development of immunochromatographic strip tests for selective
TRANSCRIPT
Makara J. Sci. 18/3 (2014), In Press
doi: 10.7454/mss.v18i3.3722
September 2014 | Vol. 18 | No. 3 96
Development of Immunochromatographic Strip Tests for Selective and
Quantitative Detection of Melamine
Wiyogo P. Wicaksono
1, Tribidasari A. Ivandini
1*, Endang Saepudin
1, and Yasuaki Einaga
2,3
1. Department of Chemistry, Faculty of Mathematics and Science, Universitas Indonesia, Depok 16424, Indonesia
2. Department of Chemistry, Faculty of Science and Technology, Keio University 3-14-1 Hiyoshi, Kohoku-ku,
Yokohama 223-8522, Japan,
3. JST-CREST, 3-14-1 Hiyoshi, Yokohama 2238522, Japan
*E-mail: [email protected]
Abstract
An immunochromatographic strip test based on the complex reaction of antigen-antibody (melamine-antimelamine) was developed
for quantitative detection of melamine. Gold nanoparticles (AuNP) were used to form AuNP-labeled antibody, which then acted as a
biosensor. Melamine quantification was performed by the determination of AuNP using anodic stripping voltammetry technique with
a boron-doped diamond as the working electrode. With sample volume of 100 µL and immunoreaction time of 7 min, the developed
immunochromatographic strip test produced a linear calibration curve for melamine concentration range of 0–0.6 mg/L, with
detection limit of 0.1 mg/L and RSD of ~5%. Furthermore, negative results were obtained for samples containing cyanuric acid and
urea, indicating that the developed immunochromatographic strip test has potential for selective and quantitative detection of melamine.
Abstrak Pengembangan Strip Test Immunokromatografi untuk Pendeteksi Melamine Secara Selektif dan Kuantitatif. Strip test
berbasis imunokromatografi berdasarkan reaksi pembentukan kompleks antigen-antibodi (melamin-antimelamin) dikembangkan
untuk mendeteksi melamin secara kuantitatif. Nanopartikel emas (AuNP) digunakan sebagai label pada antibodi untuk membentuk
antibodi berlabel AuNP yang kemudian berfungsi sebagai biosensor. Kuantifikasi melamin diperoleh dengan menentukan konsentrasi
AuNP menggunakan teknik anodic stripping voltammetry. Boron-doped diamond digunakan sebagai elektroda kerja. Dengan volume
sampel 100 µL dan waktu imunoreaksi 7 menit, strip test menghasilkan kurva linier pada kisaran konsentrasi 0–0,6 mg/L dengan
batas deteksi 0.1 mg/L dan RSD ~5%. Selain itu hasil negatif diperoleh ketika strip test diaplikasikan pada sampel yang mengandung
asam sianurat dan urea, hal tersebut mengindikasikan bahwa strip test yang dikembangkan dapat digunakan sebagai pendeteksi
melamin secara selektif dan kuantitatif.
Keywords: anodic stripping voltammetry, boron-doped diamond, gold nanoparticles, immunochromatographic strip
test, melamine
1. Introduction
Melamine, 1,3,5-triazine-2,4,6-triamine (Figure 1) is a
synthetic compound used as an industrial chemical in
the production of plastics, amino resins, and flame
retardants [1-3]. It was recently reported that melamine
was deliberately added to milk and cheese [4] to provide
a false indication of protein concentration, since the
conventional method of protein detection (Kjeldahl
method) determines nitrogen content only. Although
melamine has low oral toxicity, some studies showed
that high and continuous dietary exposure to melamine
can cause renal stones and urinary bladder tumors [5-7].
Various analytical methods have been developed for the
detection of melamine, such as gas chromatography, high-
performance liquid chromatography, gas chromatography-
mass spectrometry, liquid chromatography-tandem mass
spectrometry, capillary electrophoresis, and enzyme-
linked immunosorbent assay (ELISA) [8-12]. Although
these methods offer high sensitivity and specificity, they
nevertheless require expensive instruments and highly
skilled analysis.
Recently, Li et al. [7] developed a strip test for rapid
qualitative detection of melamine, based on immuno-
chromatography. In another study, the same authors
Figure 1. Structure of Melamine
Development of Immunochromatographic Strip Tests for
Makara J. Sci. September 2014 | Vol. 18 | No. 3
97
also developed an electrochemical immunosensor
device, based on a nanoparticle probe, for quantitative
detection of proteins [13].
In this work, we developed an immunochromatographic
strip test for melamine, combined with electrochemistry
technique for detection of gold nanoparticles (AuNP).
