Visualized detection of melamine in milk by supramolecular hydrogelations

Article abstract of DOI:10.1039/c4cc05826g

We reported a visualized detection system for melamine based on supramolecular dydrogelations.

Full text of DOI:10.1039/c4cc05826g

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Visualized detection of melamine in milk by
supramolecular hydrogelations†
Cite this: Chem. Commun., 2014,
50, 12873
Jianwu Zhang,a Caiwen Ou,a Yang Shi,b Ling Wang,c Minsheng Chen*a and
Zhimou Yang*bc
Received 27th July 2014,
Accepted 5th September 2014
DOI: 10.1039/c4cc05826g
www.rsc.org/chemcomm
We reported a visualized detection system for melamine based on extensive research interests. Supramolecular hydrogel-based sensors
supramolecular dydrogelations.
can not only provide an unambiguous visual change in the material
physical properties but also, in contrast to colorimetric and
Melamine, with high nitrogen content (66%) and low cost, was fluorescent sensors, there is no need of any instruments and
illegally used in adulterating milk to boost the apparent protein transporting samples to a laboratory. Such an advantage makes
level. However, melamine has a deleterious effect on human rapid, real-time and on-site monitoring possible. To date, many
body and can lead to serious tissue injury in children,1 such as supramolecular hydrogel-based sensors have been developed for
acute kidney failure, urolithiasis, bladder cancer, and even various important analytes, including enzymes,11f,12 glucose,13
death due to the formation of melamine–cyanuric acid crystals in nitric oxide14 and metal ions,15 etc. For instance, Xu and co-workers
the kidneys.2 Therefore, it is of great importance to detect melamine have reported on a simple assay for screening inhibitors of an
in foods. For detecting melamine, various methods have been enzyme (acid phosphatase) based on enzyme-triggered gel
reported, including gas chromatography-mass spectrometry formation.12a Using a similar strategy, McNeil and coworkers
(GC-MS),3 liquid chromatography-mass spectrometry (LC-MS),4 also developed a modular system for screening protease activity
enzyme-linked immunosorbent assay (ELISA),5 and fluorescence via the sol–gel phase transition and for monitoring artificial
spectroscopy,6 etc. Although the existing methods can provide blood clotting.12b Besides, Zhang and co-workers developed a
both accuracy and high sensitivity for melamine detection, all simple visual biosensor for glucose detection based on the
require expensive and complicated instruments. Moreover, the responsiveness of the peptide-based self-assembly to glucose
melamine is usually needed to be extracted from the milk to metabolism.13 Hamachi et al. reported a simple gel–sol phase
render its detection,6c,7 thus making the on-site and real-time transition catalyzed by H2O2 for the detection of several important
monitoring difficult. There is an urgent need to develop simple, enzymes.11f All these efforts advance the research progress of the
rapid, easily accessible, and cost-effective methods to detect supramolecular hydrogel-based detection systems. In this study, we
melamine in foods.
reported on a simple and visual assay for the detection of melamine
Supramolecular hydrogels are advantageous soft biomaterials based on supramolecular hydrogelation.
because of their ease of synthesis, biocompatibility, degradability,
It is well-known that melamine and cyanuric acid (CA) or its
and fast responses to external stimuli.8 They have led to diverse derivatives can form a stable complex through the interaction
applications such as tissue engineering,9 drug delivery,10 analyte between diaminopyridine and diimide moieties, exhibiting
detection,11 etc. Recently, their application in sensing attracts three complementary NH–O and NH–N hydrogen bonds.16
Such triple hydrogen bonding is considered to be particularly
useful for controlling molecular self-assembly due to the reversibility,
a Department of Cardiology, Zhujiang Hospital of Southern Medical University,
specificity, directionality, and cooperative strength of this class
Guangzhou 510280, P. R. China. E-mail: gzminsheng@vip.163.com
b State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive
of interaction.17 Inspired by this principle and the nature of the
self-assembled peptide systems, we opted to design a peptide-
based CA derivative with self-assembling properties for the
detection of melamine. The derivative could self-assemble into
nanofibers but not hydrogels due to the relatively weak inter-
fiber interactions. With the assistance of the triple hydrogen
bond from CA–melamine, the nanofibers might be cross-linked
into 3D fiber networks, leading to the dramatic visualized phase
Materials, Ministry of Education, and College of Life Sciences, Collaborative
Innovation Center of Chemical Science and Engineering, Nankai University,
Tianjin 300071, P. R. China. E-mail: yangzm@nankai.edu.cn
c College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Design,
Nankai University, Tianjin 300071, P. R. China
† Electronic supplementary information (ESI) available: Synthesis and character-
ization, chemical structures, rheological data, and TEM images. See DOI: 10.1039/
c4cc05826g
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Scheme 1 The synthetic route to Nap-FFYGK-CA.
