Detection Of Aflatoxin M1 In Milk Research Articles

Novel dual-recognition electrochemical biosensor for the sensitive detection of AFM1 in milk

Aflatoxin M1 (AFM1) is a highly carcinogenic and toxic compound that is commonly found in dairy foods such as milk. We developed an electrochemical MIP (molecularly imprinted polymer) and aptamer (Apt) based aptasensor (Apt-Au@PEIM/AFM1/MIP–Apt/AuNPs)/GCE) with dual-recognition signal amplification to detect AFM1 in milk. The MIP–Apt/AuNP/GCE sensor was prepared by synthesizing an MIP film through the electropolymerization of resorcinol on gold nanoparticles-modified electrodes. The dropwise addition of our cApt-Au@PEIM signal amplification probe could enhance the current signal response and improve the sensitivity of the sensor to detect AFM1. The AFM1 present in milk could prevent the probe from binding to the sensor, thereby drastically reducing the current signal. Under optimal experimental conditions, the sensor exhibits a linear range of 0.01–200 nM and a limit of detection of 0.07 nM (S/N = 3). The developed dual-recognition aptasensor would provide a novel and rapid method for specific detection of AFM1 in milk.

Mach-Zehnder Interferometric Immunosensor for Detection of Aflatoxin M1 in Milk, Chocolate Milk, and Yogurt.

Aflatoxin M1 (AFM1) is detected in the milk of animals after ingestion of aflatoxin B1-contaminated food; since 2002, it has been categorized as a group I carcinogen. In this work, a silicon-based optoelectronic immunosensor for the detection of AFM1 in milk, chocolate milk, and yogurt has been developed. The immunosensor consists of ten Mach-Zehnder silicon nitride waveguide interferometers (MZIs) integrated on the same chip with the respective light sources, and an external spectrophotometer for transmission spectra collection. The sensing arm windows of MZIs are bio-functionalized after chip activation with aminosilane by spotting an AFM1 conjugate with bovine serum albumin. For AFM1 detection, a three-step competitive immunoassay is employed, including the primary reaction with a rabbit polyclonal anti-AFM1 antibody, followed by biotinylated donkey polyclonal anti-rabbit IgG antibody and streptavidin. The assay duration was 15 min with limits of detection of 0.005 ng/mL in both full-fat and chocolate milk, and 0.01 ng/mL in yogurt, which are lower than the maximum allowable concentration of 0.05 ng/mL set by the European Union. The assay is accurate (% recovery values 86.7-115) and repeatable (inter- and intra-assay variation coefficients <8%). The excellent analytical performance of the proposed immunosensor paves the way for accurate on-site AFM1 determination in milk.

Development of Gold Nanoparticles Decorated Molecularly Imprinted–Based Plasmonic Sensor for the Detection of Aflatoxin M1 in Milk Samples

Aflatoxins are a group of extremely toxic and carcinogenic substances generated by the mold of the genus Aspergillus that contaminate agricultural products. When dairy cows ingest aflatoxin B1 (AFB1)−contaminated feeds, it is metabolized and transformed in the liver into a carcinogenic major form of aflatoxin M1 (AFM1), which is eliminated through the milk. The detection of AFM1 in milk is very important to be able to guarantee food safety and quality. In recent years, sensors have emerged as a quick, low–cost, and reliable platform for the detection of aflatoxins. Plasmonic sensors with molecularly imprinted polymers (MIPs) can be interesting alternatives for the determination of AFM1. In this work, we designed a molecularly–imprinted–based plasmonic sensor to directly detect lower amounts of AFM1 in raw milk samples. For this purpose, we prepared gold–nanoparticle–(AuNP)−integrated polymer nanofilm on a gold plasmonic sensor chip coated with allyl mercaptan. N−methacryloyl−l−phenylalanine (MAPA) was chosen as a functional monomer. The MIP nanofilm was prepared using the light–initiated polymerization of MAPA and ethylene glycol dimethacrylate in the presence of AFM1 as a template molecule. The developed method enabled the detection of AFM1 with a detection limit of 0.4 pg/mL and demonstrated good linearity (0.0003 ng/mL–20.0 ng/mL) under optimized experimental conditions. The AFM1 determination was performed in random dairy farmer milk samples. Using the analogous mycotoxins, it was also demonstrated that the plasmonic sensor platforms were specific to the detection of AFM1.

