DNA-Polylactide Modified Biosensor for Electrochemical Determination of the DNA-Drugs and Aptamer-Aflatoxin M1 Interactions.

DNA sensors were assembled by consecutive deposition of thiacalix[4]arenes bearing oligolactic fragments, poly(ethylene imine), and DNA onto the glassy carbon electrode. The assembling of the layers was monitored with scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. The configuration of the thiacalix[4]arene core determined self-assembling of the polymeric species to the nano/micro particles with a size of 70-350 nm. Depending on the granulation, the coatings show the accumulation of a variety of DNA quantities, charges, and internal pore volumes. These parameters were used to optimize the DNA sensors based on these coatings. Thus, doxorubicin was determined to have limits of detection of 0.01 nM (cone configuration), 0.05 nM (partial cone configuration), and 0.10 nM (1,3-alternate configuration of the macrocycle core). Substitution of native DNA with aptamer specific to aflatoxin M1 resulted in the detection of the toxin in the range of 20 to 200 ng/L (limit of detection 5 ng/L). The aptasensor was tested in spiked milk samples and showed a recovery of 80 and 85% for 20 and 50 ng/L of the aflatoxin M1, respectively.

A Novel Graphene Oxide-Based Aptasensor for Amplified Fluorescent Detection of Aflatoxin M1 in Milk Powder.

In this paper, a rapid and sensitive fluorescent aptasensor for the detection of aflatoxin M1 (AFM1) in milk powder was developed. Graphene oxide (GO) was employed to quench the fluorescence of a carboxyfluorescein-labelled aptamer and protect the aptamer from nuclease cleavage. Upon the addition of AFM1, the formation of an AFM1/aptamer complex resulted in the aptamer detaching from the surface of GO, followed by the aptamer cleavage by DNase I and the release of the target AFM1 for a new cycle, which led to great signal amplification and high sensitivity. Under optimized conditions, the GO-based detection of the aptasensor exhibited a linear response to AFM1 levels in a dynamic range from 0.2 to 10 µg/kg, with a limit of detection (LOD) of 0.05 µg/kg. Moreover, the developed aptasensor showed a high specificity towards AFM1 without interference from other mycotoxins. In addition, the technique was successfully applied for the detection of AFM1 in infant milk powder samples. The aptasensor proposed here offers a promising technology for food safety monitoring and can be extended to various targets.

Label-Free and Redox Markers-Based Electrochemical Aptasensors for Aflatoxin M1 Detection.

We performed a comparative analysis of the sensitivity of aptamer-based biosensors for detection mycotoxin aflatoxin M1 (AFM1) depending on the method of immobilization of DNA aptamers and method of the detection. Label-free electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) for ferrocene labeled neutravidin layers were used for this purpose. Amino-modified DNA aptamers have been immobilized at the surface of polyamidoamine dendrimers (PAMAM) of fourth generation (G4) or biotin-modified aptamers were immobilized at the neutravidin layer chemisorbed at gold surface. In the first case the limit of detection (LOD) has been determined as 8.47 ng/L. In the second approach the LOD was similar 8.62 ng/L, which is below of allowable limits of AFM1 in milk and milk products. The aptasensors were validated in a spiked milk samples with good recovery better than 78%. Comparative analysis of the sensitivity of immuno- and aptasensors was also performed and showed comparable sensitivity.

Lateral flow immunoassay integrated with competitive and sandwich models for the detection of aflatoxin M1 and Escherichia coli O157:H7 in milk.

Pathogens, mycotoxins, or antibiotics may exist in a food sample. Micro- and macromolecular substances must be detected quickly. A rapid and convenient lateral flow immunoassay (LFI) integrated with competitive and sandwich models was developed to detect micro- and macromolecular substances. In this study, aflatoxin M1 (AFM1) and Escherichia coli O157:H7 were selected as the micro- and macromolecular substances, respectively. Two test lines in the LFI test strip were evaluated to detect AFM1 and E. coli O157:H7 by competitive and sandwich models. Results showed that the limits of detection for detecting AFM1 and E. coli O157:H7 were 50 pg·mL-1 and 1.58 × 104 cfu·mL-1, respectively. The whole assay time was 30 min. The recoveries of gold nanoparticle-LFI ranged from 78.0 to 111.6% with coefficients of variation in the range of 3.9 to 8.5% for the detection of AFM1. For the detection of E. coli O157:H7, the range of recoveries was from 70.1 to 89.6% with coefficients of variation ranging from 4.9 to 13.0%. This study not only tested sensitivity and specificity, but also was a systematic study of location of 2 test lines of the LFI test strip integrated with competitive and sandwich models.

