Fluorescence "turn-on" detection of melamine with aggregation-induced-emission-active tetraphenylethene

Article abstract of DOI:10.1002/chem.201203081

The design and evaluation of fluorescence "turn-on" detection of melamine with cyanuric acid modified tetraphenylethene based on aggregation-induced emission, which can recognize the safe concentration level of melamine (1 ppm) in real powdered milk, is d

Full text of DOI:10.1002/chem.201203081

DOI: 10.1002/chem.201203081
Fluorescence “Turn-On” Detection of Melamine with Aggregation-Induced-
Emission-Active Tetraphenylethene
Takanobu Sanji,*[a] Mitsutaka Nakamura,[a] Shiori Kawamata,[a] Masato Tanaka,*[a]
Shotaro Itagaki,[b] and Takahiro Gunji[b]
A few years ago, melamine was found as contaminant in
pet food sold in the U.S. and Canada, as well as in milk,
milk products, and infant formula, predominantly in China.
Melamine has a number of industrial uses, but it is not ap-
proved for use in foods. In these incidents, however, mela-
mine was illegally added to the foods at some point in the
manufacturing process to boost the apparent protein content
because of its high percent mass of nitrogen (66.6%), caus-
ing renal failure in pets, as well as in infants and children in
China. The U.S. Food and Drug Administration (FDA) set a
safe concentration level of melamine at 2.5 ppm for foods
and at 1 ppm for powdered infant formula. Because there is
an increasing public concern over food safety, selective de-
tection methods for melamine in these products are highly
desirable. Instrumental methods, such as gas chromatogra-
phy mass spectrometry (GC-MS),[1] electrospray ionization
mass spectrometry (ESI-MS),[2] high-performance liquid
chromatography mass spectrometry (HPLC-MS),[3] and
high-performance liquid chromatography (HPLC),[4] are
available for the screening and quantification of melamine
and its analogues in contamination events. These techniques
are highly selective, but require expensive and complicated
instruments that are not easy to operate. Recently, a colori-
metric detection method by using Au nanoparticles has
been reported, which could offer simple and sensitive detec-
tion of melamine.[5] Herein, we report the design and evalu-
ation of alternative fluorescence “turn-on” detection of mel-
amine in solution and on membrane. This system can sense
the safe concentration level of melamine (1 ppm) in real
milk products. The proposed method showed high precision
and accuracy.[6] Thus, this fluorescence-based technique is
suitable for routine and on-site use without using complicat-
ed instruments.
Recently, we[7] and others[8] have demonstrated that aggre-
gation-induced emission (AIE)-active materials, first report-
ed by Tang and co-workers,[9] have a potential utility in sen-
sory fields.[10] AIE-active molecules show no emission in sol-
ution, but an intense emission when aggregated or in the
solid state because of restriction of intramolecular rotations.
Based on the state (aggregation)-dependent fluorescence,
we designed an AIE-active tetraphenylethene (TPE) with
cyanuric acid moieties, 1, for the fluorescence sensing of
melamine (Figure 1a). Because melamine/cyanuric acid
combine together to form a stable adduct through multiva-
lent hydrogen-bonding interactions (Figure 1b),[11] TPE inte-
grated with cyanuric acid moieties 1 is seen to display an in-
tense emission, that is, “turn-on” fluorescence, when it rec-
ognizes melamine and then forms aggregates (Figure 1c).
The TPE integrated with cyanuric acid 1 was synthesized
by the reaction of 4-bromobutoxy-substituted TPE and cya-
nuric acid (Scheme S1 in the Supporting Information).
[a] Prof. T. Sanji, M. Nakamura, S. Kawamata, Prof. M. Tanaka
Chemical Resources Laboratory
Tokyo Institute of Technology
4259 Nagatsuta, Midori-ku
Yokohama 226-8503 (Japan)
Fax :ACHTUNGTRENNUNG(+81)45-924-5277
E-mail: sanji.t.aa@m.titech.ac.jp
m.tanaka@res.titech.ac.jp
[b] S. Itagaki, Prof. T. Gunji
Department of Pure and Applied Chemistry
Faculty of Science and Technology
Tokyo University of Science
Figure 1. a) The chemical structure of 1. b) Melamine–cyanuric acid
adduct formed through multivalent hydrogen bonding. c) Schematic rep-
resentation of a “turn-on” fluorescent sensing of melamine by using cya-
nuric acid modified tetraphenylethene (TPE) based on aggregation-
induced emission (AIE).
2641 Yamazaki, Noda Chiba 278-8510 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201203081.
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Chem. Eur. J. 2012, 18, 15254 – 15257

