is not clear yet, we speculate that it may be similar to
that of Pd2+-promoted cyclization of propargylamides to 5-
oxazolecarbaldehydes.[12] However, the reaction mechanism
remains to be investigated. Additionally, the absorption and
emission spectra of compound 1 titrated with Au3+ (Fig-
ure S5 in the Supporting Information) imply that compound
1 exhibits a fluorescence turn-on response to Au3+ and may
be further modified to afford a new ratiometric probe.
We decided to incorporate a boron dipyrromethene
(BODIPY) dye into compound 1 to give the new ratiomet-
ric probe Ratio-Au (Scheme 1B). The choice of the
BODIPY dye is based on the consideration that the emis-
sion of the BODIPY fluorophore has excellent overlap with
the absorption of the ring-opening form of the rhodamine
dye. Thus, the combination of the BODIPY dye and the
rhodamine fluorophore is an appropriate FRET dyad. We
reasoned that the FRET is “off” in the free probe Ratio-Au
as the rhodamine is in the spirothiolactam form. By con-
Figure 1. Fluorescence titration spectra of Ratio-Au (5 mm) in phosphate
buffer/C2H5OH (3:7, pH 7.0) upon addition of increasing concentrations
of Au3+ (0–5.0 equiv) with excitation at 470 nm. The inset shows the
changes in the fluorescence-intensity ratio (I594/I514) with the concentra-
tion of Au3+. The spectra were recorded after incubation of Ratio-Au
with Au3+ for 15 min.
ure S8 in the Supporting Information, the ratiometric fluo-
rescence response induced by Au3+ could not be suppressed
by further addition of excess tetrabutylammonium cyanide
(a chelating agent), suggesting the irreversible nature of the
signaling process. This is in good agreement with the time-
dependent fluorescence-intensity-ratio (I594/I514) changes of
the ratiometric probe Ratio-Au (5 mm) upon incubation with
Au3+ (Figure S9 in the Supporting Information). The maxi-
mal emission ratio (I594/I514) was reached within 15 min when
Ratio-Au (5 mm) was treated with 3 equiv of Au3+. However,
the reaction was much slower when a smaller amount of
Au3+ was used (Figure S9 in the Supporting Information).
Thus, similarly to compound 1, Ratio-Au also functions by a
reaction-based mechanism, likely due to Au3+-promoted
transformation of a thioamide-phenyl-substituted alkyne to
5-ketothiazole. This is further substantiated by the result of
in situ ESI-MS titration experiment. As shown in Figure S10
in the Supporting Information, a major peak at m/z=832.3,
which corresponds to the desired reaction product, is pres-
ent in the mass spectrum. However, the attempt to isolate
the product by silica-gel column chromatography was not
successful, as the product was instable during the separation
process.
The ratiometric probe exhibited an excellent linearity be-
tween the emission ratio (I594/I514) and the Au3+ concentra-
tion in the range from 5.0ꢂ10À7 to 1.5ꢂ10À5 m (Figure S11 in
the Supporting Information) with a detection limit of 3.9ꢂ
10À7 m (signal-to-noise ratio=3), suggesting that the ratio-
metric probe Ratio-Au may be useful for the quantitative
determination of Au3+. The time-course studies indicate
that the maximal emission ratio (I594/I514) was reached within
15 min (Figure S9 in the Supporting Information). Thus, an
assay time of 15 min was chosen in the evaluation of the se-
lectivity and sensitivity of the probe toward Au3+. The pH
studies indicated that the free probe is stable over the wide
pH range of 2.0–10.0 and that the ratiometric response of
Ratio-Au to Au3+ is pH-dependent (Figure S12 in the Sup-
porting Information).
trast, upon treatment of Ratio-Au with Au3+, the Au3+
-
mediated reaction may render the rhodamine fluorophore in
the fluorescent ring-opening form. Thus, the FRET should
be switched “on”.