Aptamer functionalized magnetic nanoparticles for effective extraction of ultratrace amounts of aflatoxin M1 prior its determination by HPLC.

Aptamers, due to the inherently high selectivity towards target analytes, are promising candidate for surface modification of the nanoparticles. Therefore, aptamer-functionalized magnetic nanoparticles (AMNPs) was prepared and used to develop a magnetic solid-phase extraction procedure for clean-up of milk and dairy products samples before measuring the aflatoxin M1 (AFM1) contents by the high-performance liquid chromatography. The prepared sorbent was characterized by different instrumental methods such as FT-IR, FESEM, TEM, EDX and AGFM. The AMNPs was used in extraction and pre-concentration of ultratrace amounts AFM1 from local milk samples. The amount of sorbent, elution volume, extraction time, and salt addition were optimized. Based on the results, calibration plot is linear over the 0.3 to 1 ng·L-1 and 5 to 50 ng·L-1 AFM1 concentration ranges. The limits of detection of the developed method were obtained 0.2 ng·L-1 which is the smallest value that has been reported up to now. The results show that this new superior sorbent has a large potential to simplify the complex matrix of the samples and can used for detection, preconcentration and accurate determination of ultratrace amounts of the AFM1 from milk and dairy products.

Electrochemical monitoring of aflatoxin M1 in milk samples using silver nanoparticles dispersed on α-cyclodextrin-GQDs nanocomposite.

Aflatoxins are potential food pollutants produced by fungi. One of important toxins is aflatoxin M1 (AFM1). A great deal of concern is associated with AFM1 toxicity. In the present study, an innovative electrochemical interface for quantitation of AFM1 based on ternary signal amplification strategy was fabricated. In this work, silver nanoparticles was electrodeposited onto green and biocompatible nanocomposite containing α-cyclodextrin as conductive matrix and graphene quantum dots as amplification element. Therefore, a multilayer film based on α-cyclodextrin, graphene quantum dots, and silver nanoparticles was exploited to develop a highly sensitive electrochemical sensor for detection of AFM1. Fully electrochemical methodology was used to prepare a transducer on a glassy carbon electrode, which provided a high surface area toward sensitive detection of AFM1. The surface morphology of electrode surface was characterized by high-resolution field emission scanning electron microscope. The proposed sensing platform provides a simple tool for AFM1 detection. Under optimized condition, the calibration curve for AFM1 concentration was linear in 0.015mM to 25mM with low limit of quantification of 2µM. The practical analytical utility of the modified electrode was illustrated by determination of AFM1 in unprocessed milk samples.

Determination of aflatoxin M1 in milk and dairy products using high performance liquid chromatography-fluorescence with post column photochemical derivatization.

The determination of aflatoxin M1 in milk using high performance liquid chromatography with photochemical post-column derivatization and fluorescence detection is described. The samples were first extracted and clean-up using the immunoaffinity AFLATEST column originally targeted for aflatoxins B1, B2, G1 and G2. The separation of aflatoxin M1 were performed using C18 Hypersil gold (150mm×4.6mm, 5µm) column at 40°C under isocratic elution. Fluorescence detector (FLD) was set at 360nm and 440nm as excitation and emission, respectively. The use of methanol to replace acetonitrile as the mobile phase resulted in ∼67% peak area enhancement of AFM1. The limit of detection (LOD) and quantification (LOQ) of the analytical method after post-column derivatization without evaporation/reconstitution with mobile phase was 0.0085µgL-1 and 0.025µgL-1 respectively. However, LOD and LOQ improved to 0.002 and 0.004µgL-1 respectively with the addition of evaporation/reconstitution step. The method was statistically validated, showing linear response (R2>0.999), good recoveries (85.2-107.0%) and relative standard deviations (RSD) were found to be ≤7%. The proposed method was applied to determine AFM1 contamination in various types of milk and milk products. Only 2 samples were contaminated with aflatoxin M1 (10% incidence). However, the contamination level is below the Malaysian and European legislation limits.