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The sensory response of 1 to melamine was examined by
monitoring the fluorescence spectral change upon addition
of melamine ([1]=10 mm). As shown in Figure 2, an acetoni-
trile solution of 1 was practically nonluminescent, with a
value with a greater than approximately 200-fold enhance-
ment (Ff =0.022). However, melamine concentrations below
0.6 ppm (5 mm) did not cause an emission under this set of
conditions. The assay is qualitative or semiqualitative. As
shown in Figure 2b, the fluorescence intensity increased lin-
early with melamine concentration in the range 5–20 mm.
The line obtained could be used as a calibration curve for
quantification of the amount of melamine in the analyte.
The fluorescence enhancement upon addition of melamine
is achieved in terms of aggregation triggered by multivalent
complexation of melamine with 1, which in turn induces en-
hancement of the emission, as illustrated in Figure 1c. After
filtration through a 0.45 mm pore size membrane, the residu-
al solution showed no emission, indicating that aggregates
formed (Figure S3 in the Supporting Information).
The selectivity of this assay was confirmed by screening
its fluorescence response to relevant analytes (Figure S4 in
the Supporting Information). Again, TPE 1 (10 mm) showed
a
fluorescence response in the presence of melamine
(10 mm). No fluorescence signal increases were observed in
the presence of ammeline, ammelide, uracil, cytosine, or thy-
mine (100 mm). Thus, TPE 1 only responded to melamine,
but not to the other compounds. This is because the cyanuric
acid units on 1 specifically recognize melamine.
Most importantly, when tested in powdered infant formu-
la spiked with melamine, the assay responded at concentra-
tions of melamine above 1 ppm. As shown in Figure 3, the
“turn-on” fluorescence was clearly observable by the naked
eye (also. see Figure S5 in the Supporting Information).
Figure 2. a) Fluorescence spectra and b) changes in fluorescence intensity
(IF) monitored at l=500 nm of 1 (10 mm) upon addition of melamine to a
solution of 1 in acetonitrile (lEx =350 nm). The dotted line indicates the
safe concentration level of melamine for infant formula (1 ppm). The
inset shows photographs of 1 and the mixture with melamine (0, 1, 5, and
10 mm, from left to right) in an acetonitrile solution under irradiation
with UV light at l=365 nm.
quantum yield (Ff) of 0.002 (relative to quinine sulfate as a
standard). However, when melamine was added to the solu-
tion followed by ultrasonication, the mixture exhibited an
intense emission. Importantly, we found that this system is
well designed to identify melamine concentrations above
1 ppm (7.7 mm), which is the safe contamination level of mel-
amine for infant formula set by the FDA. For example, at
concentrations of melamine above 0.6 ppm (5 mm), the emis-
sion started to appear within a few hours and the intensity
increased as a function of melamine concentration.[12] The
emission was easily detectable by the naked eye, as can be
seen in the inset in Figure 2b. Above 2.6 ppm (20 mm) of
melamine, the fluorescence intensity reached a constant
Figure 3. Photographs of 1 (10 mm) with extracts from melamine-contami-
nated infant powdered milk (blank, 1, and 10 ppm, from left to right) in
an acetonitrile solution under irradiation with UV light at l=365 nm.
Next, with practical applications in mind, the membrane-
based sensing of melamine was demonstrated. The mem-
brane (or paper)-based sensors have attracted much atten-
tion for point-of-care testing alternative to conventional an-
alytical instrumentations, because the devices are simple,
portable, inexpensive, disposable, and use low sample
