Highly sensitive and simultaneous detection of melamine and aflatoxin M1 in milk products by multiplexed planar waveguide fluorescence immunosensor (MPWFI)

doi:10.1016/j.foodchem.2015.08.109

Food Chemistry

Volume 197, Part A, 15 April 2016, Pages 359-366

https://doi.org/10.1016/j.foodchem.2015.08.109 Get rights and content

Highlights

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A multiplexed planar waveguide fluorescence immunosensor (MPWFI) has been developed.
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Rapidly and simultaneously detect melamine and aflatoxin M1 with high recoveries in short time.
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Cross-reactivity against analytes and some compounds structurally similar to analytes is little.
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Limit of simultaneous detection of two analytes meet the requirements set by the WHO.

Abstract

Mycotoxins and industrial chemicals, such as aflatoxin M1 and melamine, now commonly exist in milk and cause potential health risks. This study presents an indirect competitive immunoassay through multiplex planar waveguide fluorescence immunosensor (MPWFI) for rapid, sensitive, and simultaneous detection and quantification of aflatoxin M1 and melamine by applying the principle of immunoreaction and total internal reflect fluorescent.

Double-channel standard curves with appropriate logistic correlation (R² > 0.99) were plotted, respectively. The working ranges (0.073–0.400 ng/mL and 26.38–270.00 ng/mL, respectively) were calculated, as well as the limit of detection (0.045 and 13.37 ng/mL, respectively), when two analytes were simultaneously detected. Both results satisfied the requirements for the maximum amount set by the WHO, which illustrated that the current method was better than some other standard methods. The recovery rates in the actual samples ranged from 85% to 103%, with relative standard deviations between 1.3% and 6.5%, which indicated high accuracy and repeatability.

Introduction

Aflatoxins are highly toxic and carcinogenic metabolites produced by Aspergillus parasiticus and Aspergillus flavus, which may contaminate various agricultural commodities and animal foodstuffs. Aflatoxin M1 (AFM1) can be found as hydroxylated metabolite of aflatoxin B1 (AFB1) in urine, blood, milk, and internal organs of animals that have ingested AFB1 contaminated feed. Currently, available research results demonstrate that aflatoxin M1 can also be present in a wide range of milk-derived or products containing milk, such as cheese, yogurt, cream, and chocolate (Pei, Zhang, Eremin, & Lee, 2009), because of the stability of aflatoxins during processes involved in preparation of these commodities. However, unlike aflatoxin M1, melamine (1,3,5-triazine-2,4,6-triamine) is a nitrogen-based industrial chemical used in the manufacture of durable plastics, melamine–formaldehyde resins, and flame-retardants, which has been illegally added in feed stuffs and milk products in some developing countries in the recent years to artificially boost crude protein value, which has caused or may cause illnesses and even death (Brown et al., 2007). Given the hepatotoxic and carcinogenic effects of aflatoxin M1 and the oral acute toxicity of melamine when unintentionally and excessively ingested, many countries have regulated the levels of aflatoxin M1 and melamine in milk. In particular, the World Health Organization has set maximum permissible levels of 0.5 ppb (0.25 ppb for baby food) for aflatoxin M1 and 2.5 ppm (1.0 ppm for infant formula) for melamine. These guidelines were also followed by the Food and Drug Administration (FDA) of the USA and the Chinese government after several accidents related to milk.

Currently, routine analysis of melamine contaminated milk powder samples are mostly performed by gas chromatography with mass spectrometry (GC–MS) (Miao et al., 2010), high performance liquid chromatography (HPLC) (Zhong, Zhang, Zhang, Liu, & Wang, 2011) or in tandem with mass spectrometry (LC–MS) (Goscinny et al., 2011, MacMahon et al., 2012), enzyme-linked immunosorbent assay (ELISA) (Lei et al., 2011, Pei et al., 2009, Zhou et al., 2012), capillary electrophoresis (CE) (Huang et al., 2012, Wen et al., 2010), Fourier-transform infrared (FTIR) spectroscopy (Mauer, Chernyshova, Hiatt, Deering, & Davis, 2009), and Raman spectrometry (Almeida et al., 2011, Cheng et al., 2010); while thin-layer chromatography (TLC), self-assembled metal-supported bilayer lipid membranes (s-BLMs) (Siontorou, Nikolelis, Miernik, & Krull, 1998), and ultrasensitive chemiluminescent enzyme immunoassay (Magliulo et al., 2005) are reported for routine analysis of aflatoxin M1 for the same milk powder samples. These analytical methods have high sensitivity and good accuracy, but are time-consuming and involve complicated techniques, require highly trained personnel in specialized laboratories, extensive preparation steps, and bulky and expensive instruments. Therefore, alternative methods have been reported to simplify and expedite the detection of melamine and aflatoxin M1, such as colorimetric (Guan et al., 2013, Li et al., 2010) and fluorescence (Le, Yan, Xu, & Hao, 2013). Colorimetric and fluorescence methods based on Au NPs or Ag NPs suffer from low selectivity and non-applicability in real time detection of melamine. Therefore, rapid, sensitive, portable, on-site, and low-cost methods to analyze melamine and aflatoxin M1 are in great demand.