Determination of Aflatoxin M1 and Chloramphenicol in Milk Based on Background Fluorescence Quenching Immunochromatographic Assay.

Harsh demanding has been exposed on the concentration of aflatoxin M1 (AFM1) and chloramphenicol (CAP) in milk. In this study, we developed a new method based on background fluorescence quenching immunochromatographic assay (bFQICA) to detect AFM1 and CAP in milk. The detection limit for AFM1 was 0.0009 ng/mL, while that for the CAP was 0.0008 ng/mL. The assay variability was determined with 3 AFM1 standards (i.e., 0.25 ng/mL, 0.5 ng/mL, and 1.0 ng/mL), and the actual detection value was 0.2497, 0.5329, and 1.0941, respectively. For the assay variability of 3 CAP standards (i.e., 0.10 ng/mL, 0.30 ng/mL, and 0.50 ng/mL), the actual detection value was 0.0996, 0.3096, and 0.4905, respectively. The recovery rate of AFM1 was 99.7%-101.7%, while that for CAP was 95.3%-97.6%. For the test stability, AFM1 and CAP showed satisfactory test stability even at month 5. Compared with the sensitivity of liquid chromatography-mass spectrometry (LC-MS) method, no statistical difference was noticed in results of the bFQICA. Our method is convenient for the detection of AFM1 and CAP in milk with a test duration of about 8 minutes. Additionally, an internal WiFi facility is provided in the system allowing for quick connection and storage in the intelligent cell phone.

Asymmetric Mach-Zehnder Interferometer Based Biosensors for Aflatoxin M1 Detection.

In this work, we present a study of Aflatoxin M1 detection by photonic biosensors based on Si3N4 Asymmetric Mach-Zehnder Interferometer (aMZI) functionalized with antibodies fragments (Fab'). We measured a best volumetric sensitivity of 104 rad/RIU, leading to a Limit of Detection below 5 × 10(-7) RIU. On sensors functionalized with Fab', we performed specific and non-specific sensing measurements at various toxin concentrations. Reproducibility of the measurements and re-usability of the sensor were also investigated.

Aflatoxin B1 and M1 in milk.

The aflatoxin M1 (AFLAM1) is a mycotoxin that results from the hydroxylation of the aflatoxin B1 (AFLAB1). It contaminates the milk of animals fed with a diet containing its precursor. In this work, we determined the occurrence of AFLAB1 and AFLAM1 in milk, as well as the chromatographic conditions to quantify these mycotoxins. The extraction and quantification of AFLAB1 and AFLAM1 in naturally contaminated and artificially spiked milk samples which are produced and marketed in the state of RS were performed using the AOAC official method and UHPLC with fluorescence detection. We obtained a separation factor of 2.3 for AFLAB1 and AFLAM1 using a mobile phase consisting of 1% acetic acid:acetonitrile:methanol (55:10:35). The analytical curves had a wide linearity range and the limit of quantification (LOQm) concentrations of AFLAB1 and AFLAM1 were equal to 0.5 and 0.25 µg L(-1), respectively. Samples of pasteurized and ultra-high-temperature processed (UHT) milk showed natural contamination, and the levels for both aflatoxins ranged from 0.7 to 1.5 µg L(-1). Raw and concentrated milk samples only contained AFLAM1, with a maximum average concentration of 1.7 µg L(-1). These concentrations, higher than permitted by legislation, confirm the existence of a health risk, as well as highlight the relevance of searching for alternatives to reduce this contamination.

Flow-based impedimetric immunosensor for aflatoxin analysis in milk products.