The synthesis of Ratio-Au is outlined in Scheme 1B. Brief-
ly, coupling the iodo compound 3 with alkyne 4 under the
Sonogashira cross-coupling reaction conditions[13] gave the
key intermediate 5, which was further treated with Lawes-
sonꢁs reagent in dry benzene to afford the target compound
Ratio-Au.
With Ratio-Au in hand, we then examined the ratiometric
response of the probe to Au3+ by absorption and emission
spectroscopy. Probe Ratio-Au (5 mm) was titrated with Au3+
in phosphate buffer/C2H5OH (3: 7, pH 7.0). As anticipated,
in the absence of Au3+, the free probe exhibited only the
absorption band of the BODIPY fluorophore at around
499 nm (Figure S6 in the Supporting Information). However,
addition of Au3+ elicited the formation of a new absorption
peak at around 570 nm ascribed to the absorption of the
ring-opening form of the rhodamine dye; this is in good
agreement with the change of the probe solution from color-
less to pink (Figure S7A in the Supporting Information).
As shown in Figure 1, upon excitation at 470 nm, the free
probe only displayed the intense characteristic emission of
BODIPY at 514 nm, but no typical emission of rhodamine
at 594 nm, suggesting that the FRET was indeed off, in ac-
cordance with the design strategy. By contrast, upon addi-
tion of Au3+ (the spectra were recorded after incubation of
the probe Ratio-Au with Au3+ for 15 min), the emission in-
tensity of the BODIPY band decreased, and simultaneously,
a significant enhancement in the rhodamine emission peak
at 594 nm was noticed, suggesting that the FRET is on. This
behavior was substantiated by the observation that the emis-
sion color of the probe solution changed from green to
orange (Figure S7B in the Supporting Information).
To shed light on the possible signaling mechanism, we de-
cided to first examine whether the reaction of Ratio-Au
with Au3+ is reversible or irreversible. As shown in Fig-
Chem. Eur. J. 2011, 17, 9066 – 9069
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W. Lin et al.
Probe Ratio-Au (5 mm) was incubated with various metal
Figure S14 in the Supporting Information and the measured
data of the fluorescence-intensity ratio (Figure S16 in the
Supporting Information). This value is in good agreement
with the value of 76 (Æ4) ppm (per gram) obtained from
the ICP-OES analysis (see the Supporting Information).[14a,c]
Notably, this is the first report on determination of AuNPs
level by using a fluorescent gold probe.
ions, namely, Au3+, K+, Na+, Ca2+, Mg2+, Cu2+, Co2+, Ni2+
Zn2+, Cd2+, Pb2+, Pd2+, Mn2+, Fe3+, Fe2+, Ag+, and Hg2+
,
,
in phosphate buffer/C2H5OH (3:7, pH 7.0) to investigate the
selectivity. As shown in Figure 2 and S13 in the Supporting
In summary, we have described a new Au3+-mediated
transformation of a thioamide-phenyl-substituted alkyne to
5-ketothiazole. This is the first report on Au3+-mediated
thioACTHNUGRTENUNGamide alkyne cyclizations. Based on this finding, we fur-
ther judiciously designed and synthesized Ratio-Au as the
first FRET-based ratiometric fluorescent probe for Au3+
The probe showed high sensitivity and selectivity to Au3+
.
.
Importantly, we have demonstrated, for the first time, quan-
titative detection of AuNPs in a real cosmetic sample by
using probe Ratio-Au. We expect that the newly discovered
Au3+-mediated reaction presented herein will be widely em-
ployed for design of fluorescent gold probes.
Figure 2. Ratiometric fluorescent response of probe Ratio-Au (5 mm) to
various metal ions (3 equiv) in phosphate buffer/C2H5OH (3:7, pH 7.0).
Excitation at 470 nm.