AuNP was used to label the melamine antibody (AuNP-
antibody), which then functioned as a biosensor to
recognize the antigen (melamine). The quantity of
melamine was determined, based on the assumption that
the quantity of AuNP involved in the system was
representative of melamine concentration. Anodic
stripping voltammetry (ASV) using boron-doped
diamond (BDD) electrode as the working electrode was
selected for the electrochemical detection of AuNP.
This method was reported by Yamada et al. [14] to be
highly suited to the detection of trace metals, providing
high selectivity and stable current responses. The results
of the present study show that the developed
immunochromatographic strip test is promising for the
selective and quantitative detection of melamine.
2. Experiment
Chemicals. Bovine serum albumin (BSA), Millipore
glass-fiber filter, and nitrocellulose membrane (5 μm
pore size) were purchased from Sigma Aldrich. Antibody
of melamine was from Beacon. Melamine, trisodium
citrate (Na3C6H5O7), hydrogen tetracholoroaurate (III)
tetrahydrate (HAuCl4.4H2O), sodium borohydrate
(NaBH4), trimethoxyborane (C3H9BO3), and other
chemicals were supplied by Wako. All chemicals were
used without purification. Pure water was obtained from
a simple water system (Direct-Q UV 3-Millipore). Pt
wire and Ag wire were from Nilaco, and a peroxidase-
antiperoxidase (PAP) pen super-liquid blocker was
obtained from Cosmo Bio.
Preparation of gold nanoparticles (AuNP) and
AuNP-labeled antibody. Colloidal AuNP was prepared
by boiling 100 mL of 0.01% HAuCl4 with constant
stirring. Then, 2 mL of 1% trisodium citrate was added,
followed by 15 min boiling. After the solution had
cooled, distilled water was added to re-adjust the
volume.
In order to prepare AuNP-labeled antibody, 10 mL of
colloidal AuNP was adjusted to pH 7 by the addition of
0.1 M potassium carbonate. Then, 500 μL of antibody
was added. After an incubation time of 10 min at
ambient temperature, BSA was added with constant
gentle stirring, followed by a further 10 min incubation.
The solution was centrifuged at 12,000 rpm for 15 min
to obtain a red precipitate of AuNP-antibody, which was
then dissolved with 2 mL of phosphate buffer solution
(PBS) pH 7.4. A UV-Vis spectrometer (Jasco, V-570)
and transmission electron microscope (TEM) (TECNAI)
were used to characterize the AuNP and AuNP-labeled
antibody.
Preparation of immunochromatographic strip test.
The immunochromatographic strip tests consisted of
several components, including a sample pad, a conjugate
pad, a nitrocellulose membrane, a test zone, and an
absorbent pad. All components were assembled on a
piece of the plastic backing, as illustrated in Figure 2.
The sample pad, the conjugated pad, and the absorbent
pad were made of glass-fiber filters, whereas the
immuno-chromatographic reaction path and the test
zone were made of nitrocellulose membrane. The
conjugate pad was prepared by carefully dropping 100
μL of AuNP-labeled antibody. After drying at room
temperature, the procedure was repeated twice.
Meanwhile, the test zone was prepared via the same
method, using 100 μL of 5% antimelamine in 0.02 M
PBS pH 7.4.
Preparation of the electrochemical device. The
electrochemical device consisted of a piece of BDD as
the working electrode, Pt wire (dia. 0.2 mm) as the
supporting electrode, and an Ag/AgCl system as the
reference electrode. BDD was prepared on a silicon
wafer using microwave plasma-assisted chemical-vapor
deposition (ASTeX Corp.) as described elsewhere [15].
A mixture of 50 mL acetone and 4 mL trimethoxyborane
solution was used as carbon and boron sources,
respectively. Raman spectra (Princeton Instrument, Acton
sp2500) and SEM images (JEOL, JSM 5400) suggested
that the prepared BDD was of high quality [16]. All
electrodes were then assembled into a set of electro-
chemical cells on a plastic backing (inset of Figure 2)
and placed under the test zone of the immunochroma-
tographic strip test as the electrochemical measurements
were performed.
Quantitative detection of melamine using immuno-
chromatographic strip test. Melamine solution (volume
100 µL) of various concentrations was introduced on
the sample pad of the strip test. The sample then moved
absorbent pad
test zone
nitrocellulose
membrane
conjugate pad
sample pad
electrochemical
device
plastic backing
reference
electrode
counter
electrode
working
electrode
control line
Figure 2. Schematic of the Immunochromatographic Strip
Test. Inset Shows the Composition of the
Electrochemical Device
Wicaksono, et al.