Label-free detection technique based on impedance was investigated for aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2) analysis in milk products. The impedance change resulting from antigen-antibody interaction was studied using a two-electrode setup made up of silver (Ag) wire. Processed milk such as drinking yogurt and flavored milk samples were analyzed in a flow-based setup. Two microflow pumps were used to construct the flow system where analytes (AFM1 and AFM2) were injected and impedance was measured using functionalized Ag wire electrodes. The flow system was optimized by adjusting both inlet and outlet flows to maintain the reaction volume optimum for impedance measurements. Using Bode plot, the matrix effect was investigated for detection of AFM1 and AFM2 in various matrices. Good recoveries were obtained even at low-AFM1 concentrations in the range of 1-100 pg/mL. The influence of AFM2 on the detection of AFM1 was also investigated. The proposed method provides good scope for online monitoring of such hazardous toxins in milk products.

Simultaneous and rapid detection of six different mycotoxins using an immunochip.

Mycotoxins are highly toxic contaminants in food, animal feed, and commodities. The study has developed an immunochip for quantifying the concentrations of six mycotoxins: aflatoxin B1, aflatoxin M1, deoxynivalenol, ochratoxin A, T-2 toxin, and zearalenone, which were added to drinking water. The complete antigens (Ags) of the mycotoxins were contact printed and immobilized onto agarose-modified glass slides with 12 physically isolated subarrays, based on the reaction of both diffusion and covalent bond. The optimal concentration of each antigen and antibody (Ab) was obtained using an Ag-Ab immunoassay. Based on the indirect competitive immunoassay for the simultaneous detection of six mycotoxins in one single chip, six standard curves with good logistic correlation (R(2)>0.97) were respectively plotted. The working ranges (0.04-1.69, 0.45-3.90, 20.20-69.23, 35.68-363.18, 0.11-1.81, and 0.08-7.47 ng/mL, respectively) were calculated, as well as the median inhibitory concentrations (0.31±0.04, 1.49±0.21, 34.54±1.30, 134.06±11.75, 0.49±0.05, and 1.54±0.22 ng/mL, respectively), when six mycotoxins were detected simultaneously. Finally, the recovery rates in drinking water generally ranged from 80% to 120% on the same chip, with an intra-assay coefficient of variation lower than 15%. We successfully established an immunochip for simultaneous detection of six mycotoxins within 4h, with advantages of using minimal samples and being visually semiquantitative with our naked eyes. In summary, the method could be developed on one single chip for detecting multiple contaminants in actual samples.

Chromatographic method for the determination of aflatoxin M1 in cheese, yogurt, and dairy beverages.

The aim of this work was to develop and validate a method to determine aflatoxin M1 (AFM1) in cheese, yogurt, and dairy beverages. The method consisted of aqueous methanol extraction, immunoaffinity column purification and isolation, RPLC separation, and fluorescence detection. The four types of cheese samples were classified according to moisture and fat content. The mean recoveries were 71% for cheese at spiked levels from 100 to 517 ng/kg, and 76% for yogurt and dairy beverages spiked at levels from 66 to 260 ng/kg. The mean RSDs were 5.9% for cheese, and 10% for yogurt and dairy beverages. The LOD was 3 ng/kg and the LOQ was 10 ng/kg for all test commodities. To test the applicability of the developed method, a small survey of the presence of AFM1 in cheese, yogurt, and dairy beverages purchased in Ribeirão Preto-SP, Brazil, was conducted. AFM1 was detected (> 3 ng/kg) in all samples. Twenty cheese samples (83%) were contaminated with AFM1 in the range of 13-304 ng/kg. In yogurt and dairy beverages, the contamination was lower (13-22 ng/kg) in five samples (42%). The results indicated that the method is adequate for the determination of AFM1 in these four types of cheese, as well as in yogurt and dairy beverages.

Development of an impedimetric aflatoxin M1 biosensor based on a DNA probe and gold nanoparticles.

The present work describes the construction and application of a new DNA biosensor for detection of aflatoxin M1. In order to immobilize a thiol-modified single stranded DNA (ss-HSDNA) probe that specifically bound aflatoxin M1, a self-assembled monolayer of cysteamine and gold nanoparticles on the SAM were prepared on gold electrodes, layer-by-layer. The assembly processes of cysteamine, gold nanoparticles, and ss-HSDNA were monitored with the help of electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. K3[Fe(CN)6]/K4[Fe(CN)6] solution was used as a redox probe for electrochemical measurements. The biosensor provided a linear response to aflatoxin M1 over the concentration range of 1-14 ng/mL with a standard deviation of ±0.36 ng/mL. Finally, the biosensor was applied to a series of real milk samples.