Rapid, on-site, and sensitive detection of aflatoxin M1 in milk products by using time-resolved fluorescence microsphere test strip

Aflatoxin M1 (AFM1), a frequently occurring mycotoxin in milk and dairy products, causes acute and chronic poisoning. In this study, we developed a time-resolved fluorescence microsphere immunochromatographic test strip (TRFM–ICTS) combined with ultraviolet light for the rapid and quantitative detection of AFM1 in milk and its products. Polystyrene microspheres enclosing time-resolved fluorescent europium (III) [Eu(III)-TRFM] were prepared as a label and then conjugated to a monoclonal antibody. Pre-incubation was introduced for competitive recognition. Under optimal conditions, TRFM–ICTS had a linear range of 0.05–2.0 ng/mL with a 50% inhibitory concentration (IC50) of 0.204 ng/mL, which was nearly five–fold lower than those of ICTS in a traditional structure with traditional pretreatment (IC50 = 1.10 ng/mL). This test strip enhanced the sensitivity from 0.3 ng/mL (by using gold nanoparticles-strip method previously reported) to 0.019 ng/mL (by using this TRFM–ICTS method). The improved sensitivity may be due to the detection signals of immunoassay probes (with each fluorescence microsphere coupled with numbers of antibodies). TRFM–ICTS exhibited good recoveries ranging from 84.6% to 119.0% with a coefficient of variation (CV) lower than 8.31% in spiked samples, including milk powder, ultrahigh-temperature (UHT) and pasteurized milk. The results obtained using TRFM–ICTS were consistent with those obtained by ultrahigh–performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) during testing of real milk samples. In addition, TRFM–ICTS has a longer storage period, which basically meets the needs of the commercial market. Owing to its high sensitivity and practicability, the proposed TRFM–ICTS can be used as a sensitive, simple, rapid, and versatile method for the quantitative detection of AFM1 in milk.

Development of Flexible Dispense-Printed Electrochemical Immunosensor for Aflatoxin M1 Detection in Milk.

Detection of mycotoxins, especially aflatoxin M1 (AFM1), in milk is crucial to be able to guarantee food quality and safety. In recent years, biosensors have been emerging as a fast, reliable and low-cost technique for the detection of this toxin. In this work, flexible biosensors were fabricated using dispense-printed electrodes, which were functionalized with single-walled carbon nanotubes (SWCNTs) and subsequently coated with specific antibodies to improve their sensitivity. Next, the immunosensor was tested for the detection of AFM1 in buffer solution and a spiked milk sample using a chronoamperometric technique. Results showed that the working range of the sensors was 0.01 µg/L at minimum and 1 µg/L at maximum in both buffer and spiked milk. The lower limit of detection of the SWCNT-functionalized sensor was 0.02 µg/L, which indicates an improved sensitivity compared to the sensors reported so far. The sensitivity and detection range were in accordance with the limitation values imposed by regulations on milk and its products. Therefore, considering the low fabrication cost, the ease of operation, and the rapid read-out, the use of this sensor could contribute to safeguarding consumers’ health.

AFM1 Detection in Milk by Fab' Functionalized Si3N4 Asymmetric Mach-Zehnder Interferometric Biosensors.

Aflatoxins (AF) are naturally occurring mycotoxins, produced by many species of Aspergillus. Among aflatoxins, Aflatoxin M1 (AFM1) is one of the most frequent and dangerous for human health. The acceptable maximum level of AFM1 in milk according to EU regulation is 50 ppt, equivalent to 152 pM, and 25 ppt, equivalent to 76 pM, for adults and infants, respectively. Here, we study a photonic biosensor based on SiN asymmetric Mach–Zehnder Interferometers (aMZI) functionalized with Fab’ for AFM1 detection in milk samples (eluates). The minimum concentration of AFM1 detected by our aMZI sensors is 48 pM (16.8 pg/mL) in purified and concentrated milk samples. Moreover, the real-time detection of the ligand-analyte binding enables the study of the kinetics of the reaction. We measured the kinetic rate constants of the Fab’-AFM1 interaction.

