rapid quantification of melamine in milk using competitive 1,1′-oxalyldiimidazole chemiluminescent...
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Rapid quantification of melamine in milk using competitive1,10-oxalyldiimidazole chemiluminescent enzyme immunoassay
JooHee Choi,ab Young-Teck Kimc and Ji Hoon Lee*b
Received 11th June 2010, Accepted 3rd July 2010
DOI: 10.1039/c0an00396d
A novel competitive 1,10-oxalyldiimidazole (ODI) chemiluminescent enzyme immunoassay (CLEIA)
was developed as a method for rapid and simple screening of melamine in milk. Fat existing in milk acts
as an inhibitor in the competitive binding interaction of melamine and anti-melamine in the presence of
melamine-conjugated horseradish peroxidase. Thus, the calibration curve and sensitivity of competitive
ODI CLEIA for the quantification of melamine in fat free milk were wider and better than those in milk
containing fat. However, a centrifuge is not a good method for removing the inhibitor because
a portion of the melamine is also removed with the fat. The incubation time (20 min) for the competitive
binding interaction of anti-melamine and melamine in 20% milk diluted with PBS buffer of pH 7.4 was
longer than that (10 min) in 100% milk even though the sensitivity of the former was better than latter.
The limit of detection (1.12 ppb) determined in rapid ODI CLEIA (dynamic range: 3.8–125 ppb) for the
quantification of melamine in 20% milk not containing fat was lower than those (6.3 and 9.0 ppb)
calculated in relatively time-consuming luminol CLEIA and enzyme-linked immunosorbent assay
(ELISA). Also, we expect that ODI-CLEIA (dynamic range: 62.5–2000 ppb) capable of directly
quantifying melamine in 100% milk without any pretreatment can be applied as a new and simple
method for rapid screening of melamine in milk.
Introduction
Melamine (2,4,6-triamino-1,3,5-triazine), a substance composed
of 66% nitrogen, was synthesized to use as an additive of
industrial products such as plastics, adhesives, countertops,
dishware, and whiteboards. Despite being a non-natural
product, the presence of melamine in various high-protein foods
such as infant formula, pet food, animal feed, and wheat gluten
has been reported since 2007. It is well-known that the contin-
uous intake of edible foods containing melamine cause critical
diseases such as stone in the kidney and bladder and epithelial
hyperplasia of urinary bladder.1 Thus, the addition of melamine
in food products is not approved by the World Health Organi-
zation (WHO), the US Food and Drug Administration (FDA),
and the European Food Safety Authority (EFSA).1 WHO
adopted the tolerable daily intake (TDI) of 0.2 mg kg-1 body
weight a day-1 for melamine. TDI of WHO is lower than that of
US FDA (0.6 mg kg-1 body weight a day-1) and EFSA (0.5 mg
kg-1 body weight a day-1).1
Several analytical methods for quantifying and screening
melamine have been reported.2–15 Melamine existing in various
materials such as tissue, pet food, and protein powders have been
quantified using high performance chromatography (HPLC) or
gas chromatography (GC) with UV1 or mass (MS) spectrom-
etry.14 The limit of detection (LOD) is dependent on the prop-
erties of analytical samples.2–15 Thus, the range of LOD
aLangley High School, 6520 Georgetown Pike, McLean, VA 22101, USAbLuminescent MD, LLC, 20140 Scholar Drive, Hagerstown, MD 21742,USA. E-mail: [email protected]; Fax: +1 301393 9092; Tel: +1301 393 9092cDepartment of Packaging Science, Clemson University, Clemson, SC29634, USA
This journal is ª The Royal Society of Chemistry 2010
determined using HPLC (or GC) with UV or MS spectrometry
have been wide (10 ppb � 200 ppm)1. HPLC-MS or GC-MS are
used for the quantification of melamine in foods by US FDA
laboratories.1 Recently, other analytical methods such as
surface-enhanced Raman spectroscopy (SERS),1 competitive
enzyme-linked immunosorbent assay (ELISA),2,3 and competi-