Occurrence of aflatoxin M1 and exposure assessment in Catalonia (Spain)

Antecedentes. La aflatoxina M1 (AFM1) es el principal derivado mono-hidroxilado de la aflatoxina B1 (AFB1), formado en el hígado y excretado en la leche. A pesar de que la AFM1 es menos tóxica que la AFB1, ha sido clasificada como un posible carcinógeno para los humanos, en el Grupo 2B por la Agencia Internacional para la Investigación del Cáncer (IARC). Objetivos. i) Determinar la incidencia de AFM1 en los principales productos lácteos consumidos en Cataluña (España), y ii) evaluar la exposición de la población catalana a esta micotoxina. Métodos. La incidencia de AFM1, se determinó en 72 composites de leche, 72 composites de queso y 72 composites de yogur procedentes de Cataluña. La concentración de AFM1 se analizó con un kit comercial de Ensayo por Inmunoabsorción Ligado a Enzimas (ELISA). En la evaluación de la exposición, se llevaron a cabo tres aproximaciones: un método determínistico y dos modelos probabilísticos mediante simulación por el método Monte Carlo. Resultados. Finalmente, se detectó en el 94,4% (68/72) de las muestras de leche, en el 2,8 % (2/72) de las muestras de yogur, y no se detectó en las muestras de queso. Las concentraciones medias de leche, queso y yogur fueron 9,29±2,61, <12,5 and 13,22±4,82ng/kg, respectivamente. Conclusiones. Según estos valores, no se debería esperar que la población catalana esté expuesta a niveles significativos de riesgo derivados de AFM1, incluyendo grandes consumidores(AU)

Background. Aflatoxin M1 (AFM1) is the main monohydroxylated derivative of aflatoxin B1 (AFB1) formed in liver and excreted into milk. Although AFM1 is less toxic than AFB1, it has been classified as a possible human carcinogen, Group 2B agent by International Agency for Research on Cancer (IARC). Objectives. The objectives of this study were (i) to determine the occurrence of AFM1 in the main dairy products consumed in Catalonia region (Spain), and (ii) to assess the exposure of Catalonian population to aflatoxin M1 through deterministic and probabilistic method. Methods. Occurrence of Aflatoxin M1 (AFM1) was determined in 72 composites of milk, 72 composites of cheese and 72 composites of yoghurt from Catalonia. AFM1 content was analysed using an Enzyme-Linked ImmunoSorbent Assay commercial kit. Three approaches to exposure assessment were conducted: one deterministic method and two probabilistic models with Monte Carlo simulations. Results. AFM1 was detected in 94.4% (68/72) of whole UHT milk samples, in 2.8% (2/72) of yoghurt samples and not detected in cheese. The maximum level was detected in one yoghurt sample with 51.58ng/kg, only this sample being over the legal EU limit of 50ng/kg. Milk, cheese and yoghurt mean concentrations were 9.29±2.61, <12.5 and 13.22±4.82ng/kg, respectively. Conclusions. According to these values, it should be expected Catalonian population is not exposed to a significant risk from aflatoxin M1 including average and high consumers(AU)

Occurrence of aflatoxins in peanuts, milk, and animal feed in Trinidad.

The prevalence of aflatoxin B1 (AFB1) in 186 peanut products (140 peanuts, 32 peanut butter, and 14 nut cakes) from supermarkets, road vendors, and sale outlets, and 40 feed samples from dairy farmers was determined using the radioimmunoassay method (Charm II) test for aflatoxins. The frequency of aflatoxin M1 (AFM1) was also determined in 175 raw milk samples from milk collection centers and 37 pasteurized milk samples obtained from supermarkets and sale outlets. Overall, from a total of 438 samples tested, 18 (4.1%) were positive for aflatoxin comprising 5 (2.2%) of 226 peanut products and feeds positive for AFB1, and 13 (6.1%) of 212 milk samples positive for AFM1. All 186 peanuts and peanut products were negative (0.0%) for AFB1 while 5 (7.4%) of 40 dairy feed samples were positive. Of the 175 raw milk samples tested, 13 (7.4%) were contaminated with AFM1 while all pasteurized milk samples were negative. The detection of AFB1 in feed and AFM1 in milk is of public health importance considering the practice of raw milk consumption by the farmers and their families in the country.