Achievements and Prospects in Electrochemical-Based Biosensing Platforms for Aflatoxin M₁ Detection in Milk and Dairy Products.

Aflatoxins, which are mainly produced by Aspergillus flavus and parasiticus growing on plants and products stored under inappropriate conditions, represent the most studied group of mycotoxins. Contamination of human and animal milk with aflatoxin M1, the hydroxylated metabolite of aflatoxin B1, is an important health risk factor due to its carcinogenicity and mutagenicity. Due to the low concentration of this aflatoxin in milk and milk products, the analytical methods used for its quantification have to be highly sensitive, specific and simple. This paper presents an overview of the analytical methods, especially of the electrochemical immunosensors and aptasensors, used for determination of aflatoxin M1.

Current Immunoassay Methods for the Rapid Detection of Aflatoxin in Milk and Dairy Products.

The presence of mycotoxins in foodstuff causes serious health problems to consumers and economically affects the food industry. Among the mycotoxins, aflatoxins are very toxic and highly carcinogenic contaminants which affect the safety of many foods, and therefore endanger human health. Aflatoxin M1 (AFM1 ) found in milk results from the biotransformation of aflatoxin B1 . Many efforts have been made to control the source of AFM1 from farmers to dairy product companies. However, AFM1 escapes ordinary methods of food treatment such as cooking, sterilization, and freezing, hence it appears in milk and dairy products. The presence of high levels of AFM1 constitutes an alarming threat as milk and dairy products contain essential nutrients for human health, especially for infants and children. For this reason, there is a pressing need for developing a fast and reliable screening method for detecting trace aflatoxins in food. Several analytical methods based on high-performance liquid chromatography (HPLC) and mass spectroscopy have been used for aflatoxin detection; however, they are expensive, time-consuming, and require many skills. Recently, immunoassay methods, including enzyme-linked immunosorbent assay (ELISA), immunosensors, and lateral flow immunoassay (LFIA), have been preferred for food analysis because of their improved qualities such as high sensitivity, simplicity, and capability of onsite monitoring. This paper reviews the new developments and applications of immunoassays for the rapid detection of AFM1 in milk.

Sensitive and rapid detection of aflatoxin M1 in milk utilizing enhanced SPR and p(HEMA) brushes

The rapid and sensitive detection of aflatoxin M1 (AFM1) in milk by using surface plasmon resonance (SPR) biosensor is reported. This low molecular weight mycotoxin is analyzed using an indirect competitive immunoassay that is amplified by secondary antibodies conjugated with Au nanoparticles. In order to prevent fouling on the sensor surface by the constituents present in analyzed milk samples, an interface with poly(2-hydroxyethyl methacrylate) p(HEMA) brush was employed. The study presents a comparison of performance characteristics of p(HEMA)-based sensor with a regularly used polyethylene glycol-based architecture relying on mixed thiol self-assembled monolayer. Both sensors are characterized in terms of surface mass density of immobilized AFM1 conjugate as well as affinity bound primary and secondary antibodies. The efficiency of the amplification strategy based on Au nanoparticle is discussed. The biosensor allowed for highly sensitive detection of AFM1 in milk with a limit of detection (LOD) as low as 18pgmL−1 with the analysis time of 55min.

Microfluidic Sample Preparation Methods for the Analysis of Milk Contaminants

In systems for food analysis, one of the major challenges is related to the quantification of specific species into the complex chemical and physical composition of foods, that is, the effect of “matrix”; the sample preparation is often the key to a successful application of biosensors to real measurements but little attention is traditionally paid to such aspects in sensor research. In this critical review, we discuss several microfluidic concepts that can play a significant role in sample preparation, highlighting the importance of sample preparation for efficient detection of food contamination. As a case study, we focus on the challenges related to the detection of aflatoxinM1in milk and we evaluate possible approaches based on inertial microfluidics, electrophoresis, and acoustic separation, compared with traditional laboratory and industrial methods for phase separation as a baseline of thrust and well-established techniques.