tive chemiluminescent enzyme immunoassay (CLEIA) using
luminol chemiluminescence (CL) detection13 have been devel-
oped for the quantification of melamine. LOD of SERS for the
screening of aqueous solution was 33 ppb.14 LOD of ELISA for
the monitoring of dog food was < 20 ppb.2 Also, LOD of
competitive CLEIA with luminol CL detection for the quantifi-
cation of melamine in milk was as low as 6.3 ppb.14
It is well-known that peroxyoxalate chemiluminescence (PO-
CL) detection is more sensitive and selective than other detec-
tions such as absorbance, electrochemical, fluorescence, and
luminol chemiluminescence widely applied in enzyme immuno-
assay (EIA).16 Unfortunately, oxalate esters such as bis(2,4-
dinitrophenyl) oxalate (DNPO) and bis(2,4,6-trichlorophenyl)
oxalate (TCPO), one of PO-CL reagents, are too unstable in
aqueous solution to be applied as CL reagents for highly sensitive
CLEIA system. Recently, chemical and physical properties of
1,10-oxalyldimidazole (ODI) derivatives formed from the reac-
tion between oxalate esters and imidazole derivatives were
studied.17,18 ODI derivatives are also unstable in aqueous solu-
tion. However, it was confirmed that ODI CL detection system in
aqueous solution is highly sensitive because ODI CL reaction is
much faster than the decomposition rate of ODI in aqueous
solution.19 This result indicated that it was possible to develop
a new CLEIA with ODI CL detection.
Using the advantages of ODI derivative CL, we developed
a rapid and simple CLEIA with ODI CL detection capable of
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Fig. 1 Procedures of ELISA, Luminol CLEIA and ODI CLEIA.
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rapidly quantifying and screening melamine in milk. Trace levels
of melamine in diluted and undiluted milk were quantified using
ODI CLEIA. In this paper, we describe in detail the character-
istics and advantages of the new CLEIA.
Experimental
Chemical and materials
Melamine, 4-methylimidazole, and phosphate buffered saline
(PBS) with 0.05% tween-20 were purchased from Sigma. 1 � 8
strip-well coated with rabbit anti-melamine and melamine-
conjugated horseradish peroixdase (HRP) were purchased from
Beacon Analytical Systems, Inc. was purchased from EMD.
Bis(2,4,6-trichlorophenyl) oxalate (TCPO) was purchased from
TCI America. Ethyl acetate (HPLC grade), water (LC/Mass
grade), and Isopropyl alcohol (ACS grade) were purchased from
J. T Baker. 10 � phosphate buffer saline solution (PBS, pH 7.4)
was from EMD. 3.0% hydrogen peroxide was purchased from
VWR. Amplex red and resorufin were purchased from AnsSpec,
Inc. Dimethyl Sulfoxide (DMSO) was purchased from Calbio-
chem. Fat free milk, 2% fat milk, and whole milk were purchased
from a local food market.
Methods
Preparation of standard solutions. Stock solution of melamine
(2000 ppm) was prepared with deionized water. It was stored
under ambient condition. Using the stock solution, 9 melamine
working solutions (0.00, 0.98, 3.90, 7.81, 15.65, 31.25, 125.00,
250.00, and 500.00 ppm) were prepared daily in PBS solution.
Preparation of diluted standard solution. 20 ml of melamine
working solution was spiked into a 1.5 ml - microcentrifuge tube
containing 980 ml of milk. Some of the microcentrifuge tubes
containing different concentrations of melamine were centri-
fuged under 2000 rpm for 10 min at room temperature (21.0 �2.0 �C). The rest were used without any centrifuging. 200 ml of fat
free milk serum obtained from the centrifugation was mixed with
800 ml of PBS buffer of pH 7.4 in a microcentrifuge tube. Also,
200 ml of milk, the mixture of protein and fat, was added in
a microcentifuge tube and mixed with 800 ml of PBS solution.
Based on the procedure described above, 10 standard solutions
containing different concentrations of melamine (0, 3.9, 15.7,
31.3, 62.5, 125, 250, 500, 1000, and 2000 ppb) were prepared.