transition from a solution to a gel. This process could be developed
into a simple, real-time, and on-site sensing method for melamine.
In order to test our hypothesis, we designed the molecule of
Nap-FFYGK-CA (Scheme 1). Many peptide derivatives based on Fig. 1 Optical images of the solutions of Nap-FFYGK-CA without (left
FF or FFY are molecules with excellent self-assembling properties.18
We envisioned that our designed molecules might self-assemble
images) or with (right images) 35 ppm, 20 ppm, 20 ppm melamine in PBS
(A and B), milk (C and D), and urine (E and F), respectively.
into nanofibers with good water dispersity. The CA moiety was used
to form a complex with melamine to increase the inter-fiber
interactions for hydrogelation. The synthetic route was described
in Scheme 1, we firstly prepared CA-acid in four steps with a total
yield of about 70%. Nap-FFYGK was produced by standard solid
phase peptide synthesis (SPPS). We then obtained Nap-FFYGK-CA
by coupling CA-acid with the peptide. The pure compound was
achieved by reverse phase high performance liquid chromatography
(HPLC).
After obtaining Nap-FFYGK-CA, we tested its self-assembling
properties by the heating–cooling process. We firstly prepared
its phosphate buffer saline (PBS, pH = 7.4) solution at a
concentration of 0.5 wt%. After a heating–cooling process, we
observed the formation of a clear solution (Fig. 1A), suggesting
that the compound itself could not form a hydrogel at this
concentration. We observed a light beam when we used a laser
pointer to shine the solution, indicating the presence of nanoscale
materials in the solution. We then tested whether the addition of
melamine would lead to hydrogelations or not. We mixed the
solution of Nap-FFYGK-CA at a final concentration of 0.5 wt% and
Fig. 2 TEM images of (A) solution of Nap-FFYGK and gels with different
equiv. of melamine: (B) 2 equiv., (C) 4 equiv. and (D) 16 equiv.
the solution of melamine with different concentrations. As shown
in Fig. 2B, the solution changed to a hydrogel within 10 minutes
when the concentration of melamine was higher than 35 ppm. We of melamine in these two samples. There are proteins and urea
also observed the hydrogel formation when the concentration of in milk and urine, respectively, which may interfere with the
melamine was 20 ppm, but the gelation took a longer time of about hydrogelation. As shown in Fig. 1C, milk without melamine
8 hours. These observations clearly indicated the success of our cannot form a hydrogel at the concentration of 0.5 wt%. With
design. As the sol–gel phase transition can be easily identified by the addition of melamine above the concentration of 20 ppm, a
the naked eye, this system might be suitable for the detection of hydrogel would form within 30 min (Fig. 1D). Similar observations
melamine without instruments.
were achieved for melamine tainted urine, and the minimum
We also tested whether our method could be used to detect detection concentration of melamine in urine was also about
melamine in foods and biological fluids. We choose melamine 20 ppm (Fig. 1E and F). Compared with other melamine detecting
tainted milk and urine because of the importance of detection systems, which need to extract melamine from the samples, our
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method was much simpler that the analytes could be detected value of 0.1 rad sꢀ1 for the gel with 2 equivalent of melamine,
directly by the naked eye. Such a convenient method is very which was about 35 times bigger than that with 0.25 equivalent
promising for the detection of melamine where no instrument of melamine. However, when the amount of melamine was more
is accessible.