Development of “Field Level” Chromogenic Assay for Aflatoxin M1 Detection in Milk

Out of eighteen Bacillus spp. screened for aflatoxin M1 (AFM1) sensitivity Bacillus brevis MTCC 3136 was found most sensitive followed by B. megaterium ATCC 9885 at 0.5 ppb (Codex limit). Bacillus brevis 3136 was found weakly positive for lipase activity while B. megaterium 9885 showed significant acetyl esterase activity. Maximum sporulation was achieved in Arret and Krishbaum sporulation agar (90.63%) followed by tryptone yeast extract broth, tryptone glucose yeast extract broth, minimal media and sucrose salt medium. A field level chromogenic assay for AFM1 in milk has been developed using spores of B. megaterium 9885 grown in Arret and Krishbaum sporulation agar. Spores were added with milk containing germinants (dextrose, sucrose, tryptone) and chromogenic substrate followed by incubation at 37EšC for 75 ± 5 min. The colour change from colourless to sky blue colour within 75 ± 5 min is indication of spore germination followed by release of enzyme and its action on chromogenic substrate. However, no colour change indicates that milk is positive for AFM1 at 0.5 ppb. The developed assay was validated with raw, pasteurized and dried milks with a 96% correlation by comparing with radioimmunoassay i.e., Charm 6602 assay. The reliability of assay has also been checked by spiking AFM1 in milk. The developed assay was also validated by certified reference materials of heavy metals and pesticides for checking the cross reactivity of these with our developed assay. The developed chromogenic assay is cost effective, portable, having no interference of antibiotics, detergents, sanitizers, heavy metals and pesticides. The developed assay can be used in dairy farm and laboratories with fewer resources for AFM1 detection in milk.

Long range surface plasmon-enhanced fluorescence spectroscopy for the detection of aflatoxin M1 in milk

A novel biosensor for the highly sensitive detection of aflatoxin M1 (AFM1) in milk was developed. This biosensor is based on surface plasmon-enhanced fluorescence spectroscopy (SPFS) which was advanced through the excitation of long range surface plasmons (LRSPs). In SPFS, the binding of fluorophore-labeled molecules to the sensor surface is probed with surface plasmons (SPs) and the emitted fluorescence light is detected. This approach takes advantages of the enhanced intensity of electromagnetic field occurring upon the resonant excitation of SPs which directly increases the fluorescence signal. For the detection of AFM1, LRSP-enhanced fluorescence spectroscopy was combined with an inhibition immunoassay in which a derivative of AFM1 was immobilized on the sensor surface and antibodies against AFM1 were used as recognition elements. The developed biosensor allowed for the detection of AFM1 in milk within 53min at concentrations as low as 0.6pgmL−1. The achieved limit of detection was about two orders of magnitude lower than the maximum AFM1 residue level in milk stipulated by the European Commission legislation.

Production of Sensitive Monoclonal Antibodies to Aflatoxin B1and Aflatoxin M1 and Their Application to ELISA of Naturally Contaminated Foods

Two new hybridoma Cell lines capable of secreting sensitive monoclonal antibodies for aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1), were produced by fusing NS-1 myeloma cells with spleen cells of BALB/c female mice immunized with AFB1- and AFM1-carboxymethyloxime bovine serum albumin conjugates, respectively. Detection limits for these antibodies in the direct enzyme-linked immunosorbent assay (ELISA) were 0.5 ng/ml for AFB1 and 0.25 ng/ml for AFM1 Concentrations of AFB1 analogs (ng/ml) required to inhibit 50% binding of AFB1,-perioxidase conjugate to AFB1 monoclonal antibody solid phase in direct ELISA were: AFB1, 2.6; AFB2, 13; AFG1, 8; AFB2, 15; AFM1, 23. Analog concentrations (ng/ml) required to inhibit 50% binding of AFB1,-perioxidase conjugate to AFM1 monoclonal antibody solid phase were: AFM1,0.8; AFM2, 700; AFB1, 0.5; AFB2, 35; AFB2a, >10,000; AFG1, 12; AFG2a, 12; AFP1, 16; and AFQ1, 9.2. These new monoclonal antibodies were applicable to both the ELISA detection AFB1 in corn, cottonseed, cottonseed meal, and mixed feed following a simple extraction in 55% methanol as well as the direct detection of AFM1 in milk.