Preparation of undiluted standard solutions. 20 ml of melamine
working solution was spiked into three 1.5 ml - microcentrifuge
tubes, each containing 980 ml of milk with different fat content
(fat free milk, 2% milk, and whole milk). 200 ml of milk con-
taining melamine was diluted with 800 ml of milk instead of PBS
buffer of pH 7.4.
Preparation of the mixture of Amplex Red and H2O2 as
a substrate of ODI CLEIA. 5.0 mg of Amplex Red was dissolved
in 5.0 ml of DMSO to prepare a stock solution. 500 ml of the
stock solution was added in a 2ml-glass vial. 10 vials containing
the stock solution were stored in a freezer (- 20.0 �C). Using 3%
H2O2 solution and PBS, 20 mM H2O2 stock solution was
prepared daily. In order to prepare the substrate used in ODI
2446 | Analyst, 2010, 135, 2445–2450
CLEIA, 25 ml of Amplex Red stock solution, 100 ml of H2O2
stock solution, and 4875 ml of PBS buffer were added in a 20 ml-
amber glass vial.
Design of ODI CLEIA. As shown in Fig. 1, the procedure of
ODI CLEIA for the quantification of melamin is similar to that
of other EIAs such as ELISA and Luminol CLEIA. However,
the incubation time for the competitive reaction between mela-
mine and melamine-conjugated HRP with anti-melamine coated
on the surface of the well in ODI CLEIA is shorter than those of
other EIAs (ELISA and Luminol CLEIA) because ODI CLEIA
is more sensitive. Also, the incubation time of the substrate
(Amplex Red) added in the well for ODI CLEIA is also shorter
than that for ELISA or same as that for Luminol CLEIA.
Measurement of light emitted from ODI CLEIA. 1.0 mM
TCPO was prepared daily in ethyl acetate as a stock solution.
Also, 10.0 mM 4MImH was prepared in ethyl acetate as a stock
solution. They were stored under ambient condition. 200 ml of
TCPO stock solution and 100 ml of 4MImH stock solution were
mixed with 39.7 ml of ethyl acetate in a 40 ml - amber glass vial.
ODI was rapidly formed from the reaction of TCPO and
4MImH in the vial at room temperature. 80 mM H2O2 was
prepared in isopropyl alcohol as a working solution. Two 40 ml-
glass vials containing ODI or H2O2 solution were placed in the
This journal is ª The Royal Society of Chemistry 2010
Fig. 2 Quantification of resorufin using ODI CL detection. Condition:
1.0 mM TCPO and 4.0 mM 4-methylimidazole (4MImH) dissolved in
ethyl acetate were used to produce ODI. 0.08 M H2O2 was prepared in
isopropyl alcohol.
Scheme 1 Quantification of resorufin formed from the reaction of
Amplex Red and H2O2 in the presence of HRP using ODI CL detection.
1. TCPO, 2. 4MImH, 3. ODI, 4. Amplex Red, 5. Resorufin in ground
state, 6. Resorufin in excited state, X: high-energy intermediate capable of
transferring energy to resorufin
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reagent holder of Lumat LB Luminometer 9507 to inject the two
solutions into a detection cell (12 � 75 mm test tube) through
two dispensers.
As shown in Fig. 2, 100 ml of the substrate used in ODI CLEIA
was added into the washed well. 10 ml of resorufin formed from
the reaction between Amplex Red and H2O2 in the presence of
melamine-conjugated HRP bound with rabbit anti-melamine in
the well for 5.0 min was transferred into a 12 � 75 mm test tube.
The test tube was placed in the sample holder of Lumat LB
Luminometer 9507.
When the start button of the luminometer was pressed, the
sample holder of Lumat LB 9507 Luminometer turned to the
detection area. Then, 25.0 ml of H2O2 and 25.0 ml of ODI were
injected into the detection cell through two dispensers at 0.7 s
intervals. When ODI was inserted into the detection cell, relative
CL intensities emitted from the detection cell were integrated
immediately for 0.5 s.
Fig. 3 CL spectrum obtained in competitive ODI CLEIA for the
quantification of 62.5 ppb melamine. Condition: [TCPO] ¼ 5.0 mM,
[4MImH] ¼ 25.0 mM, [H2O2] ¼ 80 mM.