than 2 equivalent, the G0 value of resulting hydrogels dropped
We then used transmission electron microscopy (TEM) to (Fig. 3). These results indicated that the mechanical property of
characterize the morphology of self-assembled structures in the the hydrogels might be regulated by changing the molar ratios of
solution and hydrogels with different amounts of melamine. As melamine. As the mechanical properties of hydrogels are critical
shown in Fig. 2A, the compound Nap-FFYGK-CA self-assembled for their biological applications, a hydrogel that can finely tune
into filamentous structures with the diameter of about 25 nm. the mechanical properties is highly desired.22
The density of the cross-linking point was low probably due to the
Based on the above observations, we proposed possible
lack of strong interactions between fibers. In the presence of interactions between melamine and supramolecular nanofibers
melamine, we observed higher densities of the cross-linking point in the hydrogels. As shown in Fig. 4, Nap-FFYGK-CA itself could
in the gel samples (Fig. 2B–D and Fig. S5–S8, ESI†). Compared self-assemble into aligned nanofibers with a CA moiety at their
with the fibers in the solution without melamine, the diameter of surfaces. With low density of cross-linking points, these nanofibers
those in gels was bigger. Many reports have demonstrated that the can only form solutions. Assisted by the hydrogen bonds between
hydrogen bonding and p–p stacking between melamine could melamine and the CA derivative, the nanofibers entangled with
assist the formation and tune the morphology of self-assembled each other and formed stable 3D networks for hydrogelations.
nanostructures.19 In our study, we also observed that the diameter When the concentration of melamine was low, it served as the
of fibers became bigger in the presence of more melamine and it cross-linker. With the increase of melamine, more cross-linking
was about 33, 40, 48, 53, 58, 68, and 77 nm in the presence of 0.25, points formed, resulting in higher G0 values of the gels. However,
0.5, 1, 2, 4, 8, 16 equivalent of melamine, respectively (Fig. S9, melamine could also stack with each other. When an excess
ESI†). It is well-known that in order to form supramolecular amount of melamine was present in the gel, melamine assembly
hydrogels, there should be strong or at least medium interactions formed at the surface of nanofibers, leading to the increase of
between self-assembled nanostructures.20 Our previous study had diameter of nanofibers. In such situation, melamine assembly
also showed that using a recombinant protein with multiple served as the cross-linkers to form fiber networks. Since the
binding sites, we could enhance the inter-fiber interaction by interaction between melamine was not so strong as that between
the specific protein–peptide interaction for hydrogelations.21 In melamine and CA, the mechanical property of gels with an excess
this study, with the assistance of CA–melamine complexation, amount of melamine decreased.
we could enhance the interaction between supramolecular
nanofibers, resulting in the formation of stable 3D networks detection system for melamine. Using this method, melamine
and hydrogels. could be directly visualized by the naked eye in milk and urine
In summary, we reported a supramolecular hydrogel-based
We further investigated the mechanical property of hydrogels by without pre-extraction processes and the need of any instruments.
rheology. The hot aqueous solutions containing Nap-FFYGK-CA Although the minimum detection concentration of melamine for
and different amounts of melamine were directly transferred to the our system is a little bit higher than the safety limits (2.5 ppm in
rheometer. After 2 hour incubation to achieve stable hydrogels, we the USA and EU and 1 ppm in China), we have provided a simple,
performed a dynamic strain/frequency sweep. The value of the real-time, and on-site way to detect melamine at the concentra-
storage moduli (G0, elasticity) of all samples was bigger than that of tions of higher than 20 ppm in foods and in biological samples.
their corresponding loss moduli (G00, viscosity), suggesting that all
samples behaved as viscoelastic materials. The hydrogels showed
bigger G0 values with the increased amounts of melamine when the
equivalent of melamine was lower than 2 (Fig. 3 and Fig. S10–S12,
ESI†). For instance, the G0 value reached 8728 Pa at the frequency
Fig. 3 (A) Dynamic frequency sweep at the strain of 0.1% of the hydrogels
with addition of 0.5 (circles), 2 (squares) and 8 (triangles) equiv. of
melamine (filled symbols: G0 and open symbols: G00), and (B) the G0 value
in the mode of dynamic frequency sweep at the strain of 0.1% of the gels
with different equivalents of melamine.
Fig. 4 Possible molecular arrangements in self-assembled nanofibers of
Nap-FFYGK-CA and in gels with different amounts of melamine.
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This work is supported by NSFC (51222303, 31271053).
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