Makara J. Sci. September 2014 | Vol. 18 | No. 3
98
through the nitrocellulose membrane and the conjugate
pad, respectively, taking ~7 min to reach the test zone.
Then, the test zone was underlined with two lines
(distance between lines was ~10 mm) using PAP pen. A
volume of 200 µL of 0.02 M HClO4 was added into the
test zone as supporting electrolyte, and after 2 min the
measurements were performed using the ASV method.
3. Results and Discussion
Characterization of colloidal AuNP and AuNP-
labeled antibody. UV-Vis spectrum (Figure 3) shows
that the maximum absorbance of colloidal AuNP
occurred at ~520 nm. However, when AuNP was
conjugated to antibody of melamine and BSA, the
wavelength shifted and the absorbance decreased. While
the decrease in absorbance was probably due to
increased distance between the AuNP particles, the
bathochromic shift of the wavelength indicates
increased AuNP diameter.
However, TEM images of AuNP (Figure 4a) and
AuNP-labeled antibody (Figure 4b) show that when
AuNP was conjugated to antibody and BSA, the
average diameter of the particles appeared to be similar
(~16 nm), whereas the distance between particles
increased. It seems that AuNP was surrounded by much
larger molecular size of antibody and BSA, which
affects the absorbance wavelength. The surrounding
BSA needs to be included in the labeling process to act
as the active site of the antibody (paratope). Therefore,
the antibody can interact with the antigen (melamine) in
its epitope site [17]. BSA, which is an amino acid
sequence, is expected to fill the ends of the "Y"
antibody (antigen binding site) to enable the antibody to
recognize and bind to melamine specifically [18].
Anodic Stripping Voltammetry of AuNP. Meanwhile,
ASV was examined for AuNP detection. HClO4 was
used not only for the supporting electrolyte but also for
the oxidizing agent of AuNP [14]. ASV involves two
0
0.2
0.4
0.6
0.8
1
425 475 525 575 625
Ab
sorb
an
ce
Wavelength / nm
Figure 3. UV-Vis Spectrum of AuNP in the Absence (Solid
Line) and in the Presence of Antibody and BSA
(Dotted Line)
a b
Figure 4. TEM Images of (a) AuNP and (b) AuNP-
Labeled antibody
y = 7.3535x + 11.284 R² = 0.9693
10
12
14
16
18
20
0 1/5 2/5 3/5 4/5 1 11/5
Cu
rre
nt
/ µ
A
AuNP – HCl04 ratio
5
10
15
20
25
0.5 0.7 0.9 1.1 1.3 1.5
Cu
rren
t / m
A
Potential / V vs. Ag/AgCl
HClO4
AuNP-HClO41:10
AuNP-HClO41:8
AuNP-HClO41:5
AuNP-HClO41:2
AuNP-HClO41:1
a
b
Figure 5. Anodic Stripping Voltammograms of AuNP at
BDD Electrodes in Various Concentrations of
AuNP in 0.02 mM HClO4 (a) and its
Concentration Dependence Curve (b). Deposition
Potential, Deposition Time, and Scan Rate were -
0.4 V, 240 s, and 100 mV/s, Respectively
reaction steps, including reduction or pre-concentration
of the analyte at the surface; and oxidation or stripping
of the deposited analyte. Therefore, the change Au0 of
AuNP to Au3+
ions is expected to provide suitable
condition for ASV. A well-defined oxidation peak
observed at the potential around +1.1 V seems to
comply with the requirements for ASV (Figure 5a).
The oxidation reaction during the process is proposed
below:
Step 1: Dissolution step using HClO4
AuNP Au3+
+ 3e- (1)
Step 2: Reduction or pre-concentration step
Au3+
+ 3e- Au
0 (2)
Development of Immunochromatographic Strip Tests for
Makara J. Sci. September 2014 | Vol. 18 | No. 3
99
Step 3: Oxidation or stripping step
Au0 Au
3+ + 3e
- (3)
The oxidation peak current of the voltammogram was
highest at the deposition potential of -0.4 V. Meanwhile,
the oxidation peak continuously increased with
deposition time from 60 s to 300 s. Therefore,
deposition potential of -0.4 V and deposition time of
240 s were fixed for subsequent experiments.
Furthermore, ASV for various concentrations of AuNP
in HClO4 shows linear dependence of the peak current
at +1.1 V on the concentrations of AuNP (Figure 5b),
suggesting that the peak can be utilized for AuNP
detection.