Results and discussion
Quantificationi of resorufin
Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) is widely
used to determine the concentration of horseradish peroxidase
(HRP) in fluorescence enzyme assay.20 Amplex Red is converted
to resorufin having high quantum efficiency when Amplex Red
reacts with H2O2 in the presence of HRP. In other words, the
yield of resorufin formed in this reaction depends on the activity
of HRP when the concentrations of Amplex Red and H2O2 are
constant. As shown in Fig. 2, we confirmed that low concen-
trations of resorufin dissolved in water are quantified in ODI CL
reaction. The detection limit (signal/noise ¼ 3.0) of ODI CL
analytical system for the quantification of resorufin was as low as
0.73 nM. The dynamic range of linear calibration curve (R2 ¼0.9952) was wide (3.3 � 141 nM). However, the relative CL
intensity of higher concentration of resorufin than 141 nM was
self-quenched. Fig. 1 indicates that trace levels of resorufin
This journal is ª The Royal Society of Chemistry 2010
formed from the reaction between Amplex Red and H2O2 in the
presence of HRP can be quantified with ODI CL detection
system developed based on the reaction mechanism shown in
Scheme 1.
Concentrations of CL reagents for competitive ODI CLEIA
As shown in Fig. 3, the maximum intensity measured in ODI CL
reaction was reached as quickly as 0.4 s. Thus, light emitted in
ODI CLEIA was integrated for 0.5 s. When higher concentrations
of TCPO and 4MImH than those used for obtaining Fig. 3 were
prepared, relative CL intensities measured in the absence of
melamine and in the presence of low concentrations of melamine
in ODI CLEIA were too high to measure. On the other hand,
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relative CL intensities measured in the presence of high concen-
trations of melamine were too low when lower concentrations of
TCPO and 4MImH than those shown in Fig. 3 were used in
competitive ODI CLEIA.
Relative CL intensity was increased with the increase of H2O2
concentration. However, relative CL intensities measured in the
presence of higher (up to 400 mM) concentrations than 80 mM
H2O2 were slightly higher than that shown in Fig. 3. Thus, 80
mM H2O2 was used to develop competitive ODI CLEIA capable
of detecting trace levels of melamine in milk.
Effect of incubation time on the competitive binding of melamine
and melamine-conjugated HRP with anti-melamine
As shown in Fig. 4, the sensitivity of competitive ODI CLEIA
depends on the incubation time for the competitive binding of
melamine and melamine-conjugated HRP with anti-melamine.
Fig. 4 indicates that the binding between melamine-conjugated
HRP and anti-melamine in the well is faster and more predomi-
nant than that between melamine and anti-melamine. Thus, the
dynamic range (3.9� 125 ppb) of calibration curves obtained after
20 and 30 min of incubation was wider than that (3.9 � 31.3 ppb)
determined after 10 min of incubation because additional mela-
mine molecules were able to bind with anti-melamine with the
increase of incubation time. Based on the results shown in Fig.4,
we selected 20 min as an appropriate incubation time in compet-
itive ODI CLEIA for the quantification of melamine in milk.
Effect of incubation time of Amplex Red and H2O2 in the well
As shown in Scheme 1 and Fig. 3, resorufin is formed from the
reaction of Amplex Red and H2O2 in the well containing mela-
mine-conjugated HRP bound with rabbit anti-melamine. The
yield of resorufin in this reaction depends on the incubation (or
reaction) time of substrates and melamine-conjugated HRP
bound with rabbit anti-melamine in the well. In other words,
relative CL intensity of resorufin measured after 20 min of
incubation was about 7 times higher than that observed after
Fig. 4 Three calibration curves obtained with different incubation time
on the competitive binding of melamine and melamine-conjugated HRP
with anti-melamine.
