Determination Of Aflatoxin M1 In Milk Research Articles

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.

Selection and Characterization of DNA Aptamers for Constructing Aptamer-AuNPs Colorimetric Method for Detection of AFM1.

Aflatoxin M1 (AFM1), one of the most toxic mycotoxins, is a feed and food contaminant of global concern. To isolate the ssDNA aptamer of AFM1, synthesized magnetic graphene oxide nanomaterials, 12 rounds of subtractive systematic evolution of ligands by exponential enrichment (SELEX) selection were carried out. As a result, 24 candidate aptamers were selected, and their sequence similarity exceeded 97%. Their binding affinity and specificity were further examined by fluorescence and biofilm interferometry (BLI) methods. One aptamer (Apt-5) against AFM1 with a high affinity and specificity was isolated and demonstrated to be the optimal aptamer, whose dissociation constant reached the nanomolar level, Kd = 8.12 ± 1.51 nM. Additionally, molecular docking studies were used to predict the possible binding sites and mechanisms of the two. Based on Apt-5, an unlabeled aptamer-AuNPs colorimetric method was established to detect AFM1 in milk with a linear range of 0.078–10 ng/mL, and the actual detection limit was 0.078 ng/mL. These results demonstrated that this detection technique could be useful for the quantitative determination of AFM1 in milk and dairy products.

Determination of Aflatoxin M1 in Raw Milk Using an HPLC-FL Method in Comparison with Commercial ELISA Kits-Application in Raw Milk Samples from Various Regions of Greece.

The highly toxic Aflatoxin M1 (AFM1) is most often detected in milk using an Enzyme-Linked-Immunosorbent Assay (ELISA) for screening purposes, while High-Performance Liquid Chromatography with Fluorescence Detector (HPLC-FL) is the reference method used for confirmation. The aim of the present study was the comparison between three commercially available ELISA kits and a newly developed HPLC-FL method for the determination of the AFM1 in milk samples. The developed HPLC-FL method was validated for the AFM1 and Aflatoxin M2 (AFM2), determining the accuracy, precision, linearity, decision limit, and detection capability with fairly good results. All three ELISA kits were also validated and showed equally good performance with high recovery rates. Moreover, the Limit Of Detection (LOD) and Limit Of Quantification (LOQ) values were found to be significantly lower than the Maximum Residue Limit (MRL) (50 ng kg−1). After the evaluation of all three commercial kits, the ELISA kit with the optimum performance along with the HPLC method was used for the determination of AFM1 in raw cow’s, goat’s, and sheep’s milk samples (396) obtained from producers in different regions of Greece. The evaluation of both methods showed that this ELISA kit could be considered as a faster and equally reliable alternative method to HPLC in routine analysis for the determination of AFM1 in milk.

Aflatoxin M1 in milk and milk products: a short review

Aflatoxin M1 (AFM1) is associated with carcinogenicity, genotoxicity, mutagenicity, and teratogenicity and as a result, represents a human health problem worldwide. This review will detail the toxicity, analytical methodology, occurrence, and prevention and control of AFM1 in milk and milk products. The probable daily intakes (PDI) per bodyweight (bw) worldwide ranged from 0.002 to 0.26 ng/kg bw/day for AFM1. Nevertheless, the high occurrence of AFM1 demonstrated in this review establishes the need for monitoring to reduce the risk of toxicity to humans. The recommended extraction method of AFM1 from milk is liquid-liquid with acetonitrile because of the acceptable recoveries (85-97%), compatibility with the environment, and cleanest extracts. The recommended analytical technique for the determination of AFM1 in milk is the high performance-liquid chromatography-fluorescence detector (HPLC-FLD), achieving a 0.001 µg/kg detection limit. The HPLC-FLD is the most common internationally recognised official method for the analysis of AFM1 in milk. The suggested extraction and analytical method for cheese is dichloromethane (81-108% recoveries) and ELISA, respectively. This review reports the projected worldwide occurrence of AFM1 in milk of 2010-2015. Of the 7,841 samples, 5,873 (75%) were positive for AFM1, 26% (2,042) exceeded the maximum residue levels (MRL) of 0.05 µg/kg defined by the European Union and 1.53% (120) exceeded the MRL of 0.5 µg/kg defined by the US Food and Drug Administration. The most effective way of preventing AFM1 occurrences is to reduce contamination of AFB1 in animal feed using biological control with atoxigenic strains ofAspergillus flavus, proper storage of crops, and the addition of binders to AFB1-contaminated feed. Controllable measures include the addition of binders and use of biological transforming agents such as lactic acid bacteria applied directly to milk. Though the one accepted method for the control of AFM1 in milk and milk products is the enforcement of governmental MRL.

Development of an impedimetric aptasensor for the determination of aflatoxin M1 in milk

An aptasensor was designed for the determination of aflatoxin M1 (AFM1) in milk based on DNA-aptamer recognition and electrochemical impedance spectroscopy detection. A hexaethyleneglycol-modified 21-mer oligonucleotide was immobilized on a carbon screen-printed electrode through carbodiimide immobilization, after diazonium activation of the sensing surface. Cyclic voltammetry and electrochemical impedance spectroscopy in the presence of ferri/ferrocyanide redox probe were used to characterize each step of the aptasensor development. Aptamer-AFM1 interaction induced an increase in electron-transfer resistance, allowing the determination of AFM1 in buffer in the range 2–150ng/L (LOD=1.15ng/L). Application to milk analysis showed that a preliminary treatment was mandatory. A simple filtration through a 0.2µm PTFE membrane allowed determination of AFM1 in milk for concentrations ranging from 20 to 1000ng/kg. These performances are compatible with the AFM1 levels set in European Union for milk and dairy products for adults (50ng/kg) and infants (25ng/kg).