Experimental Section
Information, the probe has a higher selectivity for Au3+
than for other metal ions tested. Furthermore, the emission
color of Ratio-Au is also metal-ion dependent (Figure S7B
in the Supporting Information), indicating that the probe
Synthesis of Ratio-Au: Compound 5 (310 mg, 0.387 mmol) and Lawes-
sonꢁs reagent (156 mg, 0.387 mmol) were dissolved in dry benzene
(25 mL). Then the reaction mixture was refluxed for 45 min under N2 at-
mosphere. After removal of benzene under reduced pressure, the residue
was purified by silica-gel column chromatography (petroleum/CH2Cl2
1:1) to afford the orange-pink product Ratio-Au (163 mg, yield 51.6%).
m.p. 122–1248C; 1H NMR (400 MHz, CDCl3): d=8.19–8.17 (m, 1H),
7.53–7.49 (m, 2H), 7.22 (d, J=8.4 Hz, 2H), 7.17–7.14 (m, 1H), 7.07 (d,
J=8.0 Hz, 2H), 6.44 (d, J=8.0 Hz, 4H), 6.27 (d, J=8.4 Hz, 2H), 5.96 (s,
2H), 4.64 (s, 2H), 3.36–3.31 (q, 8H), 2.54 (s, 6H), 1.32 (s, 6H), 1.16 ppm
(t, J=7.2 Hz, 12H); 13C NMR (100 MHz, CDCl3): d=191.4, 155.7, 153.5,
151.4, 149.3, 143.0, 141.0, 137.5, 134.5, 132.8, 131.2, 129.3, 128.6, 127.6,
125.3, 124.0, 123.3, 121.3, 108.2, 103.8, 97.8, 84.6, 81.6, 73.0, 44.4, 33.7,
14.5, 12.6 ppm; HRMS (EI): m/z: calcd for C50H50BF2N5OS: 817.3797
[M+], found 817.3834.
could be employed for convenient visual sensing of Au3+
.
Because ratiometric probes are favorable for quantitative
measurements, we decided to test the ability of Ratio-Au
for quantitative detection of Au3+ in water samples. As
demonstrated in Table S1 in the Supporting Information,
Au3+ added to water samples (Yuelu spring water) could be
accurately measured with good recovery, indicating that
Ratio-Au is effective for quantitative detection of Au3+ in
water samples.
Encouraged by this, we reasoned that the probe may be
employed for quantitative detection of AuNPs, as AuNPs
can be converted to Au3+ in aqua regia.[14] Firstly, we tried
to establish a standard calibration curve. A series of known
concentrations of AuNPs were pretreated with aqua regia.[14]
Then the resulting solution was extensively diluted with the
assay solution before incubation with the probe. A good lin-
earity between the emission ratio (I594/I514) and the concen-
trations of AuNPs was observed (Figure S14 in the Support-
ing Information), suggesting that the probe is promising for
measurements of unknown amounts of AuNPs in real sam-
ples. Notably, in the control experiments, in which either
AuNPs or aqua regia was absent, the probe had no marked
ratiometric response under the same assay conditions (Fig-
ure S15 in the Supporting Information). Now, the stage was
set to quantitatively detect an unknown amount of AuNPs
in a consumer-product sample. For proof-of-concept, a com-
merical firming-eye-lotion sample that contained an unspeci-
fied amount of AuNPs (13Æ2 nm) was selected. The con-
centration of AuNPs in the eye-lotion sample was measured
to be 71(Æ4) ppm (per gram, Table S2 in the Supporting In-
formation) based on the standard calibration curve shown in
Acknowledgements
Funding was partially provided by NSFC (20872032, 20972044), NCET
(08–0175), the Key Project of Chinese Ministry of Education (108167),
and the Doctoral Fund of Chinese Ministry of Education
(20100161110008).
Keywords: alkynes · fluorescent probes · FRET · gold ·
nanoparticles
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A FRET-based Fluorescent Probe for Gold
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Received: December 13, 2010
Revised: April 18, 2011
Published online: July 15, 2011
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9069