volume.[13,14] In the sensing study, TPE 1 was adsorbed on
hydrophobic poly(vinylidene difluoride) (PVDF) mem-
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T. Sanji, M. Tanaka et al.
brane.[15] Acetone solutions of melamine at various concen-
trations (0, 1, 5, and 10 ppm) were spotted onto the test
pieces of the membrane filter (6 mm in diameter).[16] When
tested in powdered infant formula spiked with melamine, a
blue emission was found within a few minutes at concentra-
tions of melamine above 1 ppm, as show in Figure 4.[17]
Thus, the test pieces detect the safe contamination level of
melamine for infant formula set by the FDA by the naked
eye.[18]
ing residue. After ultrasonication for 3 h, the sample was subjected to the
fluorescence measurement (lEx =350 nm).
Sample preparation for imaging detection: A typical example is as fol-
lows (10 ppm). An ethanol solution of powdered infant milk (10 mL,
1.35 g) in water was spiked with melamine (0.10 mg). After addition of
ethanol (5 mL) and stirring for 5 min, the mixture was filtered through
cotton. The filtrate was used for the sensing study.
Imaging detection of melamine on membrane filter: An acetone solution
(20 mL) of 1 (100 mm) was spotted on PVDF membrane with a diameter
of 6 mm (0.28 cm2) with a micropipet. After solvent evaporation, an ace-
tone solution (20 mL) of melamine (0, 1, 5, and 10 ppm) was spotted on
the membrane filter. Again, after solvent evaporation, spotting of the
acetone solution of melamine (20 mL) on the membrane filter and solvent
evaporation was replayed. After spotting of the melamine and solvent
evaporation, process was repeated (total five times), one drop of ethanol
was spotted to keep. Then, the membrane filter spotted with melamine
was mounted on a slide glass to subject the sensing study under UV irra-
diation of black light.
Acknowledgements
This work was supported in part by the Management Expenses Grants
for National Universities Corporations “Nano-Macro Materials, Devices
and System Research Alliance” from the Ministry of Education, Culture,
Sports, Science and Technology of Japan (MEXT). We also thank Center
for Advanced Materials Analysis, Technical Department, Tokyo Tech, for
supporting elemental analysis.
Keywords: aggregation
·
fluorescence
·
sensors
·
tetraphenylethenes
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Fluorescence “Turn-On” Detection of Melamine
COMMUNICATION
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[12] The response rate was dependent on the concentration of melamine
and also the concentration of 1. For example, a mixture of 1 (10 mm)
and melamine (10 mm) showed an emission after sonication for 1 h,
[16] The response was dependent on the solvent used for spotting of
melamine. For example, acetonitrile or ethanol was used, the test
piece only detected at concentrations of melamine above 10 ppm.
[17] Under this set of conditions, the membrane was wet with ethanol
during detection. When the membrane was dried off, a blue emis-
sion was observed despite the absence of melamine. Then, the con-
trol experiment (0 ppm) was usually required.
but
a
mixture of higher concentration of melamine (ꢀ15 mm)
[18] The procedure for our assay is complicated at the present stage. To
simplify procedures for the sensing, further study is required.
showed an emission within 30 min. On the other hand, 10 mm of 1 re-
quired 1.5 h to detect 7.7 mm (1 ppm) of melamine, but 20 mm of 1
detected it within 30 min. For details, see Figures S1 and S2 in the
Supporting Information.
Received: August 30, 2012
Published online: November 5, 2012
Chem. Eur. J. 2012, 18, 15254 – 15257
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
15257