Rapid analysis of aflatoxin M1 in milk using dispersive liquid–liquid microextraction coupled with ultrahigh pressure liquid chromatography tandem mass spectrometry

A simple, rapid, and sensitive method based on simultaneous protein precipitation and extraction of aflatoxin M1 (AFM1) followed by dispersive liquid-liquid microextraction (DLLME) and ultrahigh pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) analysis was developed for the determination of AFM1 in milk samples. In order to precipitate the proteins and extract AFM1 from milk, a sample pretreatment using acetonitrile and NaCl as the extraction/denaturant solvent and salting-out agent, respectively, was optimised. Subsequently, the acetonitrile (upper) phase, containing AFM1, was used as the disperser solvent in DLLME, and extractant (chloroform) and water were added in turn to the extract to perform the DLLME process. The main parameters affecting the extraction efficiency of the whole analytical procedure, such as acetonitrile volume, amount of salt, type and volume of extractant and water volume, were carefully optimised by experimental design. Under optimum conditions, the developed method provides an enrichment factor of 33 and detection and quantification limits (0.6 and 2.0 ng kg(-1), respectively) below the maximum levels imposed by current regulations for AFM1 in milk and infant milk formulae. Recoveries (61.3-75.3%) and repeatability (RSD < 10, n = 3), tested in different types of milk at four AFM1 levels, met the performance criteria required by EC Regulation No. 401/2006. Moreover, the matrix effect on the signal intensity of the analyte was negligible. The proposed method provides a rapid extraction and an accurate determination of AFM1 in milk and formula milk using a simple and inexpensive sample preparation procedure.

An electrochemical immunosensor for aflatoxin M1 determination in milk using screen-printed electrodes

The production and assembling of disposable electrochemical AFM1 immunosensors, which can combine the high selectivity of immunoanalysis with the ease of the electrochemical probes, has been carried out. Firstly immunoassay parameters such as amounts of antibody and labelled antigen, buffer and pH, length of time and temperature of each steps (precoating, coating, binding and competition steps) were evaluated and optimised in order to set up a spectrophotometric enzyme-linked immunosorbent assay (ELISA) procedure. This assay exhibited a working range between 30 and 160 ppt in a direct competitive format. Then electrochemical immunosensors were fabricated by immobilising the antibodies directly on the surface of screen-printed electrodes (SPEs), and allowing the competition to occur between free AFM1 and that conjugated with peroxidase (HRP) enzyme. The electrochemical technique chosen was the chronoamperometry, performed at -100 mV. Furthermore, studies of interference and matrix effects have been performed to evaluate the suitability of the developed immunosensors for the analysis of aflatoxin M1 directly in milk. Results have shown that using screen-printed electrodes aflatoxin M1 can be measured with a detection limit of 25 ppt and with a working range between 30 and 160 ppt. A comparison between the spectrophotometric and electrochemical procedure showed that a better detection limit and shorter analysis time could be achieved using electrochemical detection.

Evaluation of measurement uncertainties of an analytical method for the determination of aflatoxin M1 in milk and milk powder

The mycotoxin aflatoxin M1 (AfM1) is a serious food safety hazard for which the European Commission has already established a maximum permissible level of 0.05 μg/kg AfM1 in milk and products thereof. For control analysis laboratories are increasingly asked to submit full uncertainties of their analytical results.The evaluation of measurement uncertainties of an analytical method for the determination of AfM1 in milk and milk powder on the basis of 'in-house' validation data in compliance with the 'Guide to the Expression of Uncertainty in Measurement (GUM)' [1] and the 'EURACHEM Guide' [2] is described. A similar approach will be used to assess the performance of methods employed by laboratories participating in the certification of reference materials for AfM1 in milk powder.

Flow injection monitoring of aflatoxin M1 in milk and milk preparations using filter-supported bilayer lipid membranes.

This work describes a technique for the rapid and sensitive electrochemical flow injection monitoring of aflatoxin M1 (AFM1) using stabilized systems of filter-supported bilayer lipid membranes (BLMs). Injections of AFM1 were made into flowing streams of a carrier electrolyte solution, and a transient current signal with a duration of seconds reproducibly appeared less than 10 s after exposure of the lipid membranes to the toxin. The magnitude of this signal was linearly related to the concentration of AFM1, with detection limits at the subnanomolar level. The mechanism of signal generation was investigated by differential scanning calorimetric experiments. The technique was applied for the rapid flow injection determination of AFM1 in milk and milk preparations. The effect of potent interferences such as proteins and lipids was investigated, and the results show that interferences from these milk constituents can be eliminated by modulation of the flow rate of the carrier solution so as not to allow adsorption of these compounds in BLMs. AFM1 could be determined in continuous flowing systems with a rate of at least 4 samples min-1. Repetitive cycles of injection of AFM1 showed no signal degradation during each cycle.

Citrus Artifact Interference in Aflatoxin M1 Determination in Milk

Dried citrus waste was fed to dairy cows, their milk was extracted, and aflatoxin M1 was quantitated by using both high pressure liquid chromatography (HPLC) and thin layer chromatography (TLC). Results indicate that a compound from the citrus waste, which is excreted into the milk, interferes with the HPLC determination of aflatoxin M1 in milk and causes a false positive test. This interference can be overcome by using TLC with proper selection of developing solvents.