Electrochemical detection of melamine using
immunochromatographic strip test. When melamine
standard solution is introduced to the sample pad, the
sample moves due to the capillary nature of the
nitrocellulose membrane. At the conjugated pad,
melamine selectively interacts with the immobilized
AuNP-labeled antibody to form the complexes of
AuNP-labeled antibody-melamine. The immunoreaction
is expected to complete through the nitrocellulose
membrane toward the test zone. In the test zone, the
complexes containing melamine are captured by
capturing the antibody to form AuNP-labeled antibody-
melamine-antibody sandwich complexes, whereas the
complexes without melamine continuously move
toward the absorbent pad. Since the complexes without
melamine have much smaller size than the sandwich
complexes of melamine, theoretically, it is possible to
separate them through their velocity difference during
movement within the nitrocellulose membrane.
Movement of the red-colored AuNP from the sample
pad to the test zone was observed to take ~7 min, which
is estimated to be sufficient for the immunoreaction
time [8].
Figure 6 shows voltammograms of various concentrations
of melamine detected by immunochromatographic strip
test combined with anodic stripping voltammetry measu-
rements. The oxidation peak in the voltammograms
measured without the strip test was at +1.1 V (Figure
5a), whereas after separation with the strip test the peak
shifted to +0.9 V. This was likely due to the influence of
the antibody and BSA, which surrounded the AuNP.
However, a linear calibration curve from the concentration
range of 0–6 mg/L (R2 = 0.98) could be achieved
(Figure 6b) with a sensitivity of 0.19 μA/ppm and an
estimated limit of detection (LOD) of 0.1 mg/L (S/N =
3). Better LOD was also shown in comparison to some
other methods, as presented in Table 1 [7,19,20].
Furthermore, relative standard deviation (RSD) of ~5%
was achieved for standard solution of 0.6 mg/L
melamine, indicating good stability of the current
responses.
0.2
0.3
0.4
0.5
0.5 0.7 0.9 1.1
Cu
rre
nt
/ µ
A
Potential / V vs. Ag/AgCl
Mel 0 mg/mL
Mel 0.15 mg/mL
Mel 0.3 mg/mL
Mel 0.45 mg/mL
Mel 0.6 mg/mL
y = 0.1887x + 0.3085 R² = 0.9808
0.3
0.35
0.4
0.45
0 0.2 0.4 0.6 0.8
Pea
k C
urr
en
t / µ
A
Melamine Concentration / mg L-1
a
b
Figure 6 Anodic Stripping Voltammograms Obtained
from Immunochromatographic Strip Tests
Coupled with ASV Technique at BDD Electrodes
(a) and its Concentration Dependence Curve (b).
Other Conditions were Similar to those of Fig. 5
Table 1. Comparative Performance of AuNP Detection
Methods Applied in Immunochromatographic
Strip Test for Melamine Detection
Sample Method LOD
Milk Test-strip reader [19] 260 mg/L
Milk Spectrophotometer [7] 2 mg/L
Milk Optical reader [20] 4.47 × 10-3
mg/L
Water Present method 0.1 mg/L
Selectivity examination of samples containing cyanuric
acid and urea, which have similar chemical structures to
melamine, showed negative results. Although polyclonal
antibody of melamine was utilized in the strip tests [12],
several separation stages were involved, including
immune selectivity of the antibody, paper chroma-
tography of the strip test, and potential oxidation
selectivity of ASV. Moreover, to the best of our
knowledge, this is the first electrochemical method
applied to the quantification of melamine that utilized
immunochromatographic strip tests with AuNP as the
label. Therefore, although a strip test using a UV-Vis
optical reader shows approximately two orders better
LOD, the proposed method suggests a promising
alternative for selective detection of melamine.
Wicaksono, et al.
Makara J. Sci. September 2014 | Vol. 18 | No. 3
100
4. Conclusions
Immunochromatographic strip test for melamine was
successfully combined with ASV technique using BDD
electrodes. Based on the assumption that the concentration
of gold nanoparticles used as the label for melamine
antibody was representative of the melamine concen-
tration, a linear calibration curve of melamine could be
achieved from the concentration range of 0–0.6 mg/L,
with an estimated limit of detection of 0.1 mg/L and
RSD of ~5%. Selective detection was evidenced by the
negative results of strip tests for cyanuric acid and urea,
which have similar chemical structures to melamine.
Acknowledgement
This research was funded by Prime Research Grants,
Universitas Indonesia (DAMAS) 2013 Contract No.
0974/H2.R12/HKP.05.00/2013
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