2448 | Analyst, 2010, 135, 2445–2450
5 min of incubation. However, the dynamic range (7.8�125 ppb)
of linear calibration curve obtained after the 20-minute incuba-
tion was shorter than that (3.9�125 ppb) after the 5-minute
incubation. This is because, during the 20-minute incubation, the
relative CL intensity of saturated resorufin formed from the
reaction of Amplex Red and H2O2 in the presence of lower
concentration of melamine than 7.8 ppb is self-quenched. This
result is consistent with that in Fig. 1, which shows that the
relative CL intensity of higher concentration of resorufin than
140 nM is self-quenched. Based on the results, the 5-minute
incubation of Amplex Red and H2O2 for the quantification of
melamine in milk was selected. The limit of detection (LOD¼ I0 -
3s) determined in this condition was 1.12 ppb. I0 is the relative
CL intensity measured in the absence of melamine in milk. s is
the standard deviation for the average of I0 (n ¼ 20). LOD of
competitive ODI CLEIA is lower than those (6.3 and 9.0 ppb) of
competitive luminol CLEIA13 and competitive ELISA.2
Recovery test
In order to study the effect of fat existing in commercial milk in
competitive ODI CLEIA for the quantification of melamine, 62.5
ppb melamine was spiked in fat free milk, 2% milk, and whole
milk, respectively, purchased from a local food market. Then,
melamine in each of the three types of milk was quantified using
competitive ODI CLEIA. As shown in Table 1, the recovery of
melamine spiked in milk is dependent on the amount of fat
existing in milk. With the increase of fat in milk, the recovery of
melamine was reduced because fat acts as an inhibitor while
melamine interacts with rabbit anti-melamine in the presence of
melamine-conjugated HRP. Thus, each of the three types of milk
containing 62.5 ppb melamine was centrifuged at 2000 rpm for 10
min to remove fat in the milk. Unfortunately, the recovery of
melamine in milk centrifuged wasn’t as good as that not centri-
fuged. This result indicates that parts of melamine spiked in milk
are removed with fat during the centrifuging. Thus, the three
types of milk containing different concentrations of fat should
not be centrifuged for the detection of trace levels of melamine.
Also, the results shown in Table 1 indicate that three different
linear calibration curves obtained with fat free milk, 2% milk,
and whole milk can accurately quantify melamine existing in
each type of milk.
Table 1 Recovery test of melamine (62.6 ppb) spiked in fat free milk, 2%milk, and whole milk not centrifuged and centrifuged at 2000 rpm for 10min
Centrifuging
Fat Free milka 2% milka Whole milka
Cb R (%)c Cb R (%)c Cb R (%)c
No 62.54 100.0 56.1 89.6 53.5 85.5Yes 60.5 96.6 44.1 70.4 41.2 65.8
a Each milk sample was diluted 5 times with PBS (pH 7.4). b Theconcentration of melamine (ppb) for each of milk sample wasquantified with a calibration curve obtained using melamine standards(3.9�125 ppb) spiked in fat free milk diluted 5-fold in PBS (pH 7.4)and not centrifuged. c Recovery. d Standard used to obtaina calibration curve in this research.
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Intra-assay and inter-assay
The results shown in Table 2 indicate that competitive ODI
CLEIA for the quantification of melamine existing in fat free
milk is precise within acceptable statistical error range (confi-
dence variable, CV). Also, the average values obtained in the
intra-assay and inter-assay shown in Table 2 indicate that
competitive ODI CLEIA has good quantitative accuracy. In
order to study the inter-assay of competitive ODI CLEIA, a total
of 63 milk samples were prepared with each set of 21 samples
containing 15.6, 62.5, or 125 ppb of melamine, respectively. All
the samples were prepared with fat free milk diluted 5-fold with
PBS buffer of pH 7.4. The prepared samples were stored in
a freezer (- 20.0 �C). Three sets (n ¼ 3) of the three milk samples
containing different concentrations of melamine were quantified
every day for a week.