Changes in concentration of aflatoxin M1 during manufacture and storage of skim milk powder.

In this study, skim milk powder was produced from cow's milk contaminated artificially with aflatoxin M1 (AFM1) at two different levels, 1.5 and 3.5 microg/liter (ppb), and the effects of process stages on the AFM1 contents were investigated. Pasteurization, concentration, and spray drying caused losses of about 16, 40, and 68%, respectively, in AFM1 content of the milk contaminated with 1.5 microg/liter AFM1, and losses of 12, 35, and 59%, respectively, in the milk contaminated with 3.5 microg/liter AFM1. These losses were found to be statisticially significant at the level of P < 0.01. After 3- and 6-month storage periods, AFM1 content of the skim milk powder produced from milk with 1.5 microg/liter AFM1 decreased by 2 and 5%, respectively, whereas these rates were 2 and 4%, respectively, for the skim milk powders made from milk with 3.5 microg/liter AFM1 (after adjustment for sample weight). Changes in AFM1 content of milk powder samples were found statistically insignificant (P > 0.05 and P > 0.01) for 3- and 6-month storage periods.

Head space sensor array for the detection of aflatoxin M1 in raw ewe's milk.

A novel screening method was developed for simple and rapid detection of aflatoxin M1 contamination in raw ewe's milk samples without the need for sample pretreatment. The method was based on the use of a commercial head space sensor array system constituted by 12 metal oxide semiconductor sensors, 10 metal oxide semiconductor field-effect transistor sensors, and a pattern recognition software. Twenty-four raw milk samples collected from two different groups of ewes fed with a formulated feed that contained increasing amounts of aflatoxin B1 and six noncontaminated ewe's milk samples were analyzed. The results obtained by using the head space sensor array, processed by statistical methods, made it possible to group the samples according to the presence or the absence of aflatoxin M1. Sample classification was in complete agreement with the aflatoxin M1 content measured by an enzyme-linked immunosorbent assay procedure. This is the first report, to our knowledge, of detection of aflatoxin M1 in ewe's milk by a multisensor array.

Aflatoxin M1 in yoghurts in Portugal.

Aflatoxin M1 (AFM1) may occur in milk and milk products, resulting from the ingestion of aflatoxin B1 in feedstuffs by dairy cow. Ninety-six samples of commercial yoghurts (48 natural yoghurts and 48 yoghurt with pieces of strawberries) that are produced in Portugal were analyzed for the presence of AFM1 by immunoaffinity column extraction and high-performance liquid chromatography (HPLC). The limit of detection was 10 ng/kg. The recoveries of AFM1 from the samples spiked at levels of 10.0, 50.0, 100.0 and 150.0 ng/kg were 88.0%, 91.0%, 93.0% and 99.0%, respectively. AFM1 was detected in 18 (18.8%) of yogurt samples ranging from 19 to 98 ng/kg, and 78 samples (81.2%) did not reveal the presence of the toxin. Of the 48 natural yoghurts tested, only two (4.2%) were contaminated with 43 and 45 ng/kg of AFM1. Of the 48 yoghurts with pieces of strawberries tested, 16 samples (33.3%) contained levels ranging from 19 to 98 ng/kg; six samples (12.5%) were contaminated with low levels ranging from 19 to 35 ng/kg; four samples (8.3%) were contaminated with levels ranging from 36 to 50 ng/kg, two samples (4.2%) with levels of 51 and 65 ng/kg and four samples (8.3%) presented high contamination levels, from 90 to 98 ng/kg. This paper reports the data of the first survey on the presence of AFM1 in yoghurt in Portugal.