To date, the immunoassay-based biosensor systems have proven to be the most widely reported for biosensor applications, combining with various techniques to detect the presence of a target analyte (Paniel et al., 2010, Ricci et al., 2007). Among the immunoassay-based biosensor systems, the fluorescent immunosensor has been widely used in food analysis and monitoring of pollutant (Guo et al., 2014, Hao et al., 2014). Surface plasmon resonance has been reported for detection of melamine (Fodey et al., 2011, Wu et al., 2013) and aflatoxin M1 (Wang, Dostálek, & Knoll, 2009). To date, most of the immunosensor target only a single compound. The imaging surface plasmon resonance (iSPR) platform (Raz, Bremer, Haasnoot, & Norde, 2009) and the wavelength-interrogated optical system (WIOS) (Adrian et al., 2009) have been reported for multiplex detection of antibiotic residues, which is known as the optical waveguide light-mode spectroscopy (OWLS) (Adányi et al., 2007) suspension array (Ying et al., 2013). In addition, immune chip (Ying et al., 2012) has also been developed for simultaneous detection of mycotoxins. These methods however are limited in detecting multiplex compounds in a single class, while some need more than four hours for the analysis, which are not suitable for an on-site, low cost, and real time detection, as opposed to our present study.

In this study, the multiplexed planar waveguide fluorescence immunosensor (MPWFI) was used to determine the possibility of simultaneous assays for two compounds of different families. The objective of achieving spatially-resolved excitation and collection of fluorescence from fluorescently-labeled antibodies locally-bound at a planar interface can be met by the evanescent field excitation of a fluorophore. Excitation light was guided by total internal reflection within transducer structure resulting in an evanescent wave, which allowed the excitation of fluorophore bound to the transducer surface. The total internal reflection fluorescence (TIRF) principle allowed selective detection of surface bound fluorophore, and therefore, on-line monitoring of binding events was superior to that with direct illumination of the active area of transducers. A planar transducer was also preferred to a fiber-based system, being manufactured as an integrated part of fluidics systems (Tschmelak et al., 2005).

Meanwhile, a fast, sensitive, reliable, and simultaneous indirect competitive immunoassay was adopted to detect two low molecular weight analytes (melamine and aflatoxin M1) from different families; one is an industrial chemical while the other is a mycotoxin. The sensitivity of the method in measuring melamine and aflatoxin simultaneously both in buffer and milk products based on PWFI were demonstrated. The stability and reusability of sensing surface and its cross-reactivity were also evaluated. This method was successfully applied to determine the melamine and aflatoxin M1 in the cow milk formulas for infants. The schematic of the detection procedures is illustrated in Scheme 1.

Section snippets

Reagents and materials

The 3-mercaptopropyl-trimethoxysilane (MTS), N-(4-maleimidobutyryloxy) succinimide (GMBS), bovine serum albumin (BSA), 1-ethyl-3-(-3dimethylamino-propyl), aflatoxin M1, aflatoxin B1, and aflatoxin B2 were purchased from Sigma–Aldrich (Steinheim, Germany). All the other reagents, unless specified otherwise, were purchased from Beijing Chemical Agents. Approximately 1 mg/mL melamine and 1 μg/mL stock solutions were prepared in mixture (1:9, v/v) of methanol and deionized water (18 MΩ) and stored at 4

MPWFI-based immunoassays for simultaneous detection

Simultaneous detection of melamine and aflatoxin M1 were achieved by integrating double immunoassays on a single sensor chip. The immunoassays were implemented in an indirect competitive format. Each analyte had to compete with the antigen coating for the corresponding antibody-bioreceptor binding sites. Each measurement cycle lasted for 20 min (including the regeneration steps). After four minutes of incubation, the mixture was allowed to flow onto the chip surface. During the five-minute

Conclusion

The present work had established an effective monitoring approach based on multiplex planar waveguide fluorescence immunosensors (MPWFI), which provided a new method for simultaneously determining different compounds such as melamine and aflatoxin M1 in milk products within 20 min. The entire detection system required less sample volume because of the indirect competitive immunoassay, and provided both qualitative and quantitative data with high sensitivity. However, this technique required

Acknowledgments

This research was supported by the Major Scientific Equipment Development Project of China (2012YQ030111). We would also like to thank the special funding support of State Key Joint Laboratory of Environment Simulation and Pollution Control (12L01ESPC).

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Analytical MethodsHighly sensitive and simultaneous detection of melamine and aflatoxin M1 in milk products by multiplexed planar waveguide fluorescence immunosensor (MPWFI)

Highlights

Abstract

Introduction

Section snippets

Reagents and materials

MPWFI-based immunoassays for simultaneous detection

Conclusion

Acknowledgments

Development of immunosensor based on OWLS technique for determining aflatoxin B1 and ochratoxin A

Biosensors and Bioelectronics

Wavelength-interrogated optical biosensor for multi-analyte screening of sulfonamide, fluoroquinolone, β-lactam and tetracycline antibiotics in milk

TrAC Trends in Analytical Chemistry

Fourier-transform Raman analysis of milk powder: A potential method for rapid quality screening

Journal of Raman Spectroscopy

Development and application of solvent-free extraction for the detection of aflatoxin M1 in dairy products by enzyme immunoassay

Journal of Agricultural and Food Chemistry

Survey of the occurrence of Aflatoxin M1 in ovine milk by HPLC and its confirmation by MS

Molecular Nutrition & Food Research

Outbreaks of renal failure associated with melamine and cyanuric acid in dogs and cats in 2004 and 2007

Journal of Veterinary Diagnostic Investigation

Liquid chromatography/tandem mass spectrometric confirmatory method for determining aflatoxin M1 in cow milk: Comparison between electrospray and atmospheric pressure photoionization sources

Journal of Chromatography A

Screening melamine adulterant in milk powder with laser Raman spectrometry

Journal of Food Composition and Analysis

Development of surface plasmon resonance-based immunoassay for aflatoxin B1

Journal of Agricultural and Food Chemistry

Surface plasmon resonance immunosensor for human cardiac troponin T based on self-assembled monolayer

Journal of Pharmaceutical and Biomedical Analysis

Development of an optical biosensor based immunoassay to screen infant formula milk samples for adulteration with melamine

Analytical Chemistry

Rapid analysis of melamine residue in milk, milk products, bakery goods and flour by ultra-performance liquid chromatography/tandem mass spectrometry: From food crisis to accreditation

Food Control

Label-free colorimetric sensing of melamine based on chitosan-stabilized gold nanoparticles probes

Food Control

Highly sensitive and rapid detection of melamine in milk products by planar waveguide fluorescence immunosensor (PWFI)

Sensors and Actuators B: Chemical

Melamine detection in dairy products by using a reusable evanescent wave biosensor

Sensors and Actuators B: Chemical

Capillary electrochromatography–mass spectrometry determination of melamine and related triazine by-products using poly(divinyl benzene-alkene-vinylbenzyl trimethylammonium chloride) monolithic stationary phases

Analytica Chimica Acta

Determination of melamine in milk-based products and other food and beverage products by ion-pair liquid chromatography–tandem mass spectrometry

Analytica Chimica Acta

A novel colloidal gold-based lateral flow immunoassay for rapid simultaneous detection of cyromazine and melamine in foods of animal origin

Food Chemistry

Development of a specifically enhanced enzyme-linked immunosorbent assay for the detection of melamine in milk

Molecules

Visual detection of melamine in raw milk using gold nanoparticles as colorimetric probe

Food Chemistry

Analytical Methods
Highly sensitive and simultaneous detection of melamine and aflatoxin M1 in milk products by multiplexed planar waveguide fluorescence immunosensor (MPWFI)