Competitive binding of melamine and melamine-conjugated HRP
with anti-melamine
The results shown in Table 3 indicate that the binding between
melamine and anti-melamine in 40.0% milk sample diluted
2.5-fold with PBS is faster than that in lower percentage milk
sample than 40.0%. Thus, the concentration of melamine in
40.0% milk sample determined using competitive ODI CLEIA
looks higher than that in the lower percentage milk sample even
though each of the three types of milk samples shown in Table 3
equally contains 62.5 ppb of melamine. In other words, the
concentration of melamine existing in 5.0% milk sample deter-
mined using competitive ODI CLEIA looked to be lower than
that in a higher percentage milk sample even though all four milk
samples containing 62.5 ppb of melamine were incubated in the
well for 20 min to bind with anti-melamine in the presence of the
Table 2 Intra-assay (n ¼ 7) and inter-assay (n ¼ 21) of competitive ODICLEIA for the quantification of melamine in fat free milk not centrifuged
Spiked (ppb)
Intra-assay Inter-assay
Measured (ppb) CV (%) Measured (ppb) CV (%)
15.6 15.4 2.55 15.3 4.6862.5 62.3 1.58 62.7 5.13125.0 125.5 1.77 125.4 4.38
Table 3 Quantification of melamine (62.5 ppb) spiked in milk dilutedfrom 2.5 to 20 times with PBS (pH 7.4)
Milk (%) a CL b Concentration c Recovery (%)
5.0 1.61 19.98 31.9110.0 1.35 32.23 51.4820.0 1.00 62.50d 100.0040.0 0.79 91.62 145.31
a Fat free milk was diluted from 2.5 to 20 times with PBS (pH 7.4).b Relative CL intensity measured for each type of milk sample wasnormalized with that measured with the 5-fold diluted milk sample.c The concentration of melamine (ppb) existing in each of milk samplewas quantified with a linear calibration curve obtained using melaminestandards (3.9�125 ppb) spiked in fat free milk diluted 5-fold in PBS(pH 7.4) and not centrifuged. d Standard used to obtain a linearcalibration curve in this research.
This journal is ª The Royal Society of Chemistry 2010
same concentration of melamine-conjugated HRP. Thus,
recovery of melamine spiked in 5.0 and 10.0% milks was less than
that of melamine spiked in 20% milk. Inversely, the recovery of
melamine spiked in 40.0% milk was 145.31%.
In conclusion, the results shown in Table 3 indicate that trace
levels of melamine existing in undiluted milk can be quantified
rapidly with the reduction of incubation time necessary for
binding melamine with anti-melamine.
Quantification of melamine in undiluted milk
Based on the results shown in Table 3, we were able to rapidly
quantify relatively high concentrations of melamine spiked in
undiluted milk with short incubation time (10.0 min) for binding
melamine with anti-melamine in competitive ODI CLEIA. The
incubation time necessary for binding melamine with rabbit anti-
melamine in competitive ODI CLEIA for the quantification of
melamine in undiluted milk was a half of that in 20% milk. In
addition, the three different calibration curves shown in Fig. 5
indicate that the amount of fat existing in milk is an important
factor in determining the sensitivity of competitive ODI CLEIA
using undiluted milk. This result is the same as that observed
with diluted milk with PBS (see Table 1). Thus, Table 4 shows
that the linear dynamic range of calibration curve capable of
quantifying melamine in fat free milk is wider than that in 2.0%
milk and in whole milk, because the fat in the milk acts as an
interference in competitive ODI CLEIA. The competitive ODI
CLEIA for the quantification of melamine in undiluted milk is
faster, more sensitive, and simpler than other conventional
analytical methods1,14 such as HPLC/MS/MS and GC/MS. Thus,
we expect that the competitive ODI CLEIA can be applied for
Fig. 5 Calibration curve for the quantification of melamine in undiluted
milk containing three different % concentrations of fat.
Table 4 Dynamic range of calibration curve for the quantification ofmelamine existing in undiluted fat free milk, 2.0% milk, and whole milk
Milk Dynamic range (ppb) Equation R2
Fat free 62.5–2000 y ¼ �6169ln(x) + 60619 0.9962.0% 125–1500 y ¼ �9669ln(x) + 88803 0.996Whole 250–1000 y ¼ �13878ln(x) + 127439 0.998
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the rapid screening of melamine in undiluted milk. As shown in
Fig. 5, however, the sensitivity of competitive ODI CLEIA for
the quantification of melamine in undiluted milk isn’t as good as
that for the quantification of melamine in 20% milk with PBS.
