Rapid quantification of melamine in milk using competitive 1,1′-oxalyldiimidazole chemiluminescent enzyme immunoassay

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  • PAPER www.rsc.org/analyst | Analyst

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    View Article Online / Journal Homepage / Table of Contents for this issueRapid 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/c0an00396dA 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: 3.8125 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: 62.52000 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.215 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.215 Thus, the range of LODaLangley High School, 6520 Georgetown Pike, McLean, VA 22101, USAbLuminescent MD, LLC, 20140 Scholar Drive, Hagerstown, MD 21742,USA. E-mail: jhlee@luminescentmd.com; Fax: +1 301393 9092; Tel: +1301 393 9092cDepartment of Packaging Science, Clemson University, Clemson, SC29634, USA

    This journal is The Royal Society of Chemistry 2010determined using HPLC (or GC) with UV or MS spectrometry

    have been wide (10 ppb 200 ppm)1. HPLC-MS or GC-MS areused 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 ofAnalyst, 2010, 135, 24452450 | 2445

    http://dx.doi.org/10.1039/c0an00396dhttp://pubs.rsc.org/en/journals/journal/ANhttp://pubs.rsc.org/en/journals/journal/AN?issueid=AN135009

  • Fig. 1 Procedures of ELISA, Luminol CLEIA and ODI CLEIA.

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    View Article Onlinerapidly 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 8strip-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 ODI2446 | Analyst, 2010, 135, 24452450CLEIA, 25 ml of Amplex Red stock solution, 100 ml of H2O2stock 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 theThis journal is The Royal Society of Chemistry 2010

    http://dx.doi.org/10.1039/c0an00396d

  • 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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    View Article Onlinereagent holder of Lumat LB Luminometer 9507 to inject the two

    solutions into a detection cell (12 75 mm test tube) throughtwo 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] 5.0 mM,[4MImH] 25.0 mM, [H2O2] 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 3.0) of ODI CLanalytical system for the quantification of resorufin was as low as

    0.73 nM. The dynamic range of linear calibration curve (R2 0.9952) was wide (3.3 141 nM). However, the relative CLintensity of higher concentration of resorufin than 141 nM was

    self-quenched. Fig. 1 indicates that trace levels of resorufinThis journal is The Royal Society of Chemistry 2010formed 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,Analyst, 2010, 135, 24452450 | 2447

    http://dx.doi.org/10.1039/c0an00396d

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    View Article Onlinerelative 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 H2O2concentration. 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 ob...

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