Conclusion
Rapid and simple competitive ODI CLEIA capable of detecting
trace levels of melamine in milk was developed. The novel
competitive ODI CLEIA is more sensitive than other EIAs such as
competitive ELISA and competitive luminol CLEIA. Also, we
reported for the first time that melamine in milk sample containing
a certain amount of fat should be quantified and monitored with
a calibration curve obtained with milk containing the same
amount of fat existing in the milk sample. Using competitive ODI
CLEIA, we also confirmed that melamine in milk sample can be
directly quantified without the centrifuging of milk sample and the
dilution of milk sample with a buffer solution. Finally, we expect
that the novel CLEIA with ODI CL detection can be applied in
a wide variety of research fields such as biochemistry, clinical
chemistry, environmental science and engineering, pathology and
toxicology.
Acknowledgements
This research was performed based on the intern program (LST-
2009-5) of Luminescent MD, LLC.
2450 | Analyst, 2010, 135, 2445–2450
Notes and references
1 Y.-C. Tyan, M.-H. Yang, S.-B. Jong, C.-K. Wang and J. Shiea, Anal.Bioanal. Chem., 2009, 395, 729.
2 E. A. E. Garber, J. Food Prot., 2008, 71, 590.3 H. Lei, Y. Shen, L. Song, J. Yang, O. P. Chevallier, S. A. Haughey,
H. Wang, Y. Sun and C. T. Elliott, Anal. Chim. Acta, 2010, 665, 84.4 I.-L. Tsai, S.-W. Sun, H.-W. Liao, S.-C. Lin and C.-H. Kuo, J.
Chromatogr., A, 2009, 1216, 8296.5 J. Rima, M. Abourida, T. Xu, I. K. Cho and S. Kyriacos, J. Food
Compos. Anal., 2009, 22, 689.6 J. Yu, C. Zhang, P. Dai and S. Ge, Anal. Chim. Acta, 2009, 651, 209.7 B. Kim, L. B. Perkins, R. J. Bushway, S. Nesbit, T. Fan, R. Sheridain
and V. Greene, J. AOAC Int., 2008, 91, 408.8 L. Chen, Q. Zeng, X. Du, X. Sun, X. Zhang, Y. Xu, A. Yu, H. Zhang
and L. Ding, Anal. Bioanal. Chem., 2009, 395, 1533.9 Q. Cao, H. Zhao, L. Zeng, J. Wang, R. Wang, X. Qiu and Y. Hea,
Talanta, 2009, 80, 484.10 J. Wang, L. Jianga, Q. Chu and J. Ye, Food Chem., 2010, 121, 215.11 Z. Wang, D. Chen, X. Gao and Z. Song, J. Agric. Food Chem., 2009,
57, 3464.12 X. Wang and Y. Chen, J. Chromatogr., A, 2009, 1216, 7324.13 C. Zhai, W. Qiang, J. Sheng, J. Lei and H. Ju, J. Chromatogr., A,
2010, 1217, 785.14 J. Choi, Y.-T. Kim, J. H. Lee, Anal. Chim. Acta., submitted.15 H. Miao, S. Fan, Y.-N. Wu, L. Zhang, P.-P. Zhou, J.-G. LI, H.-
J. Chen and Y.-F. Zhao, Biomed. Environ. Sci., 2009, 22, 87.16 M. Tsunoda and K. Imai, Anal. Chim. Acta, 2005, 541, 13.17 J. H. Lee, J. C. Rock, S. B. Park, M. A. Schlautman and
E. R. Carraway, J. Chem. Soc., Perkin Trans. 2, 2002, 802.18 J. H. Lee, J. T. Je, M. A. Schlautman and E. R. Carraway, Chem.
Commun., 2003, 270.19 J. H. Lee, J. Je, J. Hur, M. A. Schlautman and E. R. Carraway,
Analyst, 2003, 128, 1257.20 V. Towne, M. Will, B. Oswald and O. J. Zhao, Anal. Biochem., 2004